Ion and Osmoregulation in Prenatal Elasmobranchs: Evolutionary Implications

The elasmobranchs represent a fascinating series of experimentsin the evolution of maternal support for developing embryos.In oviparous species, eggs are enclosed in a tough, fibrouscapsule. The capsule is very permeable and the embryonic tissuesare bathed in a solution ionically similar to sea water withinhours of oviposition. In the primitively viviparous speciesSqualus acanthias, early embryos in egg capsules are retainedin utero and are bathed in a solution similar to maternal plasma.Several months into the 22 month gestation period the embryosare capable of independent iono- and osmoregulation in a uterinesolution that resembles sea water. Embryos of more advancedviviparous species develop in a solution that is similar tomaternal plasma. Iono- and osmoregulation by these embryos wouldappear to beminimal. It is clear that in the oviparous elasmobranchs,the ability of the egg/embryo to maintain salts and urea atappropriate levels is present at the earliest stage of development.The ability of prenatal elasmobranch embryos to iono- and osmoregulatewould allow the evolution ofa diverse array of reproductivestrategies in the elasmobranchs.     

Mechanisms of hyperosmotic acclimation in Xenopus laevis (salt,urea or mannitol)

The acclimation of the clawed toad Xenopus laevis to hyperosmotic solutions of NaCl (balanced solution of sea salt), urea or mannitol was studied. The animals could not be acclimated to salt solutions more concentrated centrated than 400 mosm·l^-1. Urea was tolerated till 500 mmol·l^-1. Plasma osmolality was always hyperosmotic to the environmental solution, but with diminished osmotic gradient at the highest tolerated solutions. Plasma urea concentration approached 90 mmol·l^-1, similar in the three solutions of acclimation. Urine volume was very small under all conditions. Serum aldosterone and corticosterone did not differ significantly, although there was a slight tendency towards lower aldosterone in the NaCl solution. In vivo water uptake in tap water acclimated animals was very small, and was higher in the other groups. Only the salt- and urea-acclimated, but not the tap water and mannitol-acclimated groups responded with a clear increase following injection of oxytocin or theophylline. In vitro urea fluxes were similar and invariable in both directions under all conditions. No significant effect of theophylline was observed. Sodium transport measured by the short-circuit technique in vitro was lower in salt- and mannitol-acclimation conditions, and was stimulated significantly under all conditions in response to serosal oxytocin or theopylline. It is concluded that Xenopus laevis can osmoregulate at a limited range of external solutions. It is limited in the increase of its plasma urea concentration; the transport properties of the skin do not change very much upon acclimation, except for the hydroosmotic response to oxytocin.Abbreviations I _sc short circuit current - PD potential difference - SW balanced sea water - TW tap water     

Urea is Necessary for the Culture of Embryos of the Marsupial Frog Gastrotheca riobambae, and is Tolerated by Embryos of the Aquatic Frog Xenopus laevis

Urea was found in the capsular fluid that bathes Gastrotheca riobambae embryos during incubation in the maternal pouch. The urea concentration in this fluid is higher than in blood from the mother, indicating that urea is accumulated by the embryo during the period of maternal incubation. Gastrotheca tadpoles tolerate up to 500 mM urea with 86% survival after 24 hours and die in solutions of 0.5 mM ammonia. These findings suggest that urea plays a role in the adaptation of G. riobambae embryos to the conditions of water stress within the maternal pouch. To improve the in vitro culture conditions of early embryos taken from the maternal pouch, a saline solution that contains urea was designed (GRS). GRS plus 30 mM urea was used for the culture of cleavage to the neurula stage embryos of G. riobambae. During organogenesis, the urea concentration was raised to 60 mM. Early embryos of Xenopus laevis tolerate urea, and in addition, no inducing effects of urea have been detected in animal cap explants of Xenopus .     

Use of field‐portable ultrasonography reveals differences in developmental phenology and maternal egg provisioning in two sympatric viviparous snakes

A thorough understanding of the life cycles underlying the demography of wild species is limited by the difficulty of observing hidden life‐history traits, such as embryonic development. Major aspects of embryonic development, such as the rate and timing of development, and maternal–fetal interactions can be critical features of early‐life fitness and may impact population trends via effects on individual survival. While information on development in wild snakes and lizards is particularly limited, the repeated evolution of viviparity and diversity of reproductive mode in this clade make it a valuable subject of study. We used field‐portable ultrasonography to investigate embryonic development in two sympatric garter snake species, Thamnophis sirtalis and Thamnophis elegans in the Sierra Nevada mountains of California. This approach allowed us to examine previously hidden reproductive traits including the timing and annual variation in development and differences in parental investment in young. Both species are viviparous, occupy similar ecological niches, and experience the same annual environmental conditions. We found that T. sirtalis embryos were more developmentally advanced than T. elegans embryos during June of three consecutive years. We also found that eggs increased in volume more substantially across developmental stages in T. elegans than in T. sirtalis, indicating differences in maternal provisioning of embryos via placental transfer of water. These findings shed light on interspecific differences in parental investment and timing of development within the same environmental context and demonstrate the value of field ultrasonography for pursuing questions relating to the evolution of reproductive modes, and the ecology of development.     

Changes in electrolytes,urea and free amino acids of Poecilia reticulata embryos following high salinity adaptation of the viviparous female

Vitellogenic or pregnant females of the viviparous Teleost Poecilia reticulata were gradually adapted to hypertonic saline with an ionic concentration approximating that of sea water. After 2 or 7 days adaptation, the total content in electrolytes, urea and free amino acids was determined in the bodies of the females and in their oocytes and intrafollicular embryos. Significant changes in electrolytes levels were only detected in females as a noticeable drop (30–40%) in Na⁺ concentration and a sharp rise in K⁺ level. Slower Cl⁻ increase was evidenced. The urea concentration appeared rather high in mature oocytes (30 mM/kg) and in young embryos. Electrolytes but also urea and amino acid pool level changes were displayed in oocytes and embryos after adaptation of the females to concentrated saline. A sharp but transitory increase in K+ content (40–120%) was noticed in embryos. The urea concentration rose up to 50 mM/kg (i.e. +85%) in young embryos and amino acid pool concentration reached 75 mM/kg (i.e. +140%) in embryos at more advanced stages. The significance of urea synthesis and accumulation in mature oocytes and embryos is discussed together with the mechanisms of response to osmotic changes of electrolytes, urea and amino acid pool level control systems through the maternal orgnism.     

Changes in Composition During Embryo Development of the Gulper Shark,Centrophorus Granulosus (Elasmobranchii,Centrophoridae): An Assessment of Maternal-embryonic Nutritional Relationships

Centrophorus granulosus is a deep sea shark that reproduces through aplacental viviparity. Its fecundity is one of the lowest described with only one embryo in a pregnancy lasting about two years. Its mature ovarian egg reaches one of the largest cellular sizes (> 350g) described for any animal species. A previous report suggested a loss of organic matter during development of about 50% (Ranzi 1932), the highest rate reported for any elasmobranch. We measured the amounts of water, organic and inorganic matter in a complete series of embryos, by drying and later incinerating separately the external yolksac, eviscerated body, internal yolksac, liver and digestive tract. Wet weight of uterine ova ranged from 143.2–370.4g, was positively and significantly correlated with maternal size, and the size of full-term embryos increased with maternal size. A graphical method was developed to allow weight comparison between uterine ova and full-term embryos while taking into account the initial variability in uterine ova size. Total wet weight of the embryo system increased during development by +31/+34%. Changes in percentage composition (from initial values) were: water +99/+101%; organic matter –18/–25%; inorganic matter +114/+170%. The rate of decrease of organic matter was much lower than previously suggested, and was similar to values described for oviparous species. These results suggest that C. granulosus is a strictly lecithotrophic species, with no maternal contribution of organic matter during development, although the female does provide both water and inorganic material. Other factors that might influence the accuracy of this assessment are discussed.     

A new organelle related to osmoregulation in ultrarapidly frozenPelvetia embryos

Freeze-fracture electron microscopy of the cortical cytoplasm of unfixed, uncryoprotected, ultrarapidly frozen embryos of the marine brown algaPelvetia fastigiata has demonstrated the presence of numerous 0.5-m diameter, disc-shaped vesicles lying adjacent and nearly parallel to the plasma membrane. Some vesicles are fused with the plasma membrane through a narrow connection; this however appears to be a reversible attachment rather than an intermediate stage in the incorporation of the vesicle into the plasma membrane. The distribution of these connections in the plane of the membrane is not uniform; they tend to occur in patches. The fraction of vesicles that is fused with the plasma membrane at any one time appears to be related to a cell's perception of a stressful hypotonic imbalance between the internal and external concentrations of osmotically active compounds. Thus, a sudden 5% decrease in osmolarity of the artificial seawater medium just before freezing leads to a 38% increase in connections per unit membrane area, while a 20% decrease in osmolarity leads to a 75% increase in connections per unit area. Based on these findings and the corresponding ion-transport studies of R. Nuccitelli and L.F. Jaffe (1976, Planta131, 315–320), we postulate that the disc-shaped vesicles mediate short-term osmoregulation inPelvetia embryos by reversibly inserting chloride channels into the plasma membrane.Abbreviations ASW artificial sea water - IMP intramembrane particle - EF fracture face of a freeze-fractured exoplasmic membrane leaflet - PF fracture face of a protoplasmic membrane leaflet     

Plasma membrane proteins of embryo cells of various sea urchin species and hybrid embryo]

Differences are observed in plasma membrane proteins of S. intermedius and S. droebachiensis sea urchin embryo cells isolated at middle blastula stage by means of acrylamide-gel electrophoresis in presence of SDS, urea or non-ionic detergents--Triton X-100 or Brij 35. Electrophoretic mobilities of plasma membrane proteins of sea urchin hybrid embryo malemale S. intermedius X femalefemale S. droebachiensis were identical with electrophoretic mobilities of plasma membrane proteins of maternal species S. droebachiensis. Three sea urchin embryo species under study had just the same biosynthesis of plasma membrane proteins at middle blastula stage detected by 14C-aminoacids pulse-labeling followed by membrane isolation, electrophoresis and gel-autoradiography.     

Effect of salinity on oxygen consumption in fishes: a review

The effect of salinity on resting oxygen uptake was measured in the perch Perca fluviatilis and available information on oxygen uptake in teleost species at a variety of salinities was reviewed. Trans‐epithelial ion transport against a concentration gradient requires energy and exposure to salinities osmotically different from the body fluids therefore imposes an energetic demand that is expected to be lowest in brackish water compared to fresh and sea water. Across species, there is no clear trend between oxygen uptake and salinity, and estimates of cost of osmotic and ionic regulation vary from a few per cent to >30% of standard metabolism.     

Guppies,toadfish, lungfish,coelacanths and frogs: a scenario for the evolution of urea retention in fishes

Synopsis The question of how (and why) the ureosmotic strategy, characteristic of Latimeria chalumnae and the chondrichthians evolved is addressed. There are three requirements for ureosmotic regulation: urea synthesis via the ornithine-urea cycle, urea tolerance involving biochemical and physiological adjustments, and urea retention that requires renal, branchial, metabolic and reproductive adaptations. Several examples of lower vertebrates in which urea plays a physiological role are considered to see whether they might provide insight into the origin of ureosmotic regulation. The guppy shows high urea synthesis and retention during embryonic development, and it is possible that a developmental role of urea is a general phenomenon in fishes. The toadfish, thought to be an enigma with high urea synthesis in the absence of an obvious physiological role of urea, is ureotelic under some conditions. Its urea excretion is likely related to renal function and/or parental care. In lungfish high ureogenesis is associated with estivation in periodically dry habitats. The resultant hyperuremia prevents ammonia toxicity, inhibits water loss and may repress metabolism. Latimeria is a classic marine ureosmotic regulator in which urea is used as an osmolyte that allows osmotic equilibrium with sea water while maintaining low ion levels. Adults of the frog, Rana cancrivora, are also ureosmotic regulators in brackish water. A scenario is proposed that suggests how ureosmotic regulation could have evolved in Latimeria and other fishes. The ornithine-urea cycle (composed of an arginine synthetic pathway and a second pathway that splits arginine into urea) occurred in fossil anadromous agnathans. Here the first pathway functioned in the ammocoete-like larvae for the generation of arginine to supplement a protein-deficient diet of algae, whereas the arginase pathway was important in the embryo for vitellin catabolism. Gnathostome evolution was associated with trends towards large eggs and prolonged development, requiring a complete ornithine-urea cycle for ammonia detoxification in embryos. Retention of a complete ornithine-urea cycle throughout adult life (via paedomorphosis) would preadapt any relatively large, sluggish, euryhaline fish for ureosmotic regulation when it was exposed to sea water. It is suggested that ureosmotic regulators evolved from freshwater or anadromous ancestors that entered the marine habitat. Once early ureosmotic regulators were established in the sea there would have been strong selection for internal fertilization and development, as is seen in Latimeria and many elasmobranchs. It is suggested that ureosmotic regulation was a common strategy in Paleozoic marine gnathostomes.     

Effects of dehydration on water relations and survival of lumbricid earthworm egg capsules

Earthworm egg capsules of five species were compared with regard to survival and water relations upon exposure to controlled dehydration at 20°C. Cocoons of the investigated species all contained about 3.5 g water·g^-1 dry weight when fully hydrated. Approximately 18% of this does not readily freeze upon cooling to -40°C and is referred to as osmotically inactive water. Cocoons exposed to desiccation lose a large proportion of the osmotically active water over 1–4 days until water in the cocoon fluids has equilibrated with surrounding water vapour. The amount of osmotically inactive water, on the other hand, is only reduced by 10–20%. Dendrobaena octaedra was the species most tolerant to drought, its tolerance limit coinciding with loss of practically all osmotically active water. For the five species investigated, there seemed not to be any clear correlation between drought tolerance and microhabitat. Previous investigations have suggested a very close relation between tolerance to dehydration and to subzero temperatures in overwintering earthworm cocoons. Survival at a given level of dehydration at room temperature is less than at temperatures below 0°C, and the tolerance of room temperature dehydration is not closely correlated with cold hardiness across the range of the species studied.Abbreviations dw dry weight - DSC differential scanning calorimetry - fw _pd fresh weight of partially dehydrated cocoons - OAW osmotically active water - OIW osmotically inactive water - Osm osmolality - water potential - R universal gas constant - T absolute temperature - V specific volume of water     

Chloride and water secretion and absorption by the gills of the eel

Summary Perfusion experiments with the heart-gill preparation of the common eel, Anguilla vulgaris, are presented. Various concentrations of perfusion medium were used with eels from both sea water and fresh water. The external medium was sea water in the case of the sea water eels and fresh water in the other case.Perfusate was collected over a period of hours in each experiment and the exchange of chloride and water was studied.In the early experiments it was shown that normal permeability of the gills to these substances is small. The development of an extremely precise chloride method made it possible to evaluate the magnitudes of the exchanges in accurate quantitative terms.Experiments with fresh water as external medium showed that there is a small but perfectly significant change in chloride concentration in the perfusion medium as it passes through the gills so that it becomes more dilute. Reasons are adduced from which it is concluded that the dilution is due to the diffusion of water into the internal medium through the surface of the gills. This branchial water intake is calculated to be something like 40 cc. per kilogram of eel per day. This figure is in substantial agreement with Smith's measurements of the rate of urine formation in fresh water fishes.It was found that when the external medium is sea water, the chloride concentration of the internal medium decreases at a considerably faster rate than in the case where fresh water is present outside the gills. Experiments in which the external medium was analysed showed that chloride was being secreted into the concentrated sea water. It was found possible to measure volume changes in the external medium as well as the changes of chloride concentration in both mediums. These experiments demonstrated beyond doubt that a concentrated chloride solution is secreted by the gills in opposition to a large concentration gradient.Calculations made from several different bases showed that the measurements of volume and of chloride concentration consistently agree in yielding similar values for the volume and concentration of the chloride solution secreted by the gills of the sea water eel.It is shown that the magnitude of the concentration changes effected are related to the concentration of the internal medium used so that a small increase in the concentration of the perfusion medium results in a large increase in the amount of chloride concentration change occurring in the perfusion. Below a certain concentration of the internal medium the gills no longer give evidence of doing concentration work, and may even become permeable as evidenced by the internal medium increasing in concentration as it passes through the gills.Similarities between the activities of the gill of the sea water eel and of the normal mammalian kidney are pointed out. A rough calculation indicates that the work performed by the two organs is of the same order when computed in terms of grams of tissue involved.Experiments in which urea analyses were made showed that the gills of the eel are permeable to urea and that the amount of urea excreted through the gills as determined by direct measurement agrees quite well with Smith's figures for extfa-renal urea excretion.Possible objections to some of the conclusions are brought forth and their untenability demonstrated.Fellow of the National Research Council of America.     

Comparative distribution and embryotoxicity of hydroxyurea in pregnant rats and rhesus monkeys.

Hydroxyurea was given to pregnant rhesus monkeys and pregnant rats in regimens adjusted to produce similar degrees of teratogenicity, for the purpose of comparing the distribution of the drug in the females and their embryos. According, in rats 137 mg/kg/day ip on days 9-12 resulted in a drug half-life in maternal plasma of about 15 min and in embryos about 85 min, after the last injection; and in monkeys 100 mg/kg/days iv on days 23-32 resulted in drug half-life in maternal plasma estimated to be 120 min and in embryos 265 min, after the last injection. Using as a baseline of biological effects the minimal concentration known to inhibit DNA synthesis in rat embryos and cancer cells, namely 10(-4) M, it was calculated that the rat embryos in the present study were exposed to this level or more for approximately 12 h whereas the monkey embryos were exposed for approximately 100 h. Although the teratogenic effects were not identical in the two species, these data are interpreted to mean that rat embryos are teratogenically much more sensitive to hydroxyurea than monkey embryos. These observations have important implications in the selection of appropriate species for tests to estimate human teratogenic risks. The rat, which is currently the most widely used animal for such tests, displays sizeable differences from rhesue monkeys, which is one of the animals thought to be most like man in teratogenic susceptibility.     

Nitrogen distribution and excretion during embryonic post-yolk sac development inZoarces viviparus

In the viviparous teleostZoarces viviparus (L.) embryonic post-yolk sac development in the ovary is characterized by significant increases in dry weight and nitrogen per embryo, thus indicating an extensive matrotrophic relationship. In the ovarian fluid surrounding the embryos during their intraovarian development, sources of nitrogen were shown to derive from amino acids, urea, ammonia and various cellular components. The level of urea in the ovarian fluid increased significantly from 3.68±0.25 mol urea-N·ml^-1 during early post-yolk sac embryonic development to 6.14±0.44 in late development. The corresponding level of ammonia-N in the ovarian fluid increased from 0.25 to 0.45 mol·ml^-1. An estimation of embryonic nitrogen loss was made by measuring urea and ammonia-N excretion in vitro by post-yolk sac embryos or larvae (i.e. seawater-acclimated embryos). Urea-N constituted an average of 65% of the total nitrogen excreted by the embryos into the ambient medium during a 5-h time-course compared to only 35% in the larvae. Urea-N was excreted at maximum rates during the first hour of the experiment, 0.54±0.09 mol N·g^-1·h^-1 by embryos and 0.35±0.02 by larvae, and then declined to lower levels in both embryos and larvae. A decline after 1 h was also observed for excretion rates of ammonia. In conclusion, the capacity for urea excretion by post-yolk sac embryos ofZ. viviparus may be of adaptive significance for their prolonged stay in ovario. The capacity for excretion of urea seems to decrease after acclimation to sea water.Abbreviations aa amino acid(s) - bm body mass - dw dry weight - NPS ninhydrin-positive substances - sw sea water - ww wet weight     

Chloride extrusion in the isolated perfused teleost gill

Summary Chloride extrusion is examined in the isolated perfused gill of the pinfish,Lagodon rhomboides. In both sea water and Ringer's baths, the Cl efflux from the isolated gill is 45% that of the intact animal. The transepithelial electrical potential (TEP) across the isolated gill in sea water is equal to that in vivo, in Ringer's the gill TEP is slightly less than in vivo. Cl efflux is linearly dependent upon afferent flow of the perfusate. Furosemide, added to the perfusate inhibits 57% of the Cl efflux in gills bathed bilaterally by Ringer's. Ouabain causes a marked vasoconstriction and increase in afferent pressure. Removal of Na from the perfusate produces an inhibition of the Cl efflux that is not potential mediated. Net extrusion of Cl is inhibited in isolated gills bathed bilaterally by sodium free Ringer's.     

Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

Features of the organization of low-molecular weight proteins from the blood and tissue fluid of the common stingray <Emphasis Type="Italic">Dasyatis pastinaca</Emphasis> (Chondroichthyes: Trygonidae)

Low-molecular weight proteins were revealed in the blood serum and tissue fluid from the muscles of the common stingray Dasyatis pastinaca. In the absence of urea in the reaction medium in vitro, these proteins aggregated into supramolecular complexes; 8 M urea caused the disintegration of these complexes into individual proteins. The role of urea as a component of an organism’s fluids in the formation of the structural arrangements of blood plasma proteins and other biological fluids of cartilaginous fishes is discussed. It is shown that the extracellular fluids of the stingray also contain low-molecular weight proteins as osmotically active compounds, along with urea, trimetilaminoxid, and salts.     

Effects of salt loading on salt gland function in the euryhaline turtle,Malaclemys terrapin

Summary The estuarine turtle,Malaclemys terrapin is able to ionregulate when acclimated to fresh water, 55% sea water or 100% (full strength) sea water, but when in 100% sea water it does not volume regulate successfully. Orbital gland secretions collected by a new eye cup method are very low in animals from all three salinities without salt load. After salt loading the animals from all three groups produce an orbital gland secretion with a sodium concentration greater than sea water. The concentration of ions and kinetics of the response are similar in all three groups. Orbital gland secretion returns to control preload levels well before the injected load is excreted. There is no correlation between the plasma sodium concentration and any of the parameters of the orbital gland response. There is also no correlation between the concentration of sodium in the tear fluid or the rate of sodium excretion and the level of K⁺-stimulatedp-nitrophenylphosphatase activity in the gland. Some of these unexpected results may relate to the estuarine habitat occupied byMalaclemys.Abbreviations K ⁺–NPPase potasium stimulated p-nitrophenylphosphatase - Na–K-ATPase sodium, potassium stimulated adenosine triphosphatase     

中国水蛇雌性生殖系统的血液循环

取12条处于不同生殖期的雌性中国水蛇作血管单注射,观察其生殖系统血液循环特点。结果表明,卵巢处于静止期的个体的生殖系统循环模式与雄体相似,卵子处于迅速生长期,卵巢血管变得十分发达,布满于正在生长的卵子表面,卵子处于成熟期,卵巢血管又回复到模式状态;受精卵落入子宫中发育时,子宫血管变得十分发达,子宫壁及卵黄膜均十分薄,子宫腔中有液体浸润胚胎,提示了胚胎与母体之间可能通过这些液体进行某些物质交换。     

Concanavalin A and wheat germ agglutinin binding to sea urchin embryo basal laminae

Summary The basal laminae and inner extracellular matrices of Lytechinus pictus and Arbacia punctulata embryos were characterized on the basis of lectin binding. Developmental stage specific patterns of lectin binding were observed after microinjection of Con A-FITC and WGA-FITC. Lectin-specific patterns differed between control, sulfate free sea water (SFSW) and tunicamycin treated embryos. Con A injection resulted in the rounding-up of cells in the epithelium and was most pronounced in embryos cultured in the presence of tunicamycin. Basal laminae were isolated by Triton X-100 extraction of whole embryos. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose and incubated in biotinylated lectins. Lectin-binding glycoproteins were detected with avidin peroxidase. The electrophoretic pattern of Con A-binding proteins in early developmental stages of Arbacia was similar with several low molecular weight species appearing at gastrulation in control and SFSW embryos. WGA-binding in Arbacia and Lytechinus control embryos was limited to a 125,000 Mr glycoprotein (gp125). In addition to gp125, several high molecular weight WGA-binding glycoproteins were also detected in SFSW embryos. The evidence suggests that mesenchyme migration and gastrulation are correlated with changes in the molecular composition of the ECM.