BIO 404 CSUSM Biology Main Steps of Gas Exchange Questions
Homework Practice Questions (20 points total, but I’ve indicated the points each question would be worth on an exam) Q1. There are two main steps of gas exchange between the environment and embryo in an amphibian egg. Describe what these two steps are and how each step may help or hinder oxygen available to the embryo. (6pts) Q2. How does energy use during development compare in a reptile egg (such as the common dwarf skink) that is similar in size to an amphibian egg? How is this related to gas exchange? (8pts) Conductance (ml g-1 h-1 °C-1) Body Temperature (°C) O2 Consumption (ml g-1 h-1) Q3. Describe how oxygen consumption, body temperature and conductance change with ambient temperature when sugar gliders are 79 days old (top graphs) and 99 days old (bottom graphs). How does this show us how we can use a decrease in ambient temperature to determine thermoregulatory ability in developing mammals? (10pts) The figure shows O2 consumption, 79 days; 19 g body temperature, and conductance of sugar gliders at 99 days; 79 days of age (top panel) and 50 g 99 days of age (bottom panel) as ambient temperature is decreased from Ambient Ambient Ambient 30°C to 15°C. Temperature (°C) Temperature (°C) Temperature (°C) Q4. Fetal circulation differs from adult circulation due to the presence of three shunts: the ductus venosus, ductus arteriosis and foramen ovale. Describe the location and purpose of each of each of these. (6pts) Q5. Discuss how it is possible for hypoxia to be beneficial and detrimental for a developing mammalian fetus. (10pts) Jelly layers Vitelline membrane Perivitelline fluid Embryo Yolk . VO2 = GO2 (PO2out – PO2in) . VO2 = rate of O2 consumption GO2 = O2 conductance (permeability) of capsule O2 PO2out = PO2 outside the egg PO2in = PO2 inside the egg capsule 0.08 0.009 0.07 0.008 0.06 0.007 0.006 0.05 0.005 0.04 0.004 0.03 0.003 0.02 0.002 0.01 0.001 0 0 0 10 20 Age (Days) 30 40 GO2 (µmol h-1 kPa-1) Internal PO2 (kPa) 20 18 16 14 12 10 8 6 4 2 0 VO₂ (µmol hˉ¹) Bibron’s toadlet Forms early in fish development but gradually in amphibians Cutaneous cilia and embryonic movements create convective movement within the fluid Cilia first appear at end of neural tube formation Peak near hatching and then disappear after hatching Pickerel frog Perivitelline PO₂ (kPa) 20 18 16 14 12 10 8 6 4 with convection without convection 2 0 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Distance from embryo (mm) 1.8 2 Single Egg Perivitellline Perivitelline space space Egg Mass O2 Jelly Jelly Embryo Embryo Vitelline Vitelline membrane membrane Embryos competing for O2 Jelly helps to separate eggs Aquatic gelatinous egg masses need O2 from all sides Suspend in water with a foam raft Egg masses contain 200-300 embryos Only a couple inches wide PO2 (kPa) Depth (cm) 0 5 10 15 20 1 2 3 4 PO2 (kPa) Depth (cm) 0 5 10 15 20 1 2 PO2 (kPa) Depth (cm) 0 1 2 3 5 10 15 20 Larger Egg Masses – 1000-2000 eggs Need convection to deliver O2 to eggs in middle of nest PO2 (kPa) 0 Depth (cm) 2 4 6 8 10 12 14 5 10 15 20 Wood Frog Egg mass External Currents Wind-generated agitation Changes in density of water Sunlight can heat egg masses so temperature > surrounding water At night, the convection currents reverse and water descends through mass Amphibian Reptile Vitelline Membrane Perivitelline Space Subembryonic Space Yolk Jelly Layers Albumen ? Shell Membranes (novel) Oxygen Diffusion Pathways O2 Jelly Amphibian Egg Perivitelline Space Vitelline Membrane O2 Bird Egg Shell Pore Shell Membranes Chorioallantois Oxygen diffuses 300,000 times faster through air than through jelly Amniote Embryo Yolk Sac Amnion Chorioallantois (Chorion + Allantois) Inner Shell Membrane Outer Shell Membrane Air Cell Shell Embryo3 Yolk sac Ostrich 1 week Allantois O2 Consumption Rate (ml h-1) Mallee Fowl normoxia hyperoxia Internal Gas Pressures P V = G Internal Gas Pressures V = G VO2 P VO2 pre-hatch PO2 in Incubation Time (days) Incubation Time (days) Incubation Time (days) VO2 pre-hatch VCO2 VO2 PCO2 in (ml/day) Hatch VCO2 (ml/day) PO2 in PCO2 in Incubation Time (days) Hatch Incubation Time (days) 179 Incubation Time (days) CAM respiration Internal pipping External pipping Mallee Fowl Brush Turkey Buried eggs experience: Higher humidity Lower water loss compared to other bird eggs Only have a small bubble of gas under shell rather than air cell No air cell to help in transition to lung respiration Cannot breathe until shell and membranes break and fluid drains away around head Higher CO2 & lower O2 Nest atmosphere depends on: Rates of respiration of eggs Decomposition of organic matter Resistance to diffusion in the surrounding material Eggs heated by sun (in excavations in the sand), geothermal activity or organic decomposition of surrounding litter . Egg VO2 = 20 ml h-1 20 eggs in. mound Total VO2 = 400 ml h-1 C Po 2 PCO Pco 2 °C PO 2 2 156 20.8 144 19.2 133 17.7 131 17.5 130 132 17.3 133 17.7 133 17.7 134 17.9 00 2020 15 2.0 2525 24 3.2 3030 24 23 23 3.1 21 35 21 2.8 3030 23 3.1 GROUND LEVEL 2525 0 0 20 50 40 100 60 150 13.7 L h-1 is consumed in the nest Embryos contribute just 3% to O2 consumption 80 inches 200 Above ground = selective pressure to reduce eggshell conductance to avoid excessive evaporation Underground = selection for increased shell conductance in hypoxia and hypercapnia Mallee fowl Brush Turkey Chicken O2 conductance (ml day-1 kPa-1) 162 357 116 Compared to Predicted 35% higher 120% higher Mound PCO2 (kPa) 4 9 5 (in egg) Brush turkey 0.34 Chicken 0.40 Mallee fowl 0.27 Thickness (mm) Thin shell is less costly for adults to make and helps with rapid hatching Eggs are usually incubated around 30°C Many eggs take up water during incubation from substrate Opposite of most bird eggs Water vapor can diffuse in both directions Higher nest humidity = higher eggshell water vapor conductance compared to bird eggs Individual egg O2 consumption is lower compared to birds But clutches of 30 – 200 eggs and the hatchlings are large Overall O2 consumption of the clutch is quite high 19 Loggerhead turtle . Partial Pressure (kPa) VO2 (ml day-1) 16 13 10 7 4 1 May be a limit on clutch size Green turtle Embryonic metabolism and gas exchange Embryonic amphibians and fishes require oxygen from the environment for survival, development and growth Amphibians and fishes are collectively titled anamniotes, as the embryo does not produce an amnion Eggs are not contained within a shell but surrounded by an egg capsule consisting of a vitelline membrane and jelly layers through which liquid water can move Oxygen then moves through the capsule via diffusion An important trait of diffusion to remember is that it is slow We can describe diffusive movement by the Fick equation V̇O2 = rate of oxygen consumption (but also the rate of oxygen diffusion) Conductance (GO2) = ease of movement of oxygen through the jelly Depends on egg capsule morphology and Krogh’s coefficient of oxygen diffusion (KO2, a measure of oxygen permeability – KO2 of egg capsule jelly is 75% of water A good way to think about conductance is as the inverse of resistance Changes during development – amphibian egg V̇O2 and GO2 and internal oxygen conditions Example of an Australian frog – called Bibron’s toadlet which lays eggs terrestrially One of my study species during my PhD ̇VO2 increases throughout development until hatching – as the embryos grow the overall demand for oxygen increases Conductance (calculated from measuring the internal and external diameters of the egg capsule) tracks along with the increase in V̇O2 – so should help to supply O2 to the embryo (refer to Fick equation) It is possible to look at this by calculating the PO2 inside the egg or directly measuring it Internal PO2 does drop later in embryonic development – so even though GO2 increases, there is still a potential O2 limitation This was measured in the middle of the egg (this will become an important in coming slides) Think about what the embryo might do to overcome this decreased internal oxygen (remembering that diffusion is slow) Perivitelline fluid convection Cutaneous cilia and embryonic movements create convective movement within the perivitelline fluid inside the egg First appearance of ciliation differs between species, but it generally develops around neural tube formation, peaks near hatching and disappears after hatching Suggests that the appearance of ciliated cells matches the increasing respiratory demands during embryonic development When the embryo hatches it can swim to mix the surrounding water as well as actively seek oxygenated environments Fluid movement within the perivitelline space is further enhanced once embryos develop the ability for muscular movements The direction of flow and current velocity within fluid was measured in pickerel frog eggs But when I was doing my PhD no one had looked at how convective flow influenced overall gas exchange – my goal was to quantify its contribution Convection and Internal PO2 Bibron’s toadlet lacks external gills, so must rely on a well-mixed perivitelline fluid to deliver oxygen to the embryo’s surface for diffusion across the skin My approach = prevent convection in the perivitelline space Creates an egg in which the embryo is directly surrounded by jelly and diffusion is only process of gas exchange The graph shows internal PO2 on the y axis and distance from the embryo on the x axis, so the brown circle is the embryo and the membrane and jelly are on the right. When the eggs do have convection internal PO2 drops from about 17.5 to 15.5 kPa, so barely any change at all When convection is stopped internal PO2 drops from 18 kPa down to below 12 kPa next to the embryo So how does the lack of convection influence the PO2 gradient? What does this tell you about the role of convection in gas exchange inside the egg? Egg masses So far, we have focused on individual eggs, but many amphibian species develop in egg masses Embryos may compete for O2 with other embryos in the mass Some species overcome this by laying strings, sheets, isolated clusters of eggs But some lay globular egg masses in which interior embryos may be limited for O2 In individual eggs the jelly coat may reduce oxygen uptake, but in an egg mass, it may facilitate oxygen uptake – how? Spotted marsh frog – aquatic gelatinous egg masses Can have lots of embryos if O2 can diffuse from all directions Spotted marsh frog egg mass suspended in water with a foam raft O2 profile of floating egg mass shows O2 diffusing from all directions If foam raft is removed – egg mass sinks to the bottom and anoxic conditions occur even in well oxygenated water Embryos more than 1.3cm from water are oxygen deprived, developmentally delayed and often die Some populations of spotted marsh frog do not create foam rafts but instead use vegetation to support the mass Wood frog Some other frog species lay eggs masses that are larger than 10cm in diameter and contain over 10,000 eggs Some have embryos in center of mass that are developmentally delayed, in others the O2 remains high in the center of the mass and embryos develop at same rate as exterior embryos Diffusion can only supply superficial part of mass, so these egg masses must also have convection Convection is possible when there are water channels between eggs Wood frog – inject dye and you can see the dye flow around the eggs – revealing the spaces between the eggs through which water can move – helping to create convective movement of oxygen But how does the water flow? Could be by external current, wind-generated agitation, or changes in density of water Sunlight can heat egg masses to be a few degrees above surrounding water Water then moves by upward convection currents through the mass At night, the convection currents reverse and water descends through mass Birds and reptiles = amniote eggs Bird and reptiles have similar structures to amphibian and fish eggs, but with one obvious difference – an egg surrounded by a shell Birds tend to have a hard eggshell, while most reptiles have softer eggshells some exceptions – crocodiles, geckos and some tortoises lay hard eggs Bird eggs are heated by the parent Reptiles eggs are heated by surrounding environment, not by parent so can be softer as do not need to worry about being squashed Reptile eggs are generally all hidden (buried) and so are all white (don’t need cryptic color like you see in some bird eggs) Bird and reptile eggs can be much larger than amphibian and fish eggs Because now convection takes over for gas exchange rather than diffusion O2 diffusion pathway Diffusion distance is the thickness of the eggshell Diffusion occurs through the pores of the eggshell This is important because oxygen in particular diffuses 300,000 times faster through the air than through jelly Amniote eggs The eggshell and chorioallantoic membrane (CAM) are what have allowed amniote embryos to be larger in size than the fish and amphibians Diffusion alone would be inadequate, CAM allows convective gas exchange in larger eggs Remember from our developmental biology lecture, bird and reptile embryos have 4 extraembryonic membranes Yolk sac, amnion (surrounds the embryo and not present in fish and amphibians), chorion and allantois The last two form the chorioallantois = main gas exchange organ during embryonic development before lungs function On day 12 in the chicken the distance between capillaries in the CAM and the inner shell membrane is just 1 um (short diffusion distance!) Picture of 1 week old ostrich embryo showing the embryo sitting on top of yolk sac and the blood vessels that have already formed that help transfer energy from yolk to embryo Also see the forming of the allantois – which will expand each day and eventually grow around the entire inside surface of the eggshell Attaches to shell around day 10 in chicken, fully formed by day 12 (of a 21 day incubation) O2 consumption rate Just like we see in most fishes and amphibians – as embryonic development progresses the oxygen consumption rate of bird and reptile embryos increases O2 consumption rate increases, but permeability of shell to gases does not change throughout development Internal gas partial pressures How might this change the levels of oxygen and carbon dioxide inside the egg? Think about the Fick equation here: V̇O2 = GO2 (PO2out – PO2in) Transition Near Hatching By day 17 body rotations move the embryo so that the beak is in contact with the inner membrane of the air cell Eventually through wearing and tearing the membrane is pierced = internal pipping Peak then pierces the eggshell = external pipping Coordinated backward movements of the head that working with the beak tooth acts as a saw and cuts the shell Tertiary bronchi in the lungs are aerated after the onset of breathing movements At first, breathing is irregular and sporadic, short bursts with variable breath-to-breath time intervals Early post hatch – breathing is shallow and rapid – similar to postnatal hours of newborn infant mammal But slowing the lungs take over as the main site of oxygen uptake while the CAM degrades (and is gone upon complete hatching) Underground nesting – birds Many birds lay in nests or on the ground But many birds and reptiles also lay their eggs underground – this can have significant implications for gas exchange Megapode birds (e.g. mallee fowl, brush turkey) lay eggs in mounds or underground Buried eggs experience higher humidity, higher CO2 and lower O2 High humidity means lower water loss compared to other bird eggs Only have a small bubble of gas under shell rather than air cell Don’t have air cell to help in transition to lung respiration Megapode birds cannot breathe until shell and membranes and broken and fluid drains away around head Nest atmosphere depends not only on rates of respiration of eggs, but also decomposition of organic matter and resistance to diffusion in the material surrounding eggs Eggs heated by sun (in excavations in the sand), geothermal activity or organic decomposition of surrounding litter Brush turkey mound Nests vary in size and shape – related to the degree of reliance on organic decomposition as a heat source Despite various sources of heat, megapode eggs are at similar temperatures to other bird eggs (31 – 39 C, average 36C) Central temperature maintained remarkably constant by males Dig down to eggs to check temp with head or beak Open or close mound as needed Organic decomposition is aerobic – occurs by actions of microorganisms and respiration by the plant material itself Looking at the temperature and O2 and CO2 profiles indicate that both heat and gases move through the mound primarily by diffusion What contributes to O2 consumption in the nest? If we use a mean V̇O2 of a brush turkey egg throughout incubation = 20 ml/hr With up to 20 eggs in the mound total V̇O2 of eggs = 400 ml/hr Measurements have indicated that 13.7 ml/hr is consumed in the nest So embryos contribute just 3% to O2 consumption, rest comes from mound material So mound material is important both for determining temperature and O2 levels Adaptations of shells For eggs above ground there is a balance between high eggshell conductance to obtain oxygen but low conductance to prevent excessive evaporation This selective pressure is relaxed below ground In nests that can be hypoxic and hypercapnic there is selection for increased shell conductance E.g. Mallee fowl shell conductance is 35% higher than predicted for a normal egg in air (based on egg mass and incubation time) Brush turkey conductance is 120% above predicted Alter conductance by having thinner shells Thin shell is not a liability in mound environment (won’t lose a lot of water), Thin shell is also less costly for adults (less energy and material for shell) Also helps with rapid hatching Underground nesting – reptiles All marine and most semi-aquatic and terrestrial turtles Some large lizards and all alligators and crocodiles Individual egg O2 consumption is lower in reptiles compared to birds But reptiles often have egg clutches of 30 – 200 eggs and the hatchlings are large, overall O2 consumption of the clutch is quite high In nests of green and loggerhead turtles PO2 falls to 11-15 kPa and PCO2 increases to 4-7 kPa Turtle egg gas exchange Increased shell conductance in buried eggs may be viewed as an adaptation for the soil or plant material resistance to diffusion Another important factor determining gas levels is the metabolic rate of the eggs It is possible for a nest of eggs to be so large that late in development diffusion would become inadequate Thus, there might be a limit on clutch size This might explain why marine turtles are so selective with where to lay eggs Often choose beaches with suitable gas transport properties What do you think are the key points/concepts to focus on from this PowerPoint? Differences in how energy is partitioned compared to adults E.g. Developing animals allocate more energy to growth Mass-specific metabolic rates are higher Early life stages operate under tighter energy constraints than older animals Particularly true for egg developing animals Subtle differences in energy use during early life can have a significant impact on overall fitness Knowledge of developmental energetics can contribute to our understanding of developmental events Most organ systems are directly or indirectly involved in energy metabolism Energy use can have a big impact on when and in what order organs form Most studies in this area focus on fishes, birds and reptiles Growth dominates embryonic energy budgets in fishes Around 80% of energy is allocated to growth Leaves just 20% for all other activities Eggs are closed systems with respect to energy Yolk is used to support processes such as cell proliferation and growth, cell differentiation, morphogenesis, organogenesis, internal transport, nerve function, osmoregulation, skeletal muscle activity Can try to model how energy is partitioned between different processes A=P+R+E A = Assimilated energy Assumed to be energy of yolk consumed P = Production Energy content of tissue produced R= Total amount of energy expended on metabolic processes E = Excretory losses of energy Mainly related to nitrogenous wastes Small fraction of yolk energy and so is often not measured/mentioned A=P+R+E Production (P) High early in life Most species display a sigmoid curve where mass-specific growth rate declines with size and age A=P+R+E Metabolic Energy (R) Amount of energy mobilized during development Not stored as production or used for external work Energy available to carry out life processes Instantaneous Allocation of metabolic energy: Rt = Rg + Rm + Ra Rt = total metabolic energy Rg = Cost of growth Rm = Cost of maintenance Ra = Cost of other activities Cumulative Rt = Rg + Rm + Ra Growth (Rg) Often includes differentiation Some more complex mathematical models separate growth and differentiation Rt = Rg + Rm + Ra Maintenance (Rm) Energy use associated with upkeep and repair of existing tissue under optimal conditions Protein turnover, maintenance of ionic and electrical gradients, cell repair, substrate recycling Separate from costs associated with growth, development and locomotion Different from adult animals = energy use when animal neither gains nor loses weight Expect maintenance to increase as organ systems are formed and begin to function Rt = Rg + Rm + Ra Activity (Ra) Highly variable = depends on species, stage of development and environmental conditions Two types of activities of embryos and larvae: 1. Neuromuscular activity = body movement Generally low in early embryonic stages Increases in free swimming fish larvae 2. Activities associated with maintenance of whole-body homeostasis under less than optimal conditions Technically maintenance but not under optimal conditions State of development at hatching can be very different Altricial Precocial Naked Can’t leave the nest Closed eyes Must be fed and kept warm Covered in down Open eyes Capable of thermoregulation, locomotion & feeding Altricial < Precocial 30-50% Procellariiformes (seabirds) = 105 kJ Precocial = 93 kJ Altricial = 52 kJ 50g egg Higher energy demand in precocials is reflected in energy in egg at laying Altricial = 6% lipids (7 kJ/g) Precocial = 10% lipids (5 kJ/g) Goose Cattle Egret MR = a × GR + b × M MR = metabolic rate GR = absolute growth rate M = embryo mass (wet mass) Assume cost of growth and maintenance do not vary with the composition of the embryo Can have different % water content at different times a = coefficient of energy cost of growth b = coefficient that represents cost of maintenance Goose Altricial cattle egret: Growth rate, cost of growth, and total MR all increase throughout incubation Precocial goose: Late incubation plateau of MR results from a decrease in energy expenditure for growth as growth rate declines Cattle Egret Higher energy cost of development in precocial species Results from more rapid growth early in incubation 70% of way through incubation period precocial embryos are on average 35% larger than altricial embryos More mass to maintain Must be other adaptive significance of precocial mode of development if it is more costly hatch = 16d 45 25 °C O₂ Consumption (nmol h¯¹) 40 hatch = 29d 20 °C 35 30 25 hatch = 72d 20 15 °C 0.160 mL 15 0.233 mL 10 0.358 mL 5 0 0 10 20 30 40 Age (Days) 50 60 70 Total oxygen consumed (mL) Hatchling gut-free dry mass (mg) Cost of development (mL mg-1 ) 15 ˚C 20 ˚C 25 ˚C 0.358 0.233 0.160 ×2 1.74 ± 0.2 0.206 = 1.65 ± 0.5 = 1.71 ± 0.5 0.141 ×2 0.094 Cost of embryonic development (mL mg-1) 0.30M0.22 ± 0.13 r2 = 0.52 1.00 B. nimbus O. tshawytscha P. bibronii G. morhua P. americanus C. nasus C. xerampelina H. hippoglossus C. chanos 0.10 O. mykiss G. vitellina C. georgiana 61% 42% 28% S. alpinus N. forsteri M. salmoides Amphibians Fishes 0.01 0.01 0.10 1.00 Hatchling gut-free dry mass (mg) 10.00 Cost of development (mL mg¯¹) 3 birds reptiles amphibians and fishes 0.3 Difficult to directly study the comparison of anamniote eggs versus amniote eggs because sizes don’t overlap 0.03 0.01 0.1 1 10 100 1000 Gut-free dry hatchling mass (mg) 10000 100000 One of the smallest lizard eggs @ ~100 mg Do reptilian embryos have higher energy cost compared to amphibian embryos from similar sized eggs? 12.3 ml Bomb Calorimetry = energy content determined by combusting material & measuring heat produced Total energy density: Fresh eggs = 608 J Hatchlings = 365 J 243 J lost during incubation All nutrients required for development are contained in the egg Only exchange respiratory gases and water with environment Sum of energy content of the hatchling and energy lost by metabolism should equal that of the egg when it was laid Calculations based on 12.3 ml of O2 consumed Using cost of development data for other reptiles, we can predict cost based on egg mass or hatchling mass Cost (kJ) = 2.141 * egg mass(g)0.818 Cost (kJ) = 2.647 * hatchling mass(g)0.886 Common dwarf skink: Cost based on egg mass (0.105 g) = 339 J Cost based on hatchling mass (0.068 g) = 245 J Almost identical to that measured with bomb calorimetry (243 J) Maximum rates of O2 consumption in amphibian eggs of similar size = only 1/3 those measured in the skink Eggs of Monterey ensatina (lungless salamander) are bigger Max. O2 consumption rate of ~14 µL h-1 (compared to ~30 µL h-1 in skink) Why can reptiles have such higher energy use? Energy use during development Energy is the common currency of life How an animal allocates energy to various tasks provides insights into the relative importance of those activities We know less about how energy is partitioned during development than later in life What we do know, is that there are some major differences in energy use during development versus later in life For example – developing animals allocate more energy to growth Energy constraints during early life stages particularly true for eggs as energy that is available is fixed when the eggs are laid Mass-specific metabolic rates (how much O2 consumed per unit tissue) tends to be much higher in embryos compared to adults Energy partitioning Early studies in this area showed that growth dominates embryonic energy budgets somewhere around 80% of energy is allocated to growth leaves just 20% for all other activities Eggs are closed systems with respect to energy Yolk is used to support processes such as cell proliferation and growth, cell differentiation, morphogenesis, organogenesis, internal transport processes, nerve function, osmoregulation, skeletal muscle activity Partitioning of energy is often expressed in terms of an energy balance equation: A = P+R+E A = assimilated energy Assumed to be energy content of yolk consumed P = energy content of tissue produced R= total amount of energy expended on metabolic processes E = excretory losses or energy (mainly related to nitrogenous wastes) Small fraction of yolk energy and so is often not measured/estimated/mentioned Production (P) Tissue production is high early in life Most species display a sigmoid curve Metabolic energy (R) Amount of energy mobilized during development that is not stored as production or used to perform external work Energy available to carry out life processes Do you remember from BIOL 353 in what form this is ultimately lost from an animal? Can look at energy allocation with metabolic energy Rt = Rg +Rm + Ra Rt = total metabolic energy Rg = energy for growth Rm = energy for maintenance Ra = energy for other activities Can think about this as instantaneous energy use – what is the animal using at any one time, versus cumulative energy use – what animal is using over a certain time period (all of embryonic development, gastrulation etc.) How would we measure instantaneous energy use? To measure cumulative energy use we can integrate metabolic rate over time (are under curve) Growth Often referred to as the cost of growth but does often include differentiation (the process of developing rather than just getting larger) The graph shows a model in which oxygen consumption of Bibron’s toadlet embryos and tadpoles is divided into the process of growth and maturation (differentiation) and maintenance of the tissue (do not worry about assimilation) Maintenance Energy use associated with upkeep and repair of existing tissue under optimal conditions Includes protein turnover, maintenance of ionic and electrical gradients, cell repair, substrate recycling Separate from costs associated with growth and development and locomotion Different from adult animals In adults, maintenance is defined as rate of energy when animal neither gains nor loses weight Expect maintenance to increase as organ systems are formed and begin to function Times during development when maintenance might significantly drop – like diapause or suspended development No protein turnover, no cell repair/replacement Activity Highly variable depending on species, stage of development and environmental conditions Two types of activities of embryos and larvae Neuromuscular activity Body movement Generally low in early embryonic stages, but increases as embryos become more active, and increases again in free swimming larvae Activities associated with maintenance of whole-body homeostasis under less than optimal conditions Technically maintenance but not under optimal conditions Energy use in bird embryos State of development at hatching ranges from nearly helpless altricial chicks to highly precocial chicks Intermediates are common Patterns of metabolism are different Precocial pattern Exemplified by the chicken or duck egg V̇O2 nearly exponentially until about 80% of the way through incubation Relatively constant after, until embryo pierces inner shell membrane – internally pips (IP) Metabolic rate suddenly increases at IP and then again at EP (hatching) End up with a sigmoidal type shape – V̇O2 increases most rapidly in the middle of embryonic development In the large eggs and slow developing embryos of ratites – ostrich, rhea (native to S. America), emu – metabolic rate declines for 7-10 days before hatching Altricial pattern V̇O2 increases more slowly than precocial species, but increases continuously and at an accelerating rate throughout incubation At 80% of incubation, metabolic rate is near 100% of the pre-IP rate in precocial species But only 65% of pre-IP rate of altricial species Difference between precocial and altricial means for eggs of similar size and incubation length, altricial species use less energy during embryonic development Determine this by integrating metabolic rate over the entire incubation period Or by determining energy in egg at the beginning and end of incubation Energy cost of development Plot the energy cost against initial egg mass on log-log axes and the altricial and precocial species fall on different lines Altricial species line is 30-50% below the line for precocial species For example, 50g egg – altricial species uses 52kJ, precocial species uses 93 kJ Birds of the order procellariiformes (seabirds – including albatrosses and petrels) are a special case Altricial, but energy use above regression line for either altricial or precocial species May be due to their extremely long incubation periods Higher energy demand for precocial development is reflected in energy stored at time the egg is laid Altricial eggs contain 4-7% lipids (equates to ~7.03 kJ/g), precocial contain 9-12% lipids (~5.09 kJ/g) Patterns of embryonic growth are really what explain these differences Time course of growth is different in altricial v precocial Precocial (goose) = embryo mass approaches hatchling mass as early as 80% of the way through incubation Growth rate declines near end of incubation Altricial (cattle egret) = embryo mass and growth rate increase continuously throughout incubation Overall: altricial v precocial birds Higher energy cost of development in precocial species compared with altricial results from more rapid growth early in incubation 70% of way through incubation period – precocial embryo averages 35% larger than altricial embryo more to maintain must be other adaptive significance to this mode of development if it is more costly What might be some of these benefits? Australian lungfish cost of development Maintenance costs tend to be related to long incubation times See this in my lungfish embryo cost of development study Lungfish embryos incubated at 3 temperatures At warmer temperature oxygen consumption increases, but because development rate also increases the total oxygen consumed by the embryos to hatching is lower at warmer temperatures (numbers in shaded areas under curves on figure) In fact, the total oxygen consumed is 2X higher at 15°C compared to 25°C Little change in hatching gut-free dry mass with temperature Therefore cost of development (total oxygen consumed/hatchling gut-free dry mass) also decreases with temperature When this cost of the lungfish is compared to other fishes and amphibians it shows that the species has a relatively low cost When trying to compare across different animals groups, it is harder to compare cost of development because the sizes of the hatchlings do not overlap much Common dwarf skink Bringing together the last two lectures with this case study One of the world’s smallest lizard eggs Our last PPT about gas exchange in amphibian/fish and bird/reptile eggs talked about the differences between these groups Most studies of reptilian eggs have been in those larger than amphibians So cannot do a direct comparison on energy use for similar sized eggs Common dwarf skink provides a great opportunity to use small reptilian eggs to test the hypothesis that metabolic rate is greater in reptile eggs than in amphibian eggs of similar size Closed system respirometry used to measure O2 consumption and CO2 production Chamber = glass jar with a metal lid – sound familiar to those in lab? Rates measured periodically throughout incubation (5-9 measurements per egg) O2 consumption increased and total O2 consumed averaged 12 ml Direct calorimetry Energy content of fresh egg and hatchlings determined by calorimetry (bomb calorimetry, basically combust the material and measure the heat) Energy density of hatchings was 23 J/mg, which is significantly less than eggs at laying, which is 29 J/mg Total energy density is 608 J in fresh egg and 365 J in hatchings – so 243 J lost during incubation All nutrients required for development are contained in the egg Only exchange respiratory gases and water with environment So, sum of energy content of the hatchling and energy lost by metabolism should equal that of the egg when it was laid 243 J lost in form of metabolic energy Is this supported by the data? Estimated that 12.3 ml of O2 consumed Respiratory exchange ratio (CO2 production/O2 consumption) = 0.765 Suggests energy being used is ~45% lipid and 55% protein So consumption of one liter of O2 is = to 19.15 kJ of energy This results in the O2 consumed during development equally 235 J – very close to the 243 J based on the bomb calorimetry Comparison with amphibians Using cost of development data for other reptiles, we can predict cost based on egg mass or hatchling mass Cost (kJ) = 2.141 * egg mass0.818 Cost (kJ) = 2.647 * hatchling mass0.886 Put in egg mass of common dwarf skink in first equation = 339 J Hatchling mass of common dwarf skink in second equation = 241 J (second number almost identical to that measured with bomb calorimetry Maximum rates of O2 consumption in amphibian eggs of similar size to common dwarf skink are only about 1/3 those measured in the skink Even eggs of some lungless salamanders such as the Monterey ensatina are bigger But have an max O2 consumption rate of ~14 uL/h (which is half that of max rate in skink) Seems unlikely that egg capsule seen in amphibian eggs would be able to supply the O2 needed in late stage skink embryos Why can reptiles have such higher energy use? Think about the differences in egg structures and gas exchange What do you think are the key points/concepts to focus on from this PowerPoint? The heart is the first organ to function within an embryo In humans, begins at beginning of the fourth week Nutritional and oxygen requirements of the growing embryo can no longer be met by diffusion from the placenta. The heart initially forms from two tubes located on either side of the embryo in the cranial (head) region Form early blood vessels Disc-like embryo undergoes folding, in which both the cranial and lateral parts of the embryo fold ventrally (forwards) This brings the heart-forming region to a ventral (frontal) position The heart tubes fuse in the ventral midline to form a single primordial heart tube The mesenchyme surrounding the tube condenses to form the myoepicardial mantle (the future myocardium) Gelatinous connective tissue called cardiac jelly separates the mantle from the endothelial heart tube (the future endocardium) A series of constrictions divide the heart tube into sections From cranial to cordal: Truncus arteriosus Becomes pulmonary vein & aorta Bulbus cordis Becomes part of right ventricle Primordial ventricle Primordial atrium Sinus venosus Becomes the ends of the major veins carrying blood to the heart and part of the atria 3 paired veins drain into heart Vitelline veins drain blood from yolk sac Associated with formation of liver Umbilical veins bring oxygenated blood from chorion (early placenta) Intersegmental arteries form branches off dorsal aorta (mostly transient – some form vertebral arteries Vitelline arteries to the yolk sac and later to primitive gut Three remain in adults – celiac, superior mesenteric and inferior mesenteric arteries 3 sets of paired arteries Umbilical arteries carry O2-depleted blood to placenta Looping of the heart tube allows the straight heart tube to form a more complex structure reminiscent of the adult heart Steps of looping: 1. The straight heart tube begins to elongate with simultaneous growth in the bulbus cordis and primitive ventricle 2. This forces the heart to bend ventrally & rotate to the right Forms a C-shaped loop 3. The ventricular bend moves caudally and the distance between the outflow and inflow tracts decreases 4. The atrial and outflow poles converge and myocardial cells are added, forming the truncus arteriosus An S-shape is formed First bend of the ‘S’ = large ventricular bend Second bend = junction of the atrium and sinus venosus The internal heart undergoes significant changes in order to form the atria and ventricles of the adult heart 1. Cells from the dorsal and ventral (back and front) walls of the heart grow and form endocardial cushions These grow towards each other and fuse to Ventral form the left and right atrioventricular canals Dorsal 2. Within the primordial atrium a septum (the septum primum) grows towards the endocardial cushions The space between the cushions and septum is known as the foramen (or ostium) primum Ventral view of heart 3. A second septum (septum secundum) develops on the right of the septum primum Ventral view of heart 4. A primordial muscular ridge exists in the floor of the ventricle As the left and right ventricles grow, their medial (midline) walls fuse to form the interventricular septum 5. Small ridges develop within the bulbus cordis and truncus arteriosus (which form the outflow tract) They are continuous throughout the outflow tract and form a spiral shape 6. As these ridges fuse they create a spiral shaped septum throughout the outflow tract The original outflow tract is separated into both the aorta and pulmonary trunk Heart begins to resemble adult form (2 atria, 2 ventricles, aorta connected with left ventricle, pulmonary trunk connected with right ventricle) Differs from adult circulation predominantly due to the presence of 3 major vascular shunts The main function of these shunts is to redirect oxygenated blood away from the lungs, liver and kidney Whose functions are performed by the placenta Ductus venosus Shunts a portion of the left umbilical vein blood flow directly to the inferior vena cava Oxygenated blood from the placenta bypasses liver Ductus arteriosus Shunts blood from pulmonary artery (leaving right ventricle) to aorta Blood bypasses lungs Foramen ovale Shunts blood from right atrium to left atrium Directs mixture of oxygenated blood from umbilical vein and deoxygenated blood from lower limbs and abdominal organs back out the to the body (bypasses lungs) The alveoli fill with air The pulmonary vessels dilate and decrease the pressure in the pulmonary system Decreased pressure in the pulmonary arteries prevents blood from being shunted through the ductus arteriosus Usually closes within 10 to 15 hours of birth Blood flowing back into the left atrium from the lungs increases the pressure in that chamber Presses the septum primum against the septum secundum, which closes the foramen ovale At the same time, spontaneous constriction of the umbilical vessels (or clamping) cuts off the exchange of blood with the placenta Important genes, transcription factors, & signalling molecules in steps of cardiovascular development Tbx5 & its role in septation Expressed throughout developing amphibian heart Birds and mammals have a steep gradient High in left ventricle, low in right ventricle Reduced dosages of Tbx5 in humans and mice leads to defects in the interventricular septum Reptiles are useful for examining the role of certain genes in heart development Incomplete separation of two ventricles During heart looping: Once chambers more differentiated Tbx5 expression decreased in right ventricle, still high in left Gradient not as high as in chicken Evolutionary intermediate between amphibian and avian hearts in ventricular development Delete Tbx5 from left ventricle of developing mouse heart Lack morphological distinction between ventricles (optical projection tomography) Nppa (marker for differentiation of myocardium) and Bmp10 (important for growth of heart tissue) spread into left ventricle Distinction between left and right ventricle maintained, but no ventricular septation Gene expression differences between ventricles maintained But missing septum markers Abnormalities in heart structure or function that arise before birth 19-75 of every 1000 human births Classified into three broad categories Cyanotic heart disease Deoxygenated blood bypassing the lungs and entering the systemic circulation Infants appear blue (cyanosis) E.g. Transposition (switching) of aorta and pulmonary artery Right to left shunting Left-sided obstruction defects Flow is obstructed Defect of heart valve or in the arteries/veins near the heart themselves E.g. Aortic stenosis Persistence of tissue in aorta that normally degenerates Causes hypertrophy of left ventricle & heart murmurs (sound as blood flows through stenosis) Septation defects Affect septation of atria or ventricles ‘Hole in the heart’ Atrial septal defect Ventricular septal defect Other types of congenital defects that do not fit neatly into the three main categories: Bicuspid aortic valve (BAV) Two of the aortic valve leaflets fuse during development Valve is bicuspid, instead of the normal tricuspid configuration Valve doesn’t function properly Back flow of blood into heart Can function for years without symptoms Patent ductus arteriosus (PDA) When ductus arteriosus (connection between pulmonary artery and aorta) fails to close at birth Oxygenated blood flows from aorta (higher pressure) to pulmonary artery (lower pressure) Causes shortness of breath Additional fluid returning to the lungs increases lung pressure Increases energy required to inflate the lungs Mutations in regulators of heart development Environmental influences may also be important E.g. Prenatal exposure to angiotensin-converting enzyme inhibitors (help to lower blood pressure) increases risk of several congenital malformations Role of transcription factors in heart development is well established Such as the T-box family of genes But also, regulation of chromatin-remodelling complexes and histone modification Histone deacetylases characterized as having a role in hypertrophy, but also important in heart development MicroRNAs Small (21-nucleotide) RNAs modulate protein function by binding to target messenger RNA Represses translation of target mRNA Some of miRNAs function in the heart E.g. miR-1 important in the embryonic heart development Targets the cardiac transcription factor HAND2 Important for growth of embryonic heart Controls other regulators of cardiac development Deletion of miR-1 results in: Ventricular septal defects Increased cardiomyocyte proliferation All examples of what? Cardiovascular Development The cardiovascular system is the first system to function in the embryo It is functioning by the end of the third week of development in humans The heart has a nervous system all its own = conduction system Cells that make up the conduction system have a very highly developed power of spontaneous rhythmicity and conduction that is more highly developed than in the rest of the heart The ventricles and the atria have innate powers of spontaneous contractility independent of any nervous influence We know this because The fetal heart is beating before the conduction system or the nervous system is established Isolated cardiac cells contract rhythmically when viewed in culture The human heart continues to beat even when removed from the body (as in heart transplant operations) Human heart development (day 18) The heart initially forms from two tubes located bilaterally (on either side) of the embryo in the cranial (head) region Multiple blood islands dispersed throughout the embryo will form the early blood vessels At the most cranial end of the embryonic disc these blood islands are actually the primitive heart tube Heart development (day 20-22) Embryonic folding Lateral and cranial folding of the embryo over several days brings the endocardial tubes together and tucks them ventrally in the thoracic region at the base of the yolk sac Endocardial tubes fuse together into primary heart tube through which blood eventually flows Fusion of the heart tubes begins cranially and extends caudally and is facilitated by apoptosis The mesenchyme (mainly mesodermal embryo tissue) surrounding the tube condenses to form the myoepicardial mantle (the future myocardium (cardiac muscle)) Gelatinous connective tissue called cardiac jelly separates this mantle from the endothelial heart tube (the future endocardium – membrane that lines heart chambers). A series of constrictions divide the heart tube into sections from cranial to cordal: Truncus arteriosus Bulbus cordis Primordial ventricle Primordial atrium Sinus venosus, into which the common cardinal veins, the umbilical veins and the vitelline veins drain The heart is beating at day 22 Contractions are of myocardic origin and are likened to peristalsis. Embryonic Circulation (Day 22) Three veins drain into the heart of the embryo during the fourth week: Vitelline veins drain blood from the yolk sac; their formation has associations with formation of the liver and the portal system Umbilical veins bring oxygenated blood from the chorion (early placenta) There are two at the start – the right umbilical vein degenerates and disappears The left umbilical vein persists, carrying all blood from the placenta to the fetus The umbilical vein degenerates after it is cut at birth Common cardinal veins return blood to the heart from the body of the embryo Three sets of paired arteries are evident in the embryo The first two carry blood to portions of the developing organism while the third carries blood away from the organism to the placenta The intersegmental arteries form 30-35 branches off the dorsal aortae Many of these are transient structures, but some persist to form the vertebral arteries The vitelline arteries pass to the yolk sac and later to the primitive gut which forms from the yolk sac Three of the vitelline arteries remain that provide blood to the gut in the adult These include: the celiac artery, the superior mesenteric artery and the inferior mesenteric artery The umbilical arteries carry oxygen-depleted blood to the placenta Heart looping (Days 23-28) Most cardiac looping occurs during the fourth week and completes during the fifth week of development. The steps in looping can be summarized as: 1. The straight heart tube begins to elongate with simultaneous growth in the bulbus cordis and primitive ventricle 2. This forces the heart to bend ventrally and rotate to the right, forming a C-shaped loop with convex side situated on the right 3. The ventricular bend moves caudally and the distance between the outflow and inflow tracts diminishes 4. The atrial and outflow poles converge and myocardial cells are added, forming the truncus arteriosus An S-shape is formed with the first bend of the ‘S’ being the large ventricular bend while the bend at the junction of the atrium and sinus venosus forms the second ‘S’ bend Try to describe heart looping in your own words in a few sentences Septation of the heart (early week 5) Internal heart to form the atria and ventricles can be summarized as: 1. Cells from the dorsal and ventral (back and front) walls of the heart grow and form two protrusions called the endocardial cushions These grow towards each other and fuse to form the left and right atrioventricular canals 2. Within the primordial atrium a septum (the septum primum) grows towards the endocardial cushions The space between the cushions and septum is known as the foramen primum As the foramen primum decreases in size a second opening forms in the septum: the foramen secundum. 3. A second septum (septum secundum) develops on the right of the septum primum 4. A primordial muscular ridge exists in the floor of the ventricle As the left and right ventricles grow, their medial (midline) walls fuse to form the interventricular septum 5. Within the bulbus cordis and truncus arteriosus, which form the outflow tract, small ridges develop They are continuous throughout the outflow tract and form a spiral shape. 6. As these ridges fuse they create a spiral shaped septum throughout the outflow tract The original outflow tract is therefore separated into both the aorta and pulmonary trunk. The heart begins to resemble the adult heart in that it has two atria, two ventricles and the aorta forming a connection with the left ventricle while the pulmonary trunk forms a connection with the right ventricle Note: there is some terminology here in relation to the different septum etc. You do not need to know this terminology, but should try to describe how the different chambers are formed in a simple way Fetal circulation Differs from adult predominantly due to the presence of 3 major vascular shunts: 1. Ductus venosus – between the umbilical vein and IVC 2. Foramen ovale – between the right and left atrium 3. Ductus arteriosus – between the pulmonary artery and descending aorta The main function of these shunts is to redirect oxygenated blood away from the lungs, liver and kidney (whose functions are performed by the placenta) Oxygenated blood is carried from the placenta to the fetus in the umbilical vein, most of which then passes through the ductus venosus to the inferior vena cava (main vessel that brings blood back to heart from bottom half of body) while some blood supplies the liver via the portal vein Blood from the liver drains into the inferior vena cava through the hepatic veins The blood in the inferior vena cava is a mixture of oxygenated blood from the umbilical vein and desaturated blood from the lower limbs and abdominal organs (e.g. the liver) This blood enters the right atrium where most of it is directed to the left atrium through the foramen ovale and from here to the left ventricle and aorta The remainder of the blood in the right atrium passes with blood from the superior vena cava (from the head and upper limbs) to the right ventricle and pulmonary artery where most of it passes to the aorta via the ductus arteriosus The blood passes from the aorta to the umbilical arteries and then back to the placenta At birth The 3 vascular shunts close When the alveoli of the lungs fill with air, the pulmonary vessels dilate and decrease the pressure in the pulmonary system Decreased pressure in the pulmonary arteries prevents blood from being shunted through the ductus arteriosus, and the duct usually closes within 10 to 15 hours of birth Blood flowing back into the left atrium from the lungs increases the pressure in that chamber and presses the septum primum against the septum secundum, closing the foramen ovale At the same time, spontaneous constriction of the umbilical vessels (or clamping) cuts off the exchange of blood with the placenta Molecular mechanisms of cardiovascular development In attempts to determine the molecular mechanisms controlling heart development, scientists have focused on: Early cardiomyocyte development including the cellular movement of cardiomyocyte progenitors and the signaling mechanisms that regulate cardiomyogenesis from the blastula to gastrula stages Morphological changes that occur later in development such as looping and septation Some of the predominant molecular pathways contributing to cardiac development: Transcription Factors 1. Nk family transcription factors are expressed in all animals with contractile vascular cells and hence are crucial for myocardial development Nkx2.5 is specifically required for left ventricular chamber development. 2. Gata family transcription factors interact with Nk factors to promote differentiation of cardiomyocytes, smooth muscle cells and endoderm Gata4 regulates myocardial expression and is required for fusion of the heart tubes in the ventral midline Gata5 is required for endocardial differentiation 3. T-box genes play an important role in cardiac morphogenesis Tbx1 may play a role in neural crest proliferation/function Tbx2 plays a significant role in chamber specification Tbx5 is required for atrial septation 4. Pitx2 is a left-sided transcription factor that controls normal cardiac morphogenesis by regulating cell proliferation Tbx5 expression Focus on T-box family of transcription factors Examples of genes shared between animal groups = toolbox genes Tbx5 – expressed throughout developing amphibian heart Birds and mammals have a steep gradient – high in left ventricle, low in left Reduced dosages of Tbx5 in humans and mice leads to defects in the interventricular septum Examine Tbx5 in reptiles is useful as they are in between amphibians and birds/mammals with incomplete separation of the two ventricles (or in the case of the crocodilians, complete separation) Turtle versus chick In turtle at looping heart stages, Tbx5 is broadly expressed throughout the embryonic heart In chick, Tbx5 mRNA and protein are high in the left ventricle and restricted there Once chambers are more differentiated: Turtle = Tbx5 decreases in in right ventricle, remains high in left ventricle Creates a left-right gradient – but not as steep as in chick embryo Still a distinction between left and right components Shows that the reptilian heart, although evolved to function physiologically under conditions particular to reptilian life, is an evolutionary intermediate between amphibian and avian/crocodilian hearts in ventricular development Deletion of Tbx5 To examine the effect of Tbx5 in patterning the ventricles Delete Tbx5 from parts of the ventricles of developing mouse hearts No differentiation between left and right ventricles To determine if steep Tbx5 gradient is needed for interventricular septum formation Shift Tbx5 boundary to the left Gene expression shows distinction between left and right ventricle maintained, but no ventricular septation Mouse genetic analyses are consistent with an important role for a steep gradient of Tbx5 in chamber patterning and formation In the reptilian heart, the delayed and less pronounced establishment of this patterning may contribute to varying degrees of septation How is this related to the type of heart seen in most reptiles? Congenital heart diseases Abnormalities in heart structure or function that arise before birth 19-75 of every 1000 human birds (depends on which defects are included) Classified into three broad categories 1. Cyanotic heart disease = many different types of defects Deoxygenated blood bypassing the lungs and entering the systemic circulation Mixture of oxygenated and deoxygenated blood in systemic circulation Low O2 in blood Infants appear blue (cyanosis) due to mixing of oxygenated and deoxygenated blood E.g. right to left shunting Malposition of great arteries (aorta and pulmonary artery switched) 2. Left-sided obstruction defects Flow is obstructed Defect of heart valve or in the arteries/veins near the heart themselves E.g. pulmonary valve develops completely closed – obstructs outflow of blood from heart to lungs E.g. Aortic stenosis – Persistence of tissue that normally degenerates. Results in LV hypertrophy, heart murmurs. 3. Septation defects Affect septation of atria or ventricles ‘hole in the heart’ Other types of congenital defect that do not fit neatly into the three main categories are: Bicuspid aortic valve (BAV) Two of the aortic valvular leaflets fuse during development resulting in a valve that is bicuspid, instead of the normal tricuspid configuration Valve doesn’t function properly (back flow of blood into heart) but can function for years without symptoms Patent ductus arteriosus (PDA) Ductus arteriosus is one of the shunts that connects pulmonary artery to aorta Allows blood to bypass lungs (good for an embryo), not good after birth Ductus arteriosus fails to close at birth A PDA allows a portion of the oxygenated blood from the left heart to flow back to the lungs by flowing from the aorta (which has higher pressure) to the pulmonary artery If this shunt is substantial, the baby becomes short of breath: the additional fluid returning to the lungs increases lung pressure, which in turn increases the energy required to inflate the lungs The most common congenital heart disease is BAV, and septation defects are the next most common What causes congenital heart diseases? Although the major underlying defects that cause congenital heart disease are thought to be mutations in regulators of heart development during embryogenesis, environmental influences may also be important For example, prenatal exposure to angiotensin- converting-enzyme inhibitors increases the risk of several congenital malformations Regulation of different cell lineages must be tightly controlled so that the correct lineage differentiates at the correct time and in the correct location The role of transcription factors in heart development is well established Such as the T-box family of genes discussed above But also, regulation of the dosage of chromatin-remodelling complexes and histone modification is crucial for normal heart development Histone deacetylases have mostly been characterized as having a role in hypertrophy, but they are also important in heart development In the past few years, much excitement has resulted from a newly identified class of small noncoding RNAs called microRNAs (miRNAs) These small (21-nucleotide) RNAs modulate protein function by binding to target messenger RNA, resulting in repression of translation or in degradation of the target mRNA Some of these miRNAs have recently been shown to function in the heart Potentially of most relevance to congenital heart disease, miR-1 was shown to be important in the embryonic development of the heart It has been shown that miR-1 targets the cardiac transcription factor HAND2, which is implicated in the growth of the embryonic heart, as well as several other regulators of cardiac growth and development When miR-1 is deleted heart defects such as ventricular septal defects and increased cardiomyocyte proliferation result These causes of congenital heart disease are all examples of what? What do you think are the key points/concepts to focus on from this PowerPoint? 20.9 kPa 10 kPa 2 kPa 100 90 Survival (%) 80 15 kPa 5 kPa 70 60 50 40 30 20 10 0 0 5 10 Age (days) 15 20 45 20.9 kPa 15 kPa 40 5 kPa 35 2 kPa 30 25 20 0 5 10 Average hatching stage Stage 10 kPa 46 44 42 40 38 36 34 15 20 25 30 32 Age (days)5 kPa 10 kPa 15 kPa PO₂ treatment ambient Spotted salamander Ringed salamander Development is slowed Hatching is delayed But still hatch at an earlier stage Southern leopard frog Development is not slowed Early hatching is triggered Hatched at earlier stage Pickerel frog Period of hermaphroditism before sexual differentiation begins Hypoxia may influence sex ratios and thus act as an endocrine disruptor Hypoxia mainly affects respiratory and cardiovascular systems O₂ Consumption (nmol h¯¹) 45 20.9 kPa 15 kPa 10 kPa 5 kPa 40 35 30 25 20 15 10 5 0 20 25 30 35 Developmental Stage 40 45 Heart rate Gill ventilation rate Frequency Lung ventilation rate 5 10 15 20 3 6 9 12 15 18 3 6 9 12 15 18 Ambient PO2 (kPa) 5 10 15 Ambient PO2 (kPa) 20 Adult fish and amphibians are typically oxyregulators Maintain O2 uptake at constant level during mild hypoxia O2 Consumption (µmol g-1 h-1) Embryos and larvae quite often appear to be oxyconformers 5 10 15 Ambient PO2 (kPa) 20 Pre-metamorphic tadpoles Conformers, but low O2 uptake, so decrease in O2 uptake is small 5 10 15 Ambient PO2 (kPa) 20 Metamorphic tadpoles Lungs are functional 5 10 15 Ambient PO2 (kPa) 20 Froglets and adults Decrease in mass-specific O2 uptake in adults 18 Pcrit (kPa) 15 12 9 6 3 Pcrit = critical PO2 = PO2 below which rate of O2 uptake decreases with ambient O2 Lower Pcrit = better regulator Changes related to gas exchange organs % Change in HR % Change in resistance Early development = depression of function Developmental stage % Change in CO % Change in SV Developmental stage Early development = little CV regulation Later development = regulation (innervation of ventricle) to maintain cardiac output (CO) Later development = Heart rate (HR) still decreases, stroke volume (SV) increases & resistance increase or remains constant Changes in gene expression in zebrafish Embryos exposed to hypoxia for 24 h during development Hypoxia inducible factor Lactate dehydrogenase (pyruvic acid → lactate) Creatine kinase (helps release ATP from phosphagens) Erythropoietin (increases red blood cells) Development rate, O2 consumption rate & growth are slowed At altitude, the O2 pressure gradient between the environment and cells decreases In a bird egg a decreased O2 gradient from ambient air down the O2 cascade and into the cells Decreases rate of diffusion Adaptations at different levels of the O2 cascade to cope with hypoxia Increase egg shell conductance Increase pore area Trade-off Move chicken to altitude, after 2.5 weeks start laying eggs with greater pore area Hypoxia can induce angiogenesis to increase capillary density Short, stubbly capillaries in hyperoxia Corkscrew capillaries in hypoxia Function of: Number of red blood cells (RBC) Saturation of the hemoglobin (Hb) with O2 Depends on the Hb affinity for O2 Blood flow High altitude embryos have higher hematocrit (ratio of red blood cell volume to total blood volume) & more red blood cells Embryonic RBCs of avian embryos show rapid changes during the later stages of development Response to the hypoxia and hypercapnia which normally occur with development Hb affinity for O2 increases in response to hypoxia Embryos under hypoxia develop definitive RBC and adult Hb ∼24 h earlier than under normoxia Mammal fetuses basically develop at altitude 3–5 kPa (25-40 mmHg) in utero corresponds to 6000 – 8000m altitude The outermost borders of human life Striking parallels between some of the ‘physiological peculiarities’ of newborns and well-known adaptive mechanisms in hypoxia-tolerant animals Greater resistance of newborns compared with adult mammals when subjected to circulatory arrest or exposed to anoxia Survival time in pure nitrogen (anoxia): Immature neonatal rats = 50 min Mature neonatal guinea pigs = 7 min Adults = 3 – 5 min To survive and grow under these conditions, the mammalian fetus must make use of similar mechanisms to those known from acclimatization to high altitude Hematologic adaptations in the fetus include Polycythemia (increased hemoglobin) Increases O2 binding capacity Leftward-shift of the Hb dissociation curve Enhances O2 affinity of the blood Hypoxia tolerance of neonates associated with a very low cerebral metabolic rate Marked delay in extracellular potassium increase and hypoxic depolarization Similar to adult turtle brains that can survive anoxia Heart function also maintained due to better preservation of electrical activity and a slower decline in ATP Pregnancies at higher elevations may result in significantly depressed maternal arterial PO2 Changes in placental growth High altitude pregnancies increase the risk of intrauterine growth restriction and low birth weight Factors known to cause premature birth, infant mortality, and an increased risk of developing cardiovascular diseases Other factors that may contribute to hypoxia in utero include: Pre-existing maternal illness Pre-eclampsia Cord compression Smoking & pollution Abnormal hemoglobin Abnormal placenta development The developing heart, more than any other organ, is susceptible to hypoxia due to its enhanced metabolic demand Hypoxia causes incomplete development of the heart and fetal heart maturation is slowed in mice Simulated high altitude on pregnant rats causes ventricle septal defects in rat offspring Insufficient oxygen in utero produces myocardial thinning, ventricle dilation, and epicardium detachment Decrease in heart rate of fetal mice by 35-40% in culture and by 20% in utero Decrease in cardiac output and lower contractility in high altitude sheep fetuses Early in development partial hypoxia may be necessary for development Physiological hypoxia = naturally occurring, does not produce any pathology Hypoxia inducible factor (HIF) Transcription factor Comprised of α and β subunits (α is O2-sensitive) Three Hifα genes – Hif1α, Hif2α, Hif3α Bind with hypoxia responsive element (HRE) to regulate transcription of over 200 genes in response to hypoxia HIF has two roles: Activates genes in response to hypoxia Activates genes that regulate cellular events involved in morphogenesis By mid gestation cells low in oxygen are detected in specific regions of the embryo Including the developing heart, gut, & skeleton Developing vasculature lacks the capacity to keep pace with the growth and energy demands of the embryo during the second half of gestation Often HIF expression is high in these hypoxic regions No coincidence that HIF is important for normal development of some of these hypoxic regions Cellular hypoxia and expression of HIF components occur during heart development By 13.5 days in mouse development: Hypoxia is restricted to the myocardium of the outflow tract, interventricular septum, and atrioventricular cushions HIF1α protein localization mirrors the distribution of myocardial hypoxia during these stages Global knockout of HIF1α creates: Cardiac bifida, abnormal cardiac looping, abnormal remodeling of the aortic outflow tract and cephalic blood vessels Delete or modify HIF components and cardiac development is stopped Formation of chambers & valves Myocardial hypoxia is a feature of endocardial cushion formation Endocardial cushions = swellings that form on surface of heart tube and give rise to septa and valves Loss of HIF activity affects cushion development Epithelial → mesenchymal (stem cell) transition Mesenchymal cells in cushion consume O2 Underlying myocardium becomes hypoxic → HIF → VEGFa Inhibits epithelial → mesenchymal transition Development is driven by differences in gene expression and cell behavior in both time and space HIF activity during development is controlled in space and time by the availability of oxygen The hypoxia-dependent HIF transcription system has evolved the capacity to work with the physiological hypoxia present during normal embryonic development Uses it to undertake development of the embryo The system is limited in its ability to respond to nonphysiological hypoxia (enhanced and/or spatially extended hypoxia) during development Inducing non-physiological hypoxia in mouse and rabbit embryos leads to a variety of developmental defects Insufficient exposure to ‘normal’ hypoxia Adequate exposure to ‘normal’ hypoxia Chronic exposure to moderate ‘abnormal’ hypoxia Chronic exposure to severe ‘abnormal’ hypoxia Hypoxia & Development Hypoxia during embryonic development is quite common among vertebrates Fish and amphibians are laid in aquatic environments that can experience variation in environmental PO2 Hypoxic events are likely to increase in certain areas with climate change We’ve talked about how ground nesting birds and reptiles in particular can experience hypoxia Mammals develop in what is quite a low oxygen uterine environment Going to look at some of the effects of these groups in turn, just to give you an overview of the types of effects hypoxia can have An important thing to remember for this topic – what is the ambient oxygen level (or normoxia) in air or well-mixed water (in kPa – a unit of partial pressure)? General effects of hypoxia during development of fishes and amphibians A study performed by me using lungfish embryos – representative of a lot of fish studies Survival slowly declines as incubation PO2 declines Development rate tends to be slowed down in most fish and amphibians Can see for the lungfish that at PO2 of 10 kPa or below there is significant slowing of development by halfway through embryonic development In many species hypoxia can act as a hatch trigger, so that embryos hatch at an earlier stage than under higher O2 conditions – see this for the lungfish What might be the benefit of this? Hatch timing in amphibians There are a couple different patterns of hatch timing that can emerge with hypoxia In the spotted and ringed salamanders, their development is slowed in hypoxia (like the lungfish), but their hatching is actually delayed But they still hatch at an earlier stage, because development is so delayed So technically, in terms of where they are at in development, hypoxia causes early hatching In the southern leopard frog and pickerel frog they do not show a delay in development, but hypoxia still causes early hatching and they are hatching at an earlier stage Slightly different pattern to the salamanders, because development rate not as strongly affected Malformations and sex hormones Hypoxia can induce malformations in fish and amphibian embryos Zebrafish embryos, for example, show a significant increase in malformations when exposed to hypoxia (0.8 mg O2/L) for 96 hours of more compared to normoxia (5.8 mg O2/L) Malformations include Tails developing faster than their heads Spinal deformities (curvature of the spine) Shorter body length In this study on zebrafish they also found that hypoxia altered sex hormone concentrations Testosterone was reduced and estradiol was increased Very important in this species, in which males go through a period of hermaphroditism before sexual differentiation begins (50% develop ovaries, others develop testes) May influence sex ratios and thus acts as an endocrine disruptor With those altered hormones, in which way would the sex ratio be skewed? O2 Consumption Hypoxia mainly affects respiratory and cardiovascular system – main systems delivering oxygen to tissues Lungfish oxygen consumption decreases with hypoxia Less oxygen available, can’t consume as much Later stages of development can perhaps show more active responses to hypoxia Once nervous system function can control cardiac activity and ventilation Alterations in rates Rana tadpoles that have functioning lungs and gills when exposed to hypoxia show: increase ventilation rate of lungs interestingly, heart rate responses are variable between individuals – one shows increase, other two show decrease Gill ventilation rate initially increases, but then decreases Don’t put all energy into ventilating both lungs and gills – gills do early work, then lungs take over O2 update and hypoxia While adult fish and amphibians typically are so-called oxyregulators What is an oxyregulator? embryos and larvae quite often appear to be oxyconformers What is an oxyconformer? African clawed frog larvae NF stage 1–39 larvae (first graph on left) are oxygen conformers, but their O2 consumption even during normoxia is so low that the incremental decline with increasingly severe hypoxia is not large BY NF stage 61 (on middle graph) the tadpoles are well into metamorphosis and have functional lungs. You see that O2 uptake has increased substantially, as has their ability to regulate O2 uptake, at least at higher PO2 By the time the larvae have metamorphosed (froglets on right graph) their O2 uptake is at its highest and they can regulate well down to ~7kPa Adult O2 uptake has decreased and the ability to regulate is greatest, all the way down to ~4 kPa Can see these changes between conformity and regulation when examining the critical PO2 = the PO2 below which the rate of oxygen uptake significantly decreases This varies with development and changes are clearly related to changes in gas exchange organs Cardiovascular function In early larvae there is a depression of function probably through direct effects on cardiac muscle and smooth muscle of blood vessels No regulatory ability of CV system at these early stages In comparison, at later stages hypoxia increases stroke volume and cardiac output peripheral resistance decreases rather than increases Heart rate continues to show bradycardia as in early development Switch in response indicates there is a specific time (between stages 49-51 and 52-53) when CV regulation becomes functional in tadpoles Probably due to nervous innervation of ventricle Example of using an environmental stressor to understand the development of functional regulation Before this point both HR and SV change in the same way – so little regulation of cardiac output After this point HR drops, SV increases, so indicates maintenance of cardiac output Gene expression Not just whole animal physiological changes that occur with hypoxia – also see gene expression changes In zebrafish, embryos exposed to hypoxia for 24h show a change in many genes See down regulation in many genes involved in respiration HIF-1α – we’ll talk about in more detail later in the PPT General effects of hypoxia during development of reptiles and birds Moderate hypoxia (PO2 = 17kPa) generally does not affect development and growth of bird/reptile embryos Severe hypoxia (PO2 < 13 kPa) slows development and growth and prolongs the incubation period Seen in red-bellied turtle embryos Bird embryos at altitude Why is a high altitude environment useful for understanding the effects of hypoxia? At altitude, the O2 pressure gradient between the environment and the cells decreases So in a bird egg the decreased gradient should lower the rate of diffusion from ambient air through the shell into the air cell, then through the inner shell membrane and chorioallantoic membrane into the blood, and from the blood into the cell There can be adaptations at different levels of the oxygen cascade (different steps as O2 moves from the environment to the cell) to cope with hypoxia Increase conductance By changing pore area Trade-off: Increased water loss and CO2 losses (don’t necessary want to lose all the CO2 as this can disrupt acid-base balance of the embryo) Move a chicken to elevation, it takes around 2.5 weeks for them to start laying eggs with greater pore area Changes in the CAM Hypoxia can induce angiogenesis to increase capillary density Corkscrew capillaries in hypoxia, short stubbly capillaries in hyperoxia (high O2) – lots of empty space between vessels Once O2 in CAM it has to be transported to tissues The capacity for O2 transport from the CAM to the tissue depends on the PO2 at the different levels of the O2 cascade but it also depends on the O2 delivery by the blood Transport in blood Is a function of number of red blood cells (RBC) saturation of the hemoglobin (Hb) with O2 (which depends on the Hb affinity for O2 ) blood flow High altitude embryos have higher hematocrit and a higher number of red blood cells Embryonic RBCs of the chick and other avian embryos show rapid and extensive changes of the gas transport properties during the later stages of development This is interpreted as a response to the hypoxia and hypercapnia which normally occur with development The Hb affinity for oxygen increases substantially allowing continuous adjustment to the hypoxia When embryos are grown under hypoxic conditions definitive RBC and adult Hb appear in the circulation ∼24 h earlier than they do in normoxic controls General effects of hypoxia during development of mammals Mammal fetuses basically develop at altitude Although it is generally known that the oxygen partial pressure of the mammalian fetus is below the adult values, it is not normally recognized that the 3–5 kPa (25-40 mmHg) corresponds to 6000 – 8000 m altitude, i.e. at the outermost borders of human life Striking parallels between some of the ‘physiological peculiarities’ of newborns and well-known adaptive mechanisms in hypoxia-tolerant animals Results in greater resistance of newborn compared with adult mammals, when subjected to circulatory arrest or exposed to anoxic (no O2) atmospheres Neonatal survival time in pure nitrogen ranges from 50 min in rats (immature neonates) to 7 min in guinea pigs (mature neonates), in contrast to 3 – 5 min in adult individuals of all species studied Mammals To survive — and to grow — under these conditions, the mammalian fetus must make use of similar mechanisms to those known from acclimatization to high altitude Hematologic adaptations in the fetus include polycythemia (increased hemoglobin) increasing oxygen binding capacity Leftward-shift of the hemoglobin dissociation curve enhancing oxygen affinity of the blood Neonatal hypoxia tolerance has long been known to be associated with a very low cerebral metabolic rate (low metabolic rate of brain) and this has usually been ascribed to the state of immaturity Marked delay in extracellular potassium increase and hypoxic depolarization, as has been observed in neonatal as compared to adult brain tissue Analogous to turtle in comparison with the rat brain, as a correlate of the turtle’s striking ability to remain completely submerged for up to several months markedly delayed depolarization and a later increase in extracellular potassium in neonatal compared to adult rat neurons under hypoxic conditions During hypoxia, the neonatal heart maintains mechanical function better than the adult heart of the same species associated with a better preservation of electrical activity and a slower decline in tissue ATP More extreme hypoxia But more extreme hypoxia can be detrimental Pregnancies at higher elevations may result in significantly depressed maternal arterial PO2 and changes in placental growth when compared to the sea level High altitude pregnancies increase the risk of intrauterine growth restriction and low birth weight These factors are known to cause premature birth, infant mortality, and an increased risk of developing cardiovascular related diseases Other factors that may contribute to hypoxia in utero include pre-existing maternal illness pre-eclampsia cord compression smoking pollution abnormal hemoglobin abnormal placenta development The developing heart, more than any other organ, is susceptible to hypoxic stress due to its enhanced metabolic demand Numerous studies have shown that hypoxia causes incomplete development of the heart Simulated high altitude on pregnant rats found that hypoxia caused ventricle septal defects in rat offspring More recent studies have found that insufficient oxygen in utero produces myocardial thinning, ventricle dilation, and epicardium detachment It also slows fetal heart maturation in both chicken and mouse Also see changes in heart function Hypoxia can decrease heart rate of fetal mice by 35-40% in culture and by 20% in utero High altitude sheep models also present with altered cardiac function in response to prenatal hypoxia as demonstrated by decreased cardiac output and lowered contractility Some hypoxia is good Early in mammalian development partial hypoxia referred to as physiological hypoxia = naturally occurring, ‘physiological’ as it does not produce any pathology may actually be necessary for development What consideration from our discussion of developmental critical windows does this discussion of different levels of hypoxia bring to mind? Hypoxia plays a critical role in the formation and development of the heart Hypoxia inducible factor (HIF) Transcription factor Comprised of α and β subunits (α is O2-sensitive) Bind with hypoxia responsive element (HRE) to regulate transcription of over 200 genes in response to hypoxia HIF has two roles: When hypoxic conditions occur, HIF transcriptionally activates genes that, depending on the context, are involved in energy metabolism, autophagy, translation inhibition, erythropoiesis, and angiogenesis These genes promote tolerance of hypoxia by decreasing the cellular requirement for oxygen and increasing the supply of oxygen Secondly, because normal mammalian development occurs in a hypoxic environment, HIF activates genes that regulate cellular events, so hypoxia and HIF are therefore responsible for aspects of developmental morphogenesis Mid gestation – cells low in oxygen are still consistently detected in specific regions of the embryo, including the developing heart, gut, and skeleton (dark regions in figure) Probably because the developing vasculature lacks the capacity to keep pace with the phenomenal growth and energy demands of the embryo during the second half of gestation Often HIF expression is high in these hypoxic regions It is not coincidence that HIF important for normal development of some of these hypoxic regions, including the placenta, bone formation, heart development HIF and heart development Cellular hypoxia and expression of HIF components occur during heart development HIF activity is required for normal heart development – particularly HIf1α The myocardium is broadly hypoxic (%2% oxygen) at 9 days of mouse development, but by 13.5 days the hypoxia is restricted to the myocardium of the outflow tract, interventricular septum, and atrioventricular cushions (in figure on slide) Hif1α protein localization mirrors the distribution of myocardial hypoxia during these stages Know that HIF1a is required Global knockout of HIF-1α resulted in arrested development by day E9 and embryonic lethality by day E11 with significant cardiovascular irregularities including cardiac bifida, abnormal cardiac looping, abnormal remodeling of the aortic outflow tract and cephalic blood vessels, and mesenchymal cell death Formation of chambers and valves Myocardial hypoxia is a feature of endocardial cushion formation and there is evidence that loss of HIF activity affects cushion development. Endocardial cushions = swellings that form on the surface of the heart tube and give rise to the septa and valves We know already that development is driven by differences in gene expression and cell behavior in both time and space HIF activity during development is controlled in space and time by the availability of oxygen The hypoxia-dependent HIF transcription system has evolved the capacity to work with the physiological hypoxia present during normal embryonic development And it uses this to undertake development of the embryo However, it is important to note that this system is limited in its ability to respond to nonphysiological hypoxia—that is, enhanced and/or spatially extended hypoxia— during development For example, inducing nonphysiological hypoxia in mouse and rabbit embryos leads to a variety of developmental defects that occur in places including the heart and vertebral column Hypoxia level & fetal development Diagram summarizes the effects of insufficient hypoxia, normal hypoxia and abnormal hypoxia in mammalian embryo cardiovascular development What do you think are the key points/concepts to focus on from this PowerPoint? 34 day old Muscovy duck embryo (hatch on day 35) Q10 < 2 Some endothermic ability Q10 > 2 Like an ectotherm Altricial red-winged blackbirds Increased O2 consumption matched not by breathing faster, but by breathing more deeply 79 d 86 d 91 d 99 d 120 d > 300 d 30°C 25°C 20°C 15°C 30°C 20°C 25°C 15°C 25°C 30°C Development of Endothermy Useful background information: Take a moment to write down all that you know about endothermy and how endothermic animals respond to changes in environmental temperature (this is a review of BIOL 353) Remember the relationship between metabolic rate and temperature in endotherms (birds and mammals): Within the thermoneutral zone (TNZ) metabolic rate is constant, outside the TNZ metabolic rate increases Can explain this by heat transfer equation M = C (Tb – Ta) C = thermal conductance (inverse of insulation) In the TNZ: If metabolic rate is constant in TNZ, but Tb – Ta is changing, what has to change? Thermal conductance Animals alter thermal conductance by altering their insulation How might animals do this? Below the TNZ: It is getting cold in the environment – what do mammals do? Increase heat production – shivering and non-shivering thermogenesis (BAT) (uncoupling of oxidative phosphorylation and electron transport) Regional heterothermy – use countercurrent heat exchange in limb to keep heat in body core Above the TNZ: – Environment is getting hot – what do mammals do? Behavior, insulation can help act as barrier for heat transfer in Cycling of body temp and hyperthermia (don’t expend energy to control body temperature – let it change and reduce Tb-Ta) Evaporative cooling – sweat, pant But thermoregulatory ability takes time to development – especially in young that a born very early in development What factors might limit thermoregulatory ability of developing mammals? Decrease ambient temperature When studying thermoregulatory ability we can do things like drop the external temperature and see what happens to heat production and body temperature of the study animal E.g. In Muscovy ducks Example of precocial species (well developed at hatching) Ambient temperature is dropped from ~37.5°C to ~31.5°C on day 34 Body temperature and heat production do drop somewhat, but not down to ambient temperature, indicating a thermoregulatory ability Indicates endothermic ability before hatch Can see this ability when look at Q10 values – gives an indication of how a rate changes a change in temperature If Q10 values are below 2, this indicates that heat production does not change a lot with temperature = some endothermic ability If Q10 values are above 2, this indicates that heat production does change a lot with temperature (you can see the blue diamonds decreasing more rapidly at these cooler temperatures) = ectotherm (no endothermic ability) This shows that Muscovy ducks on day 34 have some endothermic ability as long as temperature does not drop too low E.g. Red-winged blackbirds Example of an altricial species (not well developed at hatching) Graphs show oxygen consumption and body temperature at different ages Only really at 9-10 days post hatch (dph) and at fledging does oxygen consumption increase at lower temperatures Hatchlings are increasing their heat production and maintaining body temperature better (bottom graph) The increase in oxygen consumption is matched by increased breathing rate and tidal volume (how big each breath is) at 9-10 days post hatch (dph) and at fledging Why is it important that respiration change in this way? Rest of the topic focuses on the paper on sugar gliders you read in Perusall Introduction Sugar gliders = freaking cute (because they’re Aussies)! In many small mammals thermoregulation has not fully developed Marsupials are extreme case – very altricial young – extended period of development in the pouch Sugar glider – small adults, 140-160 g Litter of one or two young between June and January – winter and early summer Hairless when born – what does this mean? By 60-68 days they cannot be contained in pouch, but still largely hairless – might experience hypothermia (especially as they are often left in the nest alone while the mother forages) Methods Gliders born in captivity Once young were partly out of pouch (age 60-68), body mass, metabolic rate and Tb were measured over a Ta range of 30C down to 15C Removed from mother and put into chamber and O2 consumption measured with open flow respirometry Tb measured rectally at end of each Ta testing (except older ones – only measured at beginning and end to reduce disturbance) Thermal conductance calculated by rearranging the heat transfer equation Measure MR, measure Ta, and control Tb, so can calculate the unknown Results – what does each figure describe? Figure 1 Body mass increased over time – sigmoid growth curve 60-110 days – significant increase in mass Eyes opened, fur grew to cover entire body, co-ordination improved Resembled young adults by 110 days – when first leave the nest Slow growth of Pb10 – mother died, probably wasn’t getting nutrients Did eventually reach adult mass – gave birth to young in second breeding season = happy ending! Figure 2 How routine metabolic rate (RMR), body temperature (Tb) and conductance change with temperature at different ages All showed same pattern in RMR, Tb and conductance (Figure 2 = representative indiv.) At 79 days – RMR increased when ambient temperature (Ta) was lowered from 30 to 25°C, but then declined Tb declined over entire Ta range RMR increased with age until 120 days, highest values observed at lowest temp Tb less dependent on Ta over time – constant by 100 days – thermoregulating! 79-91 days conductance increased when Ta was lowered from 30 to 25°C – animals shaking From 99 days conductance decreased with Ta – similar to adults – what are they doing? Figure 3 Describes change in oxygen consumption with age at four temperatures When was RMR lowest? – 55 – 80 days When was RMR highest? – 80-100 days What happened after 100 days? – declined to adult values When was peak in RMR most pronounced? – at coldest temp Figure 4 Describes changes in Tb-Ta with age at four temperatures Tb-Ta – part of heat transfer equation – increased with age and with decreasing Ta At colder temperatures the increase was more rapid from 60-100days then less steep after Figure 5 Describes change in conductance with age at 25C Declined curvilinearly – was 4-5 times higher on 50-60d than 120d What does this mean? – more heat loss – even when at moderate temp (25°C) Figure 6 Shows oxygen consumption as a function of body mass in juvenile gliders As the juveniles get larger oxygen consumption declines – it is declining to adult levels Less important figure, but does demonstrate how by day 95-100 they are like mini adults in their endothermic ability Discussion By age 56 days sugar gliders possess very limited thermoregulatory ability Shown by low O2 consumption at all temperatures (Figure 3) Shown by low Tb-Ta (figure 4) Shown by high conductance (figure 5) By age 95-100 days, gliders increased heat production above that predicted for adults (Figure 6) Gliders were able to maintain constant Tb over a Ta range (Figure 2 and figure 4) Further growth characterized by steady decrease in mass-specific metabolic rate to adult levels Shown by figure 6 and figure 3 Further growth also characterized by slight increase in Tb (plateauing in Figure 4) Due to decrease in conductance (Figure 2 and Figure 5) In the wild, young are left in nest after 70 days But results show limited thermoregulatory ability This affects the mother in that she has to have shorter foraging bouts Female behavior probably linked to thermoregulatory ability of young How is thermal stress reduced in young? Insulation of nest Help from family! Dropped off at grandmas Older siblings stick around (gliders often have 2 litters in one season) Calls of young may act as cue for older siblings to stay near nest Expense of thermoregulation 80 days old – total energy expenditure 0.5 kJ/h at Ta 15C 100 days old – 3.0 kJ/h (thermoregulating like an adult despite being smaller) Cost to females – lactating v nonlactating No difference in metabolic rate with young litters But later in lactation females show increased energetic costs in other species Due to increased energy consumption of young once they start thermoregulating What results in development of endothermy? Body size ↑ SA:V ↓ = lower heat loss Fur ↑ insulation ↑ conductance ↓ = lower heat loss Perfect timing of fur growth – right when leaving pouch (not too hot in pouch, not too cold outside it) Larger body size = increased amount of heat produced Muscle mass ↑ shivering capacity ↑ But shivering does ↑ convective heat loss – disturbs boundary layers (especially if not a lot of fur to trap heat Activation of thyroid gland is also important for development of endothermy Don’t know when this happens – but probably by 100 days old Non-shivering thermogenesis in brown adipose tissue does exist in marsupials But may be uncoupling proteins in other tissues that help produce heat Vasoconstriction in muscle tissue may also help Behavioral changes – adopted curled ball position by 70 days old ↑ insulation At onset of thermoregulation, Tb is lower than in adults Probably due to higher conductance and smaller size Keeping Tb a little lower decreases Ta – Tb (like adults that cool extremities) and this can reduce heat loss Development rate geared so young emerge in favorable conditions – moving into spring and summer This PPT focuses mainly on the paper, so focusing on the results of the sugar glider study and how they inform us about the development of endothermy is the way to go! Field data (nest data) is scarce What are the thermal challenges/opportunities encountered by embryos in natural nests? Do not completely understand the heat-seeking behavior of embryos (active thermoregulatory movements) Why do reptile embryos have higher heart rates during cooling rather than heating (opposite of adults)? Possible hypothesis = as temp increases a slower HR may keep warmer blood near embryo rather than moving it into middle of egg, during cooling and embryo may benefit from increased HR to move heat from middle of egg out to embryo Or might be changes in stroke volume that change blood flow How is blood flow changed within the egg and how might this influence temperature (and also gas exchange)? Are there differences in thermoregulation and thermal acclimation in different populations? Tropical v temperate? High v low altitude? Thermal adaptations in other animal groups (fish and amphibians)? What biochemical changes occur in response to thermal changes? How can we predict the response of embryos to climate change? What relationships exist between thermal biology & climate patterns? How do embryos respond to climate change or potentially buffer climate change effects through adaptations? How is this related to species vulnerability to climate change? Need empirical data that can be used in biophysical models to predict responses The behavioral and physiological strategies of bird and reptile embryos in response to unpredictable variation in nest temperature I. Introduction Paragraph 1 – Embryo is a critical life history stage Vulnerability to unpredictable fluctuations in environment greater in egg laying animals Amniote embryos = larger eggs = slower development = more opportunity for disruption Paragraph 2 – Given long exposure, might expect birds and reptile eggs to have evolved a suite of adaptive responses to environment both for survival and to enhance fitness-relevant phenotypic traits of hatchlings Some responses involve maternal traits Less focus on adaptive responses of embryos themselves (sort of like the niche construction idea in a way) Paragraph 3 – how embryos respond to the environment is largely unstudied Due to assumption that embryos have little control But studies show embryos have behavioral and physiological tactics Focus of review on thermal variation in the environment Aim = review embryonic responses, point out some future directions and potential challenges in the field II. Thermal challenges facing embryos Paragraph 1 – embryonic reptiles and birds experience considerable thermal variation Large fluctuations can occur across time scales – daily to seasonally In some nests, daily fluctuations can be substantial Paragraph 2 – reptile nest temperatures = 20-30°C (extreme temperatures = 10-45°C (50-113F) Australian skink nest = 9-43°C (48-109F) Bird nests less variable = 30-40°C (can drop as low as 10°C when parent away) Can get different temperatures within the nest Temperature can vary within the egg (e.g. end closest to sun-warmed soil is higher) Paragraph 3 – Incubation temperature can influence metabolic rate, development rate, hatching success, morphology, locomotor performance, behavior, growth and gender Different thermal tolerances between phylogenetic lineages of birds and reptiles Paragraph 4 – Big effect of incubation temperature on incubation period (development rate) Rapid development may be beneficial – link between hatch date and survival Temperature can also influence energy-efficiency of embryonic development Paragraph 5 – Might expect two types of adaptive responses: 1. species evolve relatively hard-wired (canalized) responses that fine-tune embryonic biology to thermal environment likely to be experienced in the nest likely through tight genetic control 2. embryos deal with thermal variation by flexible adjustments = plasticity focus of the review III. The potential tactics available to an embryo (1) Embryo can influence the temperature to which it is exposed (AVOID ON DIAGRAM) (a) Delaying the time (and stage of dev) when the embryo moves from mother’s body to nest Embryo may be able to influence time of oviposition (laying) Some females will retain eggs in suboptimal conditions – lay when embryos are at later stage Not sure how this is under embryonic control (b) Accelerating time of hatching (hatch earlier) Hatch ‘prematurely’ Modifying development rate relative to temperature Or modifying stage of development required to hatch May reduce performance (c) Redistributing water…