Blastopore Closure Research Articles

Effects of different temperatures on the embryonic development of the Lebranche mullet Mugil liza.

We herein investigated the influence of temperature on theembryonic development (from fertilisation to hatching) of Mugil liza larvae. For this purpose, oocytes (>600 μm) and sperm were obtained from breeding stock at the laboratory of marine fish culture (LAPMAR). After fertilisation, 1200 eggs were distributed in 12 cylindrical experimental units of 400 mL under four different temperatures 18, 22, 26 and 30 ºC, all in triplicate. Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment. The eggs were visualized and photographed, and the classification of embryonic stages was performed. Temperature influenced the main events of the embryonic development of M. liza. More accelerated development was observed according to the increase in temperature until the gastrula phase. At temperatures of 22 and 26 °C, embryonic development occurred from fertilisation to hatching of the larvae. In the 18 °C treatment, it was verified that most of the embryos ceased development during the final phase of cleavage and the beginning of blastula formation, while in the 30 °C treatment patterns of embryo malformation were also verified, with erratic divisions of the blastomeres, resulting in irregular cells. Unlike what was observed at a temperature of 18 °C, none of the embryos incubated at 30 °C reached the blastopore closure phase, stopping in the gastrula. The larvae hatched in the treatments at 22 and 26 °C were viable and exhibited intense swimming, with a large amount of reserve material (yolk) and an evident drop of oil.

The impact of electromagnetic fields generated by high-voltage power lines on the spatial arrangement of pike (Esox Lucius Linnaeus 1758) embryos

Urbanization and technological advancements result in the dispersion of antropogenic electromagnetic fields (EMF) that can affect on ecosystems. Therefore, it is important to understand their impact on the environment. Aquatic ecosystems are subject to EMF as part of various electricity sources, e.g., high-voltage transmission lines (HVTL). We examined the impact of EMF generated by HVTL on the spatial arrangement and survival of pike (Esox lucius) embryos. Fertilized eggs were incubated under two HVTL configurations 110 kV and 220 kV compared with a control group devoid of anthropogenic EMF. Embryo orientation and survival were monitored until blastopore closure. The control group showed dominance in the arrangement of embryos along the N-S, NNW-SSE, and NNE-SSW axes, with a slight prevalence of northern directions. EMF originating from HVTL did not exert a significant influence on the spatial arrangement of pike embryos, although some deviations from the arrangement noticed in the control group were observed. Increased embryo mortality was observed only at 110 kV site, but probably due to factors unrelated to EMF. In conclusion, EMF generated by HVTL did not significantly change pike embryo orientation or chances of survival. However, longer exposure or higher EMF levels could provoke notable reactions, requiring ongoing evaluation as power networks continue to spread more widely.

Maternal vgll4a regulates zebrafish epiboly through Yap1 activity.

Gastrulation in zebrafish embryos commences with the morphogenetic rearrangement of blastodermal cells, which undergo a coordinated spreading from the animal pole to wrap around the egg at the vegetal pole. This rearrangement, known as epiboly, relies on the orchestrated activity of maternal transcripts present in the egg, compensating for the gradual activation of the zygotic genome. Epiboly involves the mechano-transducer activity of yap1 but what are the regulators of yap1 activity and whether these are maternally or zygotically derived remain elusive. Our study reveals the crucial role of maternal vgll4a, a proposed Yap1 competitor, during zebrafish epiboly. In embryos lacking maternal/zygotic vgll4a (MZvgll4a), the progression of epiboly and blastopore closure is delayed. This delay is associated with the ruffled appearance of the sliding epithelial cells, decreased expression of yap1-downstream targets and transient impairment of the actomyosin ring at the syncytial layer. Our study also shows that, rather than competing with yap1, vgll4a modulates the levels of the E-cadherin/β-catenin adhesion complex at the blastomeres' plasma membrane and hence their actin cortex distribution. Taking these results together, we propose that maternal vgll4a acts at epiboly initiation upstream of yap1 and the E-cadherin/β-catenin adhesion complex, contributing to a proper balance between tissue tension/cohesion and contractility, thereby promoting a timely epiboly progression.

Embryonic and larval development of reciprocal diploid and triploid hybrids of Caspian trout (Salmo caspius) and rainbow trout (Oncorhynchus mykiss)

The present study aims to evaluate the viability of reciprocal crosses between Caspian trout (Salmo caspius) and rainbow trout (Oncorhynchus mykiss), and the effect of triploidization on embryonic development, growth and survival of hybrid embryos and larvae. This experiment included six groups of crosses: (1) pure O. mykiss (2nR); (2) pure S. caspius (2nC); (3) diploid and (4) triploid ♀O. mykiss×♂S. caspius hybrids (2nR × C and 3nR × C); (5) diploid and (6) triploid ♀S. caspius×♂O. mykiss hybrids (2nC × R and 3nC × R). Triploidy was induced by a thermal shock after fertilization. Fertilization, hatching, and embryonic development were studied. Hatching rate was more than 90% in 2nR and 2nC, 35% in 2nR × C group, 45% in 3nR × C group, whereas all embryos died before the eyed stage in 2nC × R and 3nC × R groups. Chromosome number ranged between 57 and 61 and 78–81 in 2nR and 2nC groups, 69–71 in 2nR × C and 99–103 in 3nR × C. The time before blastopore closure was the most important point in the embryonic phases when most deaths occurred in all groups. Hatching occurred earlier in 2nR and 3nR × C groups than in 2nC group. The highest final weight and specific growth rate were observed in 2nR and 3nR × C groups. We have demonstrated that triploidy induction can increase the survival rate of distant hybrid (♀O. mykiss×♂S. caspius) and identified the key embryonic stages that could help introduce this hybrid into aquaculture. The hybrid could be easily commercialized if they show significant improvement in growth, survival and disease resistance.

The Effect of Temperature on the Embryo Development of Cephalopod Sepiella japonica Suggests Crosstalk between Autophagy and Apoptosis.

Temperature is a crucial environmental factor that affects embryonic development, particularly for marine organisms with long embryonic development periods. However, the sensitive period of embryonic development and the role of autophagy/apoptosis in temperature regulation in cephalopods remain unclear. In this study, we cultured embryos of Sepiella japonica, a typical species in the local area of the East China Sea, at different incubation temperatures (18 °C, 23 °C, and 28 °C) to investigate various developmental aspects, including morphological and histological characteristics, mortality rates, the duration of embryonic development, and expression patterns of autophagy-related genes (LC3, BECN1, Inx4) and apoptosis marker genes (Cas3, p53) at 25 developmental stages. Our findings indicate that embryos in the high-temperature (28 °C) group had significantly higher mortality and embryonic malformation rates than those in the low-temperature (18 °C) group. Furthermore, high temperature (28 °C) shortened the duration of embryonic development by 7 days compared to the optimal temperature (23 °C), while low temperature (18 °C) caused a delay of 9 days. Therefore, embryos of S. japonica were more intolerant to high temperatures (28 °C), emphasizing the critical importance of maintaining an appropriate incubation temperature (approximately 23 °C). Additionally, our study observed, for the first time, that the Early blastula, Blastopore closure, and Optic vesicle to Caudal end stages were the most sensitive stages. During these periods, abnormalities in the expression of autophagy-related and apoptosis-related genes were associated with higher rates of mortality and malformations, highlighting the strong correlation and potential interaction between autophagy and apoptosis in embryonic development under varying temperature conditions.

Early embryonic development of green crucian carp Carassius auratus indigentiaus subsp. nov.

The early embryonic development of the green crucian carp (Carassius auratus indigentiaus subsp. nov.) was observed to study its timing and characteristics. The fertilized eggs are round, slightly yellow, and viscous demersal. The egg diameter after water swelling was 1.47 ± 0.04 mm. Embryonic development can be divided into eight stages according to its major characteristics: blastoderm formation, mitotic, blastula, gastrula, neurula, blastopore closure, organogenetic and hatching stages. Under a water temperature of 24 ± 1 °C, salinity of 35 ± 1, and pH of 7.4 ± 0.5, the blastoderm began to form 35 min after fertilization. It entered the mitotic stage at 55 min, blastula stage at 220 min, gastrula stage at 460 min, neurula stage at 675 min, blastopore closure stage at 700 min, organogenetic stage at 900 min and hatching stage at 3390 min. The total length of newly hatched larvae was 4.07 ± 0.35 mm. Regression models of growth characteristics were obtained. The full-length growth rate was fastest from 15 to 26 days, with an average of 0.396 mm/day. Compared with other cyprinid fishes, green crucian carp exhibited some distinct characteristics in certain stages of embryonic development. The eye primordium developed before the sarcomere, and the heart rate was relatively high before the member stage. Yolk fluctuation was observed during the multi-cell phase of embryonic development. The sarcomere formed after the eye primordium. The heart rate in the hatching phase was 136 beats/min. This study provides a reference for embryonic development in green crucian carp, which will assist its large-scale cultivation.

Dorsal lip maturation and initial archenteron extension depend on Wnt11 family ligands

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes during the tailbud period. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues by imaging intact fixed embryos stained for anillin and microtubules. We found that the initial extension of the archenteron is correlated with blastopore lip maturation, and archenteron extension is dramatically disrupted by decreased Wnt11 family signaling. We were aided in our interpretation of the immunofluorescence by the novel, membrane proximal location of the cleavage furrow protein anillin in the epithelium of the blastopore lip and early archenteron.

Embryonic development of the moon jellyfish Aurelia aurita (Cnidaria, Scyphozoa): another variant on the theme of invagination.

BackgroundAurelia aurita (Scyphozoa, Cnidaria) is an emblematic species of the jellyfish. Currently, it is an emerging model of Evo-Devo for studying evolution and molecular regulation of metazoans’ complex life cycle, early development, and cell differentiation. For Aurelia, the genome was sequenced, the molecular cascades involved in the life cycle transitions were characterized, and embryogenesis was studied on the level of gross morphology. As a reliable representative of the class Scyphozoa, Aurelia can be used for comparative analysis of embryonic development within Cnidaria and between Cnidaria and Bilateria. One of the intriguing questions that can be posed is whether the invagination occurring during gastrulation of different cnidarians relies on the same cellular mechanisms. To answer this question, a detailed study of the cellular mechanisms underlying the early development of Aurelia is required.MethodsWe studied the embryogenesis of A. aurita using the modern methods of light microscopy, immunocytochemistry, confocal laser microscopy, scanning and transmission electron microscopy.ResultsIn this article, we report a comprehensive study of the early development of A. aurita from the White Sea population. We described in detail the embryonic development of A. aurita from early cleavage up to the planula larva. We focused mainly on the cell morphogenetic movements underlying gastrulation. The dynamics of cell shape changes and cell behavior during invagination of the archenteron (future endoderm) were characterized. That allowed comparing the gastrulation by invagination in two cnidarian species—scyphozoan A. aurita and anthozoan Nematostella vectensis. We described the successive stages of blastopore closure and found that segregation of the germ layers in A. aurita is linked to the ’healing’ of the blastopore lip. We followed the developmental origin of the planula body parts and characterized the planula cells’ ultrastructure. We also found that the planula endoderm consists of three morphologically distinct compartments along the oral-aboral axis.ConclusionsEpithelial invagination is a fundamental morphogenetic movement that is believed as highly conserved across metazoans. Our data on the cell shaping and behaviours driving invagination in A. aurita contribute to understanding of morphologically similar morphogenesis in different animals. By comparative analysis, we clearly show that invagination may differ at the cellular level between cnidarian species belonging to different classes (Anthozoa and Scyphozoa). The number of cells involved in invagination, the dynamics of the shape of the archenteron cells, the stage of epithelial-mesenchymal transition that these cells can reach, and the fate of blastopore lip cells may vary greatly between species. These results help to gain insight into the evolution of morphogenesis within the Cnidaria and within Metazoa in general.

In preprints: buckling under pressure during gastrulation.

In preprints: buckling under pressure during gastrulation.

Characterization of convergent thickening, a major convergence force producing morphogenic movement in amphibians.

The morphogenic process of convergent thickening (CT) was originally described as the mediolateral convergence and radial thickening of the explanted ventral involuting marginal zone (IMZ) of Xenopus gastrulae (Keller and Danilchik, 1988). Here, we show that CT is expressed in all sectors of the pre-involution IMZ, which transitions to expressing convergent extension (CE) after involution. CT occurs without CE and drives symmetric blastopore closure in ventralized embryos. Assays of tissue affinity and tissue surface tension measurements suggest CT is driven by increased interfacial tension between the deep IMZ and the overlying epithelium. The resulting minimization of deep IMZ surface area drives a tendency to shorten the mediolateral (circumblastoporal) aspect of the IMZ, thereby generating tensile force contributing to blastopore closure (Shook et al., 2018). These results establish CT as an independent force-generating process of evolutionary significance and provide the first clear example of an oriented, tensile force generated by an isotropic, Holtfreterian/Steinbergian tissue affinity change.

EMBRYOGENESIS OF MYLOSSOMA DURIVENTRE (PACU-PEVA)

This paper aimed to test a semi natural reproduction and to study the morphological development of the Mylossoma duriventre embryo until hatching. We selected six females with coelomic cavity bulging and softness and eighteen males that release sperm under light belly compression. We applied two doses of luteinizing hormone (0.0125 mg/kg and 0.125 mg/kg) for females and for males no hormonal induction was made, a semi natural reproduction was performed. After fertilization the embryos were transfer to a incubator and were evaluated every at 15-min intervals during the first hours of development and at one-hour intervals until hatching. The embryos are megalecites with meroblastic division, being visualized six different stages of development: fertilization, cleavage, blastula, gastrula, segmentation and pharyngula. In gastrulation (120-min post fertilization), there is formation of "tail bud" and appearance of somites during segmentation (510-min post fertilization), after the blastopore closure. The hatching occurred with 20 hours of development, at a temperature of 24°C. It was possible to reproduce the pacu peva in captivity, and the morphological development of the embryos showed some specificity when compared to other species and its results regarding embryonic development are essential for a better understanding of biology this species.

Furry is required for cell movements during gastrulation and functionally interacts with NDR1

Gastrulation is a key event in animal embryogenesis during which germ layer precursors are rearranged and the embryonic axes are established. Cell polarization is essential during gastrulation, driving asymmetric cell division, cell movements, and cell shape changes. The furry (fry) gene encodes an evolutionarily conserved protein with a wide variety of cellular functions, including cell polarization and morphogenesis in invertebrates. However, little is known about its function in vertebrate development. Here, we show that in Xenopus, Fry plays a role in morphogenetic processes during gastrulation, in addition to its previously described function in the regulation of dorsal mesoderm gene expression. Using morpholino knock-down, we demonstrate a distinct role for Fry in blastopore closure and dorsal axis elongation. Loss of Fry function drastically affects the movement and morphological polarization of cells during gastrulation and disrupts dorsal mesoderm convergent extension, responsible for head-to-tail elongation. Finally, we evaluate a functional interaction between Fry and NDR1 kinase, providing evidence of an evolutionarily conserved complex required for morphogenesis.

Induced reproduction and early development in dourado, Salminus franciscanus Lima & Britski, 2007 (Pisces: Characiformes).

Our study aimed to establish the response of Salminus franciscanus to hypophysation and describe the main morphological events of its embryonic process. Wild fish were captured in São Francisco River and selected broodstock (females: 66.4 ± 11.1 cm and 4.04 ± 2.32 kg; males: 58.3 ± 10.2 cm and 3.62 ± 1.12 kg) were kept at 26.1 ± 0.6°C for induction of final maturation/gamete release via the hypophysation technique. In females, two doses (0.8 and 5.6 mg/kg body weight) of crude pituitary extract of common carp (Cyprinus carpio) were administered with a 14 h interval. For males, a single dose (2.7 mg/kg body weight) of crude pituitary extract was applied at the same time as the females' second dose. Oocytes and sperm were manually stripped 8 h after a females' second hormonal dose. Fertilization was carried out using the dry method. Eggs were kept in funnel-type 60 L incubators at 24.3 ± 0.3°C and were analyzed and photographed every 10 min. After hormonal induction, 60% of females and 100% of males reacted positively and no broodstock mortality was recorded. The females released an average of 385.2 ± 78.4 g of oocytes and the fertilization rate observed was 50.4 ± 12.3%. The blastopore closure occurred at 7.5 h, somite formation at 12 h and hatching at 20 h post-fertilization. In general, the results of this study improve the understanding of the reproductive biology of dourado and confirm its potential for fish farming in the neotropical region.

Spawning induction and embryonic development of Salminus hilarii (Characiformes: Characidae).

The present study aimed to evaluate two different methods of artificial reproduction induction, and characterize the embryonic development of Salminus hilarii. Different than observed for other tropical fish species, artificial reproduction induction followed by hand-stripping of gametes was considered unfeasible for S. hilarii, as no gamete fertilization was observed. However, females that were induced and spawned naturally presented a fertilization rate of 65.64 ± 0.54%. With a mean temperature of 26.20 ± 0.90ºC it was possible to clearly distinguish a large perivitelline space at 14 min after fertilization (AF) and at 49 min AF more than 50% of the embryos presented two blastomeres, and these cleavages occurred until 1 h 54 min AF. The gastrula phase was characterized at 3 h 56 min AF, and blastopore closure was observed at 8 h 31 min AF. At 9 h 1 min, organogenesis started, with a clear distinction of the yolk mass, embryonic axis, cephalic and caudal regions; at 11 h 51 min AF the embryos already had advanced segmentation and a free tail. Total hatching occurred at 21 h 17 min and after opening the mouth, which occurred at 33 h 9 min, the larvae of S. hilarii presented a strong and characteristic cannibalism. This information can be considered fundamental to improving S. hilarii production in captivity and for collaboration with a conservation programme in the upper Tietê river basin.

Histological and morphological study of the embrionary development of the capitan de la sabana (Eremophilus mutisii)

Eremophilus mutisii, due to its vulnerable condition and its potential for fish farming, generates research challenges; one of them is to know the embryonic changes of the E. mutisiibased on morphological and histological features from fer-tilization to hatching. The embryos were obtained from adult and sexually mature individuals of the Capitan de la Sabana (Eremophilus mutisii), sexually induced with carp pituitary extract. Oocytes and semen were obtained by abdominal pressure 24 hours after the last dose at 14±1°C. Sampling was performed every 60 minutes for the first twelve hours post-fertilization (HPF); from here on at hatching, the sam-ples were fixed every 6 hours. The embryos were fixed in 4% formalin for observing the morphological features and for the histological studies. Results revealed that the fertilized eggs had spherical shape, light green color and average diameter of 1270μm. The first cleavage at 2 HPF was observed, the blastulation began at 9 HPF. Epibolic movements and gastru-lation were observed from 12 HPF to 30 HPF with blastopore closure. At 60 HPF the yolk sac tail detachment was evident and at 66 HPF the first autonomous movements began. Finally, at 72 HPF, the hatching began, showing absence of pigmentation and evidencing a rudimentary digestive tract without oral and anal opening. It was observed the presence of abundant yolk sac with continuous vertical movements of the larva.

Expression of the neuropeptides RFamide and LWamide during development of the coral Acropora millepora in relation to settlement and metamorphosis

Neuropeptides play critical roles in cnidarian development. However, although they are known to play key roles in settlement and metamorphosis, their temporal and spatial developmental expression has not previously been characterized in any coral. We here describe Acropora millepora LWamide and RFamide and their developmental expression from the time of their first appearance, using in situ hybridization and FMRFamide immunohistochemistry. AmRFamide transcripts first appear in the ectoderm toward the oral end of the planula larva following blastopore closure. This oral bias becomes less apparent as the planula develops. The cell bodies of AmRFamide-expressing cells are centrally located in the ectoderm, with narrow projections to the mesoglea and to the cell surface. As the planula approaches settlement, AmRFamide expression disappears and is undetectable in the newly settled polyp. Expressing cells then gradually reappear as the polyp develops, becoming particularly abundant on the tentacles. AmLWamide transcripts first appear in ectodermal cells of the developing planula, with minimal expression at its two ends. The cell bodies of expressing cells lie just above the mesoglea, in a position distinct from those of AmRFamide-expressing cells, and have a narrow projection extending across the ectoderm to its surface. AmLWamide-expressing cells persist for most of the planula stage, disappearing shortly before settlement, but later than AmRFamide-expressing cells. As is the case with AmRFamide, expressing cells are absent from the polyp immediately after settlement, reappearing later on its oral side. AmLWamide expression lags that of AmRFamide in both its disappearance and reappearance. Antibodies to FMRFamide stain cells in a pattern similar to that of the transcripts, but also cells in areas where there is no expression revealed by in situ hybridization, most notably at the aboral end of the planula and in the adult polyp. Adult polyps have numerous staining cells on the tentacles and oral discs, as well as an immunoreactive nerve ring around the mouth. There are scattered staining cells in the coenosarc between polyps and staining cells are abundant in the mesenterial filaments. The above results are discussed in the context of our knowledge of the behavior of coral planulae at the time of their settlement and metamorphosis. Corals are facing multiple environmental threats, and these results both highlight the need for, and bring us a step closer to, a mechanistic understanding of a process that is critical to their survival.

Morphogenesis and Evolution of the Blastopore

The blastopore evolution is considered as a model example of the evolution of morphogenesis mechanism based on dipole interactions between the sources and sinks of epithelial sheet surface energy. The blastopore arises as a singular point of the vector field movement of a spherical surface having a zero velocity of the planar flow being surrounded by a toroidal surface (“border torus,” BT). The BT domain is the region of maximum difference in the planar surface flow velocity and, as a consequence, of maximum variability of the gastrulation movements. The evolution of gastrulation begins with the blastopore closure and continues through the formation of intercalary developmental stages that precede this closure leading to an increase in the blastopore diameter (retrograde evolution). Only three types of robust sink/source distributions at the BT surface are feasible irrespective to their phylogenetic origin. First, it is the “unilateral” gastrulation of Lophotrochozoa with the source and sinks at two opposite BT poles and two bilaterally symmetrical flows along BT. Then, it is the “bilateral” gastrulation of Ecdysozoa with two bilaterally symmetrical surface sources and two sinks at the opposite blastopore poles. Thirdly, it is the “radial” gastrulation of Deuterostomia including Chordates: one of the BT poles is a sink of the surface coming from two sources: from the outer BT surface adjacent to the sink and from the opposite pole of the BT circumference. In the evolution of the blastopore of chordates from the lancelet to amniotes, it is possible to trace the gradual replacement of gastrulation movements with pre-gastrulation cellular flows due to fixation of heterochronies, set out in the normal variability of morphogenesis. Since the variability of structures is reduced as they are formed, the evolution uses variability of earlier developmental stages.

Ontogenesis and embryogenesis of Megaleporinus elongatus larvae (Characiformes: Anostomidae) from the Jequitinhonha River Basin

Five females and five males of Megaleporinus elongatus captured in the Jequitinhonha River were transferred to hypophysation tanks with a constant water flow at 26°C at the Machado Mineiro Fish Farm. They were submitted to induced reproduction by the hypophysation method. Crude carp pituitary extract (Cyprinus carpio) was injected into the coelomic cavity in two doses (2.5 and 5.0 mg/kg) for males and 3 doses (2.5, 5.0, and 2.5 mg/kg) for females. The doses were applied with a 24-hr interval between the 1st and 2nd doses and a 12-hr interval between the 2nd and 3rd ones. Oocytes and semen were obtained by manual extrusion, and the dry method was used for fertilisation. The eggs were kept in funnel type 20-litre incubators with constant water flow at 23°C. Egg samples were collected every 10 min and photographed under a stereomicroscope for analysis of embryonic development until hatching. The main events of embryogenesis and their respective duration times were: cleavage (45 min), blastula (3 hr 14 min), gastrula (5 hr 25 min), closure of blastopore (6 hr 34 min), differentiation of somites (13 hr 20 min), tail release (17 hr 50 min), and hatching (24 hr 50 min). During the larval ontogenesis, the yolk sac gradually decreased until its complete reabsorption on the 7th day after hatching. The opening of the mouth and digestive tract occurred from the 5th to the 7th day, indicating that larvae could feed exogenously. These results provide information to improve the techniques of induced spawning and cultivation of M. elongatus.

Thermal stress induces heat shock protein 70 and apoptosis during embryo development in a Neotropical freshwater fish.

Fish embryos are particularly vulnerable to temperature changes, with the effects varying with developmental stage. The major aim of the present study was to analyse the relationship between apoptosis and heat shock protein (HSP) 70 during embryo development under thermal stress conditions. To this end, Prochilodus lineatus embryos at the blastopore closure stage were subjected to one of three thermal treatments for 1h (Group 1, 25°C (control); Group 2, 20°C; Group 3, 30°C) and then examined at 0, 4 and 8h posttreatment (h.p.t.). The viability of embryos was highest in Group 1 (81.33±16.65%), followed by Group 3 and Group 2 (75.33±12.10% and 68.67±16.86% respectively), with significant difference between Groups 1 and 2 (P<0.05). At 0h.p.t., embryos subjected to thermal stress (Group 3) had a significantly higher number of terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL)- and caspase-3-labelled cells, and a lower number of HSP70-positive cells than those in the control group. At 4h.p.t., there was a decrease in the TUNEL reaction and an increase in HSP70 in embryos in Group 3. At 8h.p.t., the size of Group 3 embryos was significantly smaller than that of Group 1 embryos. The results indicate a cytoprotective role for HSP70, regulating caspase-3-mediated apoptosis during embryo development of P. lineatus; however, this mechanism is not effective in controlling embryo viability and larval malformations.

Large, long range tensile forces drive convergence during Xenopus blastopore closure and body axis elongation.

Indirect evidence suggests that blastopore closure during gastrulation of anamniotes, including amphibians such as Xenopus laevis, depends on circumblastoporal convergence forces generated by the marginal zone (MZ), but direct evidence is lacking. We show that explanted MZs generate tensile convergence forces up to 1.5 μN during gastrulation and over 4 μN thereafter. These forces are generated by convergent thickening (CT) until the midgastrula and increasingly by convergent extension (CE) thereafter. Explants from ventralized embryos, which lack tissues expressing CE but close their blastopores, produce up to 2 μN of tensile force, showing that CT alone generates forces sufficient to close the blastopore. Uniaxial tensile stress relaxation assays show stiffening of mesodermal and ectodermal tissues around the onset of neurulation, potentially enhancing long-range transmission of convergence forces. These results illuminate the mechanobiology of early vertebrate morphogenic mechanisms, aid interpretation of phenotypes, and give insight into the evolution of blastopore closure mechanisms.