Surface Of Yolk Research Articles

Improved surrogate eggshell incubation system for assisted reproductive technologies such as cloning, genome modification, and de-extinction efforts

A germinal disc located on the egg yolk surface drives genetic modification. Windowed and surrogate eggshell incubation methods have been developed, but these exhibit limited abilities to generate transgenic chickens. In the present study, we investigated the frequency of observing the germinal disc according to the preincubation positioning direction and time and found that it depended on those conditions, but only a few chicks (2.8–5.6%) hatched using the windowed method. Then, we attempted to improve surrogate method via one- or two-step procedures. All eggs, including surrogates that were 10 g heavier than the donor eggs, were obtained from a poultry flock of the same age. With the one-step surrogate method, where the donor egg was transferred only once through a 3.5 cm hole on the point end, at the first day of preincubation, into the surrogate egg, the survival rate at day 4 was 30.8%, and the hatching rate was 11.8%. With the two-step surrogate method (transfer was on the 1st and 4th day of incubation), the survival rate at day 4 was improved to 90.7%, and a hatching rate of 70.0% was achieved. Therefore, this method can be effective for in ovo artificial incubation.

Effects of different light qualities on embryonic development of Sepia pharaonis

To investigate the impact of different light qualities (red, blue, green, yellow, and white light) on the embryonic development of Sepia pharaonis, this experiment studied the effects of different light qualities on the timing of embryonic development, incubation period, hatching rate, malformation rate, weight of hatchlings, and survival rate on the 7th day after hatching. The findings demonstrated a significant influence of different light qualities on the embryonic development of S. pharaonis (P<0.05). Specifically, red and blue light led to increased occurrences of phenomena such as yolk surface cracking, cell autolysis, coagulation of cells in the animal pole, yolk sac edema, malformations in endoskeleton development, and decreased pigmentation and whitening of the body surface compared to the control group (white light). Conversely, green light showed a tendency to reduce these phenomena. The hatching rate and hatching cuttlefish weight were highest under green and yellow light conditions, with significantly greater survival rate on the 7th day after hatching compared to red, blue, and white light conditions. Furthermore, the malformation rate was notably lower under green light conditions compared to other light groups. Red and blue light conditions exhibited the shortest hatching periods, significantly outpacing other light groups. In conclusion, green light appears advantageous for enhancing hatching rate, the quality and health of hatched larvae, reducing malformation occurrences and embryonic mortality, and improving overall hatching quality.

Heterochronic development of pelvic fins in zebrafish: possible involvement of temporal regulation of pitx1 expression.

Anterior and posterior paired appendages of vertebrates are notable examples of heterochrony in the relative timing of their development. In teleosts, posterior paired appendages (pelvic fin buds) emerge much later than their anterior paired appendages (pectoral fin buds). Pelvic fin buds of zebrafish (Danio rerio) appear at 3weeks post-fertilization (wpf) during the larva-to-juvenile transition (metamorphosis), whereas pectoral fin buds arise from the lateral plate mesoderm on the yolk surface at the embryonic stage. Here we explored the mechanism by which presumptive pelvic fin cells maintain their fate, which is determined at the embryonic stage, until the onset of metamorphosis. Expression analysis revealed that transcripts of pitx1, one of the key factors for the development of posterior paired appendages, became briefly detectable in the posterior lateral plate mesoderm at early embryonic stages. Further analysis indicated that the pelvic fin-specific pitx1 enhancer was in the poised state at the larval stage and is activated at the juvenile stage. We discuss the implications of these findings for the heterochronic development of pelvic fin buds.

Force measurements of Myosin II waves at the yolk surface during Drosophila dorsal closure

The mechanical properties and the forces involved during tissue morphogenesis have been the focus of much research in the last years. Absolute values of forces during tissue closure events have not yet been measured. This is also true for a common force-producing mechanism involving Myosin II waves that results in pulsed cell surface contractions. Our patented magnetic tweezer, CAARMA, integrated into a spinning disk confocal microscope, provides a powerful explorative tool for quantitatively measuring forces during tissue morphogenesis. Here, we used this tool to quantify the in vivo force production of Myosin II waves that we observed at the dorsal surface of the yolk cell in stage 13 Drosophila melanogaster embryos. In addition to providing for the first time to our knowledge quantitative values on an active Myosin-driven force, we elucidated the dynamics of the Myosin II waves by measuring their periodicity in both absence and presence of external perturbations, and we characterized the mechanical properties of the dorsal yolk cell surface.

Construction of Longan–like hybrid structures by anchoring nickel hydroxide on yolk–shell polypyrrole for asymmetric supercapacitors

To effectively avoid the “dead volume” and improve the utilization ratio of the electrode material, a novel longan-like hybrid structure has been designed and fabricated by controlled growth of Ni(OH)2 nanosheets on the surface of yolk–shell PPy nanospheres at a mild reaction temperature. The strategy takes full advantages of the hollow structure and further reduces the charge transport distance, leading to high conductivity and energy storage capacity of longan-like hybrid structures. Furthermore, the assembled asymmetric supercapacitors (ASCs) exhibits a high retention ratio of 91.5% for capacitance after 6000 continuous cycles and an impressive energy density of 34 W h kg−1 at 755 W kg−1. Remarkably, two devices in series have a higher cell-voltage output of 3.0 V compared to a single device. This work will supply a new hollow nanostructure design strategy to enhance the electrochemical performance of electrode materials.

Fabrication of CeO2-MO x (M = Cu, Co, Ni) composite yolk-shell nanospheres with enhanced catalytic properties for CO oxidation.

CeO2–MOx (M = Cu, Co, Ni) composite yolk–shell nanospheres with uniform size were fabricated by a general wet-chemical approach. It involved a non-equilibrium heat-treatment of Ce coordination polymer colloidal spheres (Ce-CPCSs) with a proper heating rate to produce CeO2 yolk–shell nanospheres, followed by a solvothermal treatment of as-synthesized CeO2 with M(CH3COO)2 in ethanol solution. During the solvothermal process, highly dispersed MOx species were decorated on the surface of CeO2 yolk–shell nanospheres to form CeO2–MOx composites. As a CO oxidation catalyst, the CeO2–MOx composite yolk–shell nanospheres showed strikingly higher catalytic activity than naked CeO2 due to the strong synergistic interaction at the interface sites between MOx and CeO2. Cycling tests demonstrate the good cycle stability of these yolk–shell nanospheres. The initial concentration of M(CH3COO)2·xH2O in the synthesis process played a significant role in catalytic performance for CO oxidation. Impressively, complete CO conversion as reached at a relatively low temperature of 145 °C over the CeO2–CuOx-2 sample. Furthermore, the CeO2–CuOx catalyst is more active than the CeO2–CoOx and CeO2–NiO catalysts, indicating that the catalytic activity is correlates with the metal oxide. Additionally, this versatile synthesis approach can be expected to create other ceria-based composite oxide systems with various structures for a broad range of technical applications.

Digestive enzyme ratios are good indicators of hatchling yolk reserve and digestive gland maturation in early life stages of cuttlefish Sepia officinalis L.: application of these new tools in ecology and aquaculture.

In Sepia officinalis (Linnaeus, 1758), the digestive gland matures during the first month post-hatching, while a shift from intracellular acid to extracellular alkaline digestion occurs. The purpose of this study was to investigate the possibility of using enzymatic ratios for the description of digestive system maturation in early life stages of S. officinalis. Second, it is intended to apply these new tools as eco-physiological indicators for understanding the impact of cuttlefish eggs' life history from different spawning sites of the English Channel on digestive performance of juveniles. An experimental rearing was performed over 35days after hatching (DAH) on juveniles from wild collected eggs in 2010 and 2011. Four digestive enzyme activities and their ratios [i.e., trypsin, cathepsin, acid (ACP), and alkaline (ALP) phosphatase, ALP/ACP, and trypsin/cathepsin] were studied along with histological features (e.g., internal yolk surface and digestive gland development). The two enzyme ratios were good indicators of digestive system maturation allowing the study of the digestive gland's development. They were highly correlated to juveniles' weight increase and histological features of the gland in early DAH. These ratios described more accurately the shift occurring between the intracellular acid and the extracellular alkaline modes of digestion in S. officinalis and were more specific than separated enzyme activities. Their application as eco-physiological tools revealed that enzyme ratios reflected yolk content and digestive gland development in new hatching juveniles. Finally, ALP/ACP ratio was shown to be a powerful tool to describe growth performance of S. officinalis which is useful for aquaculture optimization.

Reproduction, embryonic and early post-embryonic development of saffron cod &lt;i&gt;Eleginus gracilis&lt;/i&gt; (Gadidae) from the Tauyskaya Guba Bay (northern Okhotsk Sea)

New information on reproductive biology, embryonic and early post-embryonic development of saffron cod Eleginus gracilis Tilesius is presented on the base of long-term observations in the Tauyskaya Guba Bay, northern Okhotsk Sea. The species begins to spawn in this area at the age of 2 years, with the body length of 14 cm, and reaches mass maturity (when at least 50 % of fish in the age group are matured) at the age of 3 years. All fish are matured at the age of 5 year and elder. Males prevail in the first three age groups, but females dominate in elder groups. The mature fish enter to the spawning grounds in the Tauyskaya Guba Bay in December. The spawning occurs in January-March under the water temperature of -1.5 о C. The saffron cod females spawn 7.2-172.0 thousand eggs, depending on their size, weight, and age (on average 56,600 eggs or 334 eggs per 1 g of body weight). Relative fecundity of the females increases until the age of 7 years, elder females have slightly lower but stable fecundity. The eggs at the beginning of the second stage of development have the diameter of 1.00-1.58 mm, on average 1.410 ± 0.005 mm, and are distinguished by almost transparent shell and colorless yolk, with the ochre colored plasmodisc. The animal pole (were plasmodisc is formed) is in the upper position. A micropyle on the outer shell is clearly visible as a small bulge with diameter slit surrounded by two concentric beads (this structure remains during entire embryogenesis and can be considered as a taxonomic sign of the species rank). Under the incubation temperature of 2.8 о C, the eggs reach the stage of two blastomeres in 11-12 h. At the first stages of partition, numerous small fat-like inclusions are visible in the blastomeres. The second and third cycles of the zygote division with forming of 4 and 8 blastomers pass in the age interval 14-18 h. The interphase between divisions increases from 3 h between the first and second divisions to 5 h between further divisions. The eggs reach the early stage III (large cell blastula) and late stage III (small cell blastula) after 30 and 41 h of their development, respectively. By the end of the stage III, the dome of blastula is maximally developed. At the age of 75 h, the stage of gastrulation and epiboly begins. The blastoderm covers 50-60 % of the yolk surface after 123 h. Bead-shaped embryo is formed after 177 h of development. In the next 309, 417, 459 h of incubation, the embryos undergo successively the stages of tail separation from the yolk sac, mobility beginning, and heart functioning. In experiments, prelarvae of saffron cod hatched after 849 h of incubation. Their size in the Tauyskaya Guba population is the largest known in scientific literature: the total body length varies from 5.30 to 6.25 mm (on average 5.82 ± 0.05 mm); besides, they are distinguished by greater number of pigment cells on the yolk sac and nearby. Intensive pigmentation of prelarvae is known for other fish species in the northern Okhotsk Sea, as well, that presumably is their adaptation for better accumulation of solar heat in conditions of late spring warming in this area.

Vaccination of chickens decreased Newcastle disease virus contamination in eggs

Newcastle disease is an important health issue of poultry causing major economic losses and inhibits trade worldwide. Vaccination is used as a control measure, but it is unknown whether vaccination will prevent virus contamination of eggs. In this study, hens were sham-vaccinated or received one or two doses of inactivated LaSota vaccine, followed three weeks later by virulent Newcastle disease virus (NDV) challenge. Eggs were collected daily and shell, albumen and yolk were subjected to virus isolation, as were oral and cloacal swabs at 2 and 4 days post-challenge (dpc). A second experiment evaluated the distribution of the virus in the reproductive tract of non-vaccinates. All vaccinated chickens survived challenge, and the levels of virus shed from cloacal swabs were decreased significantly when compared to shams. In non-vaccinated hens, virus was detected in the ovary and all segments of the oviduct. Yolk, albumen and eggshell surface from eggs laid at day 4 and 5 post-infection by sham-vaccinated hens were positive for NDV, but eggs from LaSota vaccinated hens lacked virus in internal egg components (i.e. yolk and albumen) and had reduction in the number of positive eggshell surfaces. These results indicate virulent NDV can replicate in the reproductive tract of hens and contaminate internal components of eggs and eggshell surface, but vaccination was able to prevent internal egg contamination, reducing eggshell surface contamination, and reducing shedding from digestive and respiratory tracts in virulent NDV challenged hens.

Anterior migration of lateral plate mesodermal cells during embryogenesis of the pufferfish Takifugu niphobles: insight into the rostral positioning of pelvic fins.

In vertebrates, paired appendages (limbs and fins) are derived from the somatic mesoderm subsequent to the separation of the lateral plate mesoderm into somatic and splanchnic layers. This is less clear for teleosts, however, because the developmental processes of separation into two layers and of extension over the yolk have rarely been studied. During teleost evolution, the position of pelvic fins has generally shifted rostrally (Rosen; Nelson, 1982, 1994), although at the early embryonic stage the presumptive pelvic fin cells are initially located near the future anus region - the anterior border of hoxc10a expression in the spinal cord - regardless of their final destination. Our previous studies in zebrafish (abdominal pelvic fins) and Nile tilapia (thoracic pelvic fins) showed that the presumptive pelvic fin cells shift their position with respect to the body trunk after its protrusion from the yolk surface. Furthermore, in Nile tilapia, presumptive pelvic fin cells migrate anteriorly on the yolk surface. Here, we examined the embryonic development of the lateral plate mesoderm at histological levels in the pufferfish Takifugu niphobles, which belongs to the highly derived teleost order Tetraodontiformes, and lacks pelvic fins. Our results show that, in T.niphobles, the lateral plate mesoderm bulges out as two separate layers of cells alongside the body trunk prior to its further extension to cover the yolk sphere. Once the lateral plate mesoderm extends laterally, it rapidly covers the surface of the yolk. Furthermore, cells located near the anterior border of hoxc10a expression in the spinal cord reach the anterior-most region of the yolk surface. In light of our previous and current studies, we propose that anterior migration of presumptive pelvic fin cells might be required for them to reach the thoracic or more anterior positions as is seen in other highly derived teleost groups.

Development of the lateral plate mesoderm in medaka Oryzias latipes and Nile tilapia Oreochromis niloticus: insight into the diversification of pelvic fin position.

The position of the pelvic fins among teleost fishes has tended to shift rostrally during evolution. This positional shift seems to have led to the diversification of feeding behavior and allowed adaptation to new environments. To understand the developmental basis of this shift in pelvic fin position among teleosts, we investigated the embryonic development of the lateral plate mesoderm, which gives rise to the pelvic fins, at histological levels in the medaka Oryzias latipes (abdominal pelvic fins) and Nile tilapia Oreochromis niloticus (thoracic pelvic fins). Our histological analyses revealed that the lateral plate mesodermal cells expand not only ventrally but also rostrally to cover the yolk during embryogenesis of both medaka and Nile tilapia. In medaka, we also found that the lateral plate mesoderm completely covered the yolk prior to the initiation of the pelvic fin buds, whereas in Nile tilapia the pelvic fin buds appeared in the body wall from the lateral plate mesoderm at the thoracic level when the lateral plate mesodermal cells only covered one-third of the yolk. We discuss the relevance of such differences in the rate of the lateral plate mesoderm expansion on the yolk surface and the position of the pelvic fins.

The larval development of the Russell's snapper,Lutjanus russellii(Teleostei: Lutjanidae) reared under laboratory conditions

The larval development of the Russell's snapperLutjanus russelliiis described and illustrated for the first time. Fertilized eggs, with a mean diameter of 0.80 ± 0.04 mm and a range from 0.71 to 0.84 mm, were spherical, transparent, buoyant and unpigmented. Embryonic development lasted 26 hours 50 minutes at 24.7°C. Newly hatched larvae were 1.86 ± 0.10 mm in total length (TL) with 24 (10 + 14) myomeres and had an oil globule in the ventroanterior area of the yolk sac. Two days after hatching (DAH, 2.61 mm TL), the mouth opened. Early larvae had melanophores scattered on the head, body and yolk surface. Thirteen DAH (2.74 mm TL), the buds of the second dorsal and pelvic-fin spines had appeared. At 5.81 mm TL, the notochord was slightly flexed, and the hypural bones and caudal-fin rays had begun to develop. The ratios of the second dorsal and pelvic-fin spine lengths to TL attained their maximum, 40% and 36%, respectively. At 14.02 mm TL, all fins had the adult complement of rays and spines. The characteristic of this species, with four black stripes on the sides and with a round black spot on the upper back, was completed in juvenile of 23.1 mm TL. The larval habitat shifted from the surface and middle layers to the tank bottom. A statistical model:Y equals 2.18e0.06X, whereYis mean TL (mm) andXrepresents DAH, explained 99.34% of variation in growth (P&lt; 0.001,R2= 0.9934).

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes. In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts. In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Manipulation and Imaging of Kryptolebias marmoratus Embryos

The self-fertilizing mangrove killifish, Kryptolebias marmoratus, is an upcoming model species for a range of biological disciplines. To further establish this model in the field of developmental biology, we examined several techniques for embryonic manipulation and for imaging that can be used in an array of experimental designs. These methodological approaches can be divided into two categories: handling of embryos with and without their chorionic membrane. Embryos still enclosed in their chorion can be manipulated using an agarose bed or a methyl cellulose system, holding them in place and allowing their rotation to more specific angles and positions. Using these methods, we demonstrate microinjection of embryos and monitoring of fluorescent yolk syncytial nuclei (YSN) using both stereo and compound microscopes. For higher magnification imaging using compound microscopes as well as time-lapse analyses, embryos were dechorionated and embedded in low-melting-point agarose. To demonstrate this embedding technique, we further examined fluorescent YSN and also analyzed the yolk surface of K. marmoratus embryos. The latter was observed to provide an excellent imaging platform for study of the behavior and morphology of cells during embryonic development, for various types of cells. Our data demonstrate that K. marmoratus is an excellent model species for research in developmental biology, as methodological approaches for the manipulation and imaging of embryos are efficient and readily available.

Revealing the mechanisms of the rostral shift of pelvic fins among teleost fishes

In teleost fishes, the position of the pelvic fins shift during evolution; this positional shift seems to have diversified their locomotion and feeding behavior, thereby expanding the habitats of these fishes. Thus, such a positional shift of the pelvic fins is one of the significant features of teleost fishes from evolutionary, embryological, and taxonomic viewpoints, but no studies to date have investigated the mechanism for the rostral shift of the pelvic fins from the anal region in teleosts. Examining the fate of the prospective pelvic fin cells of the zebrafish Danio rerio and the Nile tilapia Oreochromis niloticus embryos demonstrates that the prospective pelvic fin cells are originally located near the anus, as seen in tetrapods, but their position shifts with respect to the body trunk after its protrusion from the yolk surface. In this article, we highlight such recent findings and discuss the mechanisms of pelvic fin evolution among teleost fishes.

Requirement of Npc1 and availability of cholesterol for early embryonic cell movements in zebrafish

Niemann-Pick disease, type C (NP-C), often associated with Niemann-Pick disease, type C1 (NPC1) mutations, is a cholesterol-storage disorder characterized by cellular lipid accumulation, neurodegeneration, and reduced steroid production. To study NPC1 function in vivo, we cloned zebrafish npc1 and analyzed its gene expression and activity by reducing Npc1 protein with morpholino (MO)-oligonucleotides. Filipin staining in npc1-morphant cells was punctate, suggesting abnormal accumulation of cholesterol. Developmentally, reducing Npc1 did not disrupt early cell fate or survival; however, early morphogenetic movements were delayed, and the actin cytoskeleton network was abnormal. MO-induced defects were rescued with ectopic expression of mouse NPC1, demonstrating functional gene conservation, and by treatments with steroids pregnenolone or dexamethasone, suggesting that reduced steroidogenesis contributed to abnormal cell movements. Cell death was found in anterior tissues of npc1 morphants at later stages, consistent with findings in mammals. Collectively, these studies show that npc1 is required early for proper cell movement and cholesterol localization and later for cell survival.

Ontogeny as a whole: Some developmental trends

In many animal groups, rudimentary organs of adult organisms appear at early stages of egg cleavage, when there is no trace of morphological differentiation. More than half a century ago, I. I. Schmalhausen developed a profound concept of integrity of the organism in the course of development. He emphasized that mutual adaptation of organs is based on correlations, involving the so-called nonhereditary modifications, which are transformed into hereditary traits by means of natural selection. There are many exceptions to the biogenetic law, as it was understood by de Beer. In particular, embryo of the pouched tree frog Gastrotheca shows a retarded development and is spread over the yolk surface. As a result, it becomes similar somewhat to embryos of amniotes. After the metamorphosis, Xenopus returns to ammonotelism. The integrity of ontogeny at the molecular level has much in common with the neutrality theory.

Allometric growth of the trunk leads to the rostral shift of the pelvic fin in teleost fishes

The pelvic fin position among teleost fishes has shifted rostrally during evolution, resulting in diversification of both behavior and habitat. We explored the developmental basis for the rostral shift in pelvic fin position in teleost fishes using zebrafish (abdominal pelvic fins) and Nile tilapia (thoracic pelvic fins). Cell fate mapping experiments revealed that changes in the distribution of lateral plate mesodermal cells accompany the trunk–tail protrusion. Presumptive pelvic fin cells are originally located at the body wall adjacent to the anterior limit of hoxc10a expression in the spinal cord, and their position shifts rostrally as the trunk grows. We then showed that the differences in pelvic fin position between zebrafish and Nile tilapia were not due to changes in expression or function of gdf11. We also found that hox-independent motoneurons located above the pelvic fins innervate into the pelvic musculature. Our results suggest that there is a common mechanism among teleosts and tetrapods that controls paired appendage positioning via gdf11, but in teleost fishes the position of prospective pelvic fin cells on the yolk surface shifts as the trunk grows. In addition, teleost motoneurons, which lack lateral motor columns, innervate the pelvic fins in a manner independent of the rostral–caudal patterns of hox expression in the spinal cord.

Expression of Ol-foxi3 and Na +/K +-ATPase in ionocytes during the development of euryhaline medaka ( Oryzias latipes) embryos

Osmoregulation is a vital function that is essential to all vertebrates. Ionocytes are epithelial cells responsible for this function and have been extensively studied in adult teleost fish gills. The euryhaline medaka ( Oryzias latipes) has recently emerged as an investigative model because of its ability to acclimatize easily to water presenting various salinities. However, no studies to date have focused on the development of ionocytes in medaka embryos. We first analyzed the distribution of ionocytes in the skin and gills during development, using a specific marker of differentiated ionocytes (the Na +/K +-ATPase pump, or NKA). Strikingly, we were able to identify two ionocyte domains on the yolk surface ectoderm, that we named the Vitellin Zone (VZ) and the Lateral Zone (LZ). In zebrafish, ionocyte differentiation has been shown to be controlled by two forkhead-box genes, foxi3a and foxi3b. We cloned the medaka foxi3 ortholog which appeared to be highly similar to foxi3b. Whole-mount in situ hybridizations performed on medaka embryos revealed that Ol-foxi3 is expressed in differentiated ionocytes of the pharyngeal endoderm, the branchial arches and the yolk epidermis, as well as in epibranchial placode territories. We further focused on the expression patterns of the yolk epidermis and compared the expression of Ol-foxi3 with that of the non-neural progenitor marker p63. We evidenced that Ol-foxi3 is expressed in progenitor cells which are first of all located uniformly in the VZ and then transitorily clustered in the LZ. Taken together, these data contribute to a clearer understanding of osmoregulatory tissue ontogenesis in euryhaline fish.