Late Embryonic Stages Research Articles

Heat Shock Protein 40 (HSP40) in Pacific White Shrimp (Litopenaeus vannamei): Molecular Cloning, Tissue Distribution and Ontogeny, Response to Temperature, Acidity/Alkalinity and Salinity Stresses, and Potential Role in Ovarian Development.

Heat shock proteins (HSPs), a family of conserved proteins that are produced by cells in response to stresses, are known as molecular chaperones with a range of housekeeping and cellular protective functions. The 40 kD heat shock protein (HSP40) is a co-chaperone for HSP70 in the regulation of ATP hydrolysis. Unlike its well-documented cofactor HSP70, little is currently known regarding the biological functions of HSP40 in crustacean species such as penaeid shrimp. In the present study, the cDNA encoding HSP40 (Lv-HSP40) was identified from the Pacific white shrimp Litopenaeus vannamei, a highly significant commercial culture species. The structural organization indicates that Lv-HSP40 belongs to the type-I HSP40s. The muscle, gill, and hepatopancreas are the main sites of Lv-HSP40 transcript expression. Within these tissues, Lv-HSP40 mRNA were predominantly exhibited in the myocytes, epithelial cells and hepatopancreatic cells, respectively. Under acute thermal stress in the culture environment, Lv-HSP40 transcript levels are significantly induced in these three tissues, while low pH stress only upregulates Lv-HSP40 mRNA in the hepatopancreas and gill. During ontogenesis, Lv-HSP40 transcript levels are high at early embryonic stages and drop sharply at late embryonic and early larval stages. The ovary is another major organ of Lv-HSP40 mRNA expression in female shrimp, and Lv-HSP40 transcripts were mainly presented in the follicle cells but only weekly detected in the oocytes. Ovarian Lv-HSP40 mRNA levels increase continuously during gonadal development. Silencing of the Lv-HSP40 gene by RNA interference may effectively delay ovarian maturation after unilateral eyestalk ablation. The roles of Lv-HSP40 in ovarian development are speculated to be independent of its cofactor HSP70, and the vitellogenesis factor vitellogenin (Vg) and vitellogenin receptor (VgR). Our study, as a whole, provides new insights into the roles of HSP40 in multiple physiological processes in L. vannamei: (1) HSP40 is a responding factor during stressful conditions; and (2) HSP40 participates in embryonic and ovarian development.

Cdc42 negatively regulates endocytosis during apical membrane maintenance in live animals.

Lumen establishment and maintenance are fundamental for tubular organs physiological functions. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in mammalian model systems in vivo. Here we used the salivary glands of live mice to examine the role of the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur. Depletion of Cdc42 in adult mice induced a significant expansion of the apical canaliculi, whereas depletion at late embryonic stages resulted in a complete inhibition of their postnatal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and toward the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways.

Changes in bursal B cells in chicken during embryonic development and early life after hatching

The bursa of Fabricius, the primary lymphoid organ for B cell development found only in birds, offers novel approaches to study B cell differentiation at various developmental stages. Here, we explored the changes and mechanism involved in the developmental stages of bursal B cells. The bursal B cells rapidly increased in the late embryonic stage and around hatching, which coincided with changes in specific cell surface markers. Moreover, the cells in the bursa were divided by size into small (low forward- and side-scatter) or large (high forward- and side-scatter) via flow cytometry. It is intriguing that the proportion of small and large B cells was reversed during this period. Because little is known about this phenomenon, we hypothesized that size-based B cell population could be used as an indicator to distinguish their status and stage during B cell development in chicken. The results demonstrated that large B cells are actively proliferating cells than small B cells. Additionally, large B cells showed higher mRNA expression of both proliferation- and differentiation-associated genes compared to small B cells. Taken together, these data show that large bursal B cells are the main source of proliferation and differentiation during B cell development in chickens.

Progressive divisions of multipotent neural progenitors generate late-born chandelier cells in the neocortex

Diverse γ-aminobutyric acid (GABA)-ergic interneurons provide different modes of inhibition to support circuit operation in the neocortex. However, the cellular and molecular mechanisms underlying the systematic generation of assorted neocortical interneurons remain largely unclear. Here we show that NKX2.1-expressing radial glial progenitors (RGPs) in the mouse embryonic ventral telencephalon divide progressively to generate distinct groups of interneurons, which occupy the neocortex in a time-dependent, early inside-out and late outside-in, manner. Notably, the late-born chandelier cells, one of the morphologically and physiologically highly distinguishable GABAergic interneurons, arise reliably from continuously dividing RGPs that produce non-chandelier cells initially. Selective removal of Partition defective 3, an evolutionarily conserved cell polarity protein, impairs RGP asymmetric cell division, resulting in premature depletion of RGPs towards the late embryonic stages and a consequent loss of chandelier cells. These results suggest that consecutive asymmetric divisions of multipotent RGPs generate diverse neocortical interneurons in a progressive manner.

A lipidomic perspective on the embryogenesis of two commercially important crabs, Carcinus maenas and Necora puber

Lipids play a central role in the metabolism of most organisms. These molecules are paramount during reproduction, when females transfer their internal lipid reserves to build-up oocytes' yolk. The aim of the present study was to understand neutral lipid (NL) dynamics during the embryogenesis of two commercially important crabs, Carcinus maenas (Linnaeus, 1758) and Necora puber (Linnaeus, 1767), using electrospray ionization mass spectrometry (ESI–MS) and tandem MS (ESI–MS/MS). Total lipid extracts were fractionated into NLs and polar lipids (PLs). Triacylglycerol (TAG) profile of NL and fatty acid (FA) profiles of both NLs and PLs were determined in two different embryonic stages: newly-extruded embryos (stage 1, a proxy of maternal investment) and ready-to-hatch embryos (stage 3, a proxy of newly-hatched larvae). In total, 16 molecular species of TAGs and their fatty acyl composition were identified, for the first time in marine invertebrate embryos. During embryogenesis, TAGs bearing C14 to C18 saturated and monounsaturated FAs were preferentially consumed, while TAGs with longer carbon chains and polyunsaturated FAs were selectively retained in the late embryonic stage of both crab species. The analysis of FA profiles revealed that energetic FAs were associated with NLs, while essential FAs were more abundant in PLs. These results evidence the different functions of NLs and PLs as energetic and structural lipids, respectively. The novel approach used in our study provides an unprecedented level of information of lipid dynamics at the molecular level, opening new perspectives in the study of maternal investment and embryogenesis in marine invertabrates.

Morphological and physiological changes of Octopus bimaculoides: From embryo to juvenile

Octopus bimaculoides is a holobenthic species with fishery importance in Mexico and aquaculture potential because of its high growth rate and direct development from embryo to juvenile, which increases survival in captivity. In cephalopods, transition from yolk utilization to independent active feeding is considered a critical period. Information about the digestive physiology ontogeny of O. bimaculoides is needed to determine the digestive capacity and to improve feed and feeding protocols in captivity for early life stages. Thus, this study assessed changes in morphological and digestive enzymatic activities, as well as biochemical composition of embryos and hatchlings. Internal and external yolk areas and total, mantle and arm lengths were measured in embryos; total wet weight was measured in hatchlings. Activities of lipase, acidic phosphatase, trypsin and chymotrypsin were determined by fluorometric techniques; total lipid, triglyceride and soluble protein concentrations were biochemically measured. Embryonic development lasted around 65 days at an incubation temperature of 18 °C. Embryos grew exponentially while external yolk decreased linearly throughout the embryonic developmental stages. Hatchlings grew exponentially the first 14 days after hatching (DAH). Enzymatic activities were evident since embryo stages, mainly at final development stages. Higher activities of acidic phosphatases and lipases in embryos were detected after the end of organogenesis; while trypsin and chymotrypsin activities, only were at final stages detected previous to hatching. In hatchlings, acidic phosphatase and lipase activities increased the first 12 and 20 DAH, respectively, and protease activities were erratic the first 30 DAH. Lipid concentrations did not show variations in embryo stages but decreased the first 10 DAH. Triglycerides increased in last embryo stages and decreased the first 14 DAH. Soluble proteins decreased in the last embryo stages and in the first 10 DAH. The results of lipases, acidic phosphatases, as well as lipid, triglyceride and soluble protein contents suggested that embryos started to mobilise reserves from the external to internal yolk sac at final developmental stages, and hatchlings were consuming their reserves during the first 10–14 DAH. Protease activity showed that the digestive gland started secretory activity from late embryo stages but continued maturation even after hatching. The results showed that in late embryonic development stages and first days after hatching, enzymatic activity and biochemical components respond to the mobilisation, accumulation and use of yolk reserves in O. bimaculoides.

Crinkled employs wingless pathway for wing development in Tribolium castaneum.

Crinkled is associated with embryonic denticle formation and auditory organ development in Drosophila melanogaster. However, the functions of Crinkled have not been fully investigated. Additionally, the genes that participate in the Crinkled pathway are unknown. Phylogenetic analysis indicates that crinkled exhibits a one-to-one orthologous relationship in insects. In Tribolium castaneum, the crinkled gene is 6,498bp in length and consists of six exons. Crinkled expression peaked during two phases in Tribolium: late embryonic and pupal stages. High levels of crinkled mRNA were detected in the fat body, head, epidermis, ovary, and accessory gland of late adults. Knockdown of crinkled using RNA interference (RNAi) severely affected wing morphogenesis in T. castaneum. We further showed that crinkled silencing reduced forked expression through wingless and shaven-baby, and RNAi of forked phenocopied the effects of crinkled knockdown in T. castaneum. This study investigated the development role of crinkled in postembryonic stages and indicated that forked mediates the functions of crinkled during wing morphogenesis in T. castaneum.

A tandem repeat array in IG-DMR is essential for imprinting of paternal allele at the Dlk1–Dio3 domain during embryonic development

Genomic imprinting is a phenomenon that causes parent-origin-specific monoallelic expression of a small subset of genes, known as imprinted genes, by parentally inherited epigenetic marks. Imprinted genes at the delta-like homolog 1 gene (Dlk1)-type III iodothyronine deiodinase gene (Dio3) imprinted domain, regulated by intergenic differentially methylated region (IG-DMR), are essential for normal development of late embryonic stages. Although the functions of IG-DMR have been reported by generating knockout mice, molecular details of the regulatory mechanisms are not fully understood as the specific sequence(s) of IG-DMR have not been identified. Here, we generated mutant mice by deleting a 216 bp tandem repeated sequence in IG-DMR, which comprised seven repeats of 24 bp motifs, by genome editing technologies. The mutant mice showed that paternal transmission of the deletion allele, but not maternal transmission, induces severe growth retardation and perinatal lethality, possibly due to placental defects. Embryos with a paternally transmitted deletion allele showed biallelic expression of maternally expressed genes and repression of paternally expressed genes. DNA methylation status also showed loss of methylation at IG-DMR and Gtl2-DMR, indicating that the tandem repeat sequence of IG-DMR is one of the functional sequences of IG-DMR, which is required for maintaining DNA methylation imprints of paternal allele at IG-DMR.

Relationship between Metabolic Heating and Nest Parameters in Green Turtles (Chelonia mydas, L. 1758) on Samandağ Beach, Turkey.

Acquiring information about metabolic heating is necessary for the improved understanding of nesting beaches. However, there are limited studies on metabolic heating of green turtle (Chelonia mydas, L. 1758) on Mediterranean beaches. The present study sought to determine the metabolic heating, and its effect on the feminization of green turtle hatchlings during 2016 and 2017 nesting season on Samandağ Beach, Hatay, Turkey. Moreover, the relations between metabolic heating and nest parameters, and spatio-temporal differences of metabolic heating were evaluated. Nest temperatures were recorded by using data loggers placed at the center of nests (n = 18), and the same depth was used to determine the sand temperature that was 50 cm back of the egg chamber. The mean incubation duration was calculated as 51.1 days, and the mean distance of nests from sea and the mean nest depth were measured as 32.4 meters and 67.70 cm, respectively. The mean numbers of embryo that is early, middle and late embryonic stages were found as 110.8, 93.2 and 93.05, respectively. The mean clutch size was found as 110.8. Mean metabolic heating was 0.37°C in the middle third of incubation for both years. The metabolic heating in both years was close to the as reported in the other green turtles nesting beaches in the Mediterranean. The feminization effect of metabolic heating was calculated as 6.8% in the green turtle nests on Samandağ Beach. Metabolic heating did not vary spatially, and the clutch size was the most important factor in metabolic heating and explained a large proportion of the variation in metabolic heating.

Ythdf2-mediated m6A mRNA clearance modulates neural development in mice

BackgroundN6-methyladenosine (m6A) modification in mRNAs was recently shown to be dynamically regulated, indicating a pivotal role in multiple developmental processes. Most recently, it was shown that the Mettl3-Mettl14 writer complex of this mark is required for the temporal control of cortical neurogenesis. The m6A reader protein Ythdf2 promotes mRNA degradation by recognizing m6A and recruiting the mRNA decay machinery.ResultsWe show that the conditional depletion of the m6A reader protein Ythdf2 in mice causes lethality at late embryonic developmental stages, with embryos characterized by compromised neural development. We demonstrate that neural stem/progenitor cell (NSPC) self-renewal and spatiotemporal generation of neurons and other cell types are severely impacted by the loss of Ythdf2 in embryonic neocortex. Combining in vivo and in vitro assays, we show that the proliferation and differentiation capabilities of NSPCs decrease significantly in Ythdf2−/− embryos. The Ythdf2−/− neurons are unable to produce normally functioning neurites, leading to failure in recovery upon reactive oxygen species stimulation. Consistently, expression of genes enriched in neural development pathways is significantly disturbed. Detailed analysis of the m6A-methylomes of Ythdf2−/− NSPCs identifies that the JAK-STAT cascade inhibitory genes contribute to neuroprotection and neurite outgrowths show increased expression and m6A enrichment. In agreement with the function of Ythdf2, delayed degradation of neuron differentiation-related m6A-containing mRNAs is seen in Ythdf2−/− NSPCs.ConclusionsWe show that the m6A reader protein Ythdf2 modulates neural development by promoting m6A-dependent degradation of neural development-related mRNA targets.

Molecular anatomy of the alligator dorsal telencephalon

The evolutionary relationships of the mammalian neocortex and avian dorsal telencephalon (DT) nuclei have been debated for more than a century. Despite their central importance to this debate, nonavian reptiles remain underexplored with modern molecular techniques. Reptile studies harbor great potential for understanding the changes in DT organization that occurred in the early evolution of amniotes. They may also help clarify the specializations in the avian DT, which comprises a massive, cell-dense dorsal ventricular ridge (DVR) and a nuclear dorsal-most structure, the Wulst. Crocodilians are phylogenetically and anatomically attractive for DT comparative studies: they are the closest living relatives of birds and have a strikingly bird-like DVR, but they also possess a highly differentiated reptile cerebral cortex. We studied the DT of the American alligator, Alligator mississippiensis, at late embryonic stages with a panel of molecular marker genes. Gene expression and cytoarchitectonic analyses identified clear homologs of all major avian DVR subdivisions including a mesopallium, an extensive nidopallium with primary sensory input territories, and an arcopallium. The alligator medial cortex is divided into three components that resemble the mammalian dentate gyrus, CA fields, and subiculum in gene expression and topography. The alligator dorsal cortex contains putative homologs of neocortical input, output, and intratelencephalic projection neurons and, most notably, these are organized into sublayers similar to mammalian neocortical layers. Our findings on the molecular anatomy of the crocodilian DT are summarized in an atlas of the alligator telencephalon.

Understanding the Development, Variations, and Defects of the Muscular System in Normal Human Embryos, Fetuses, and Newborns

There has been, until now, a limited knowledge on the development of the human muscular system, as most textbooks and atlases on human development described in detail the development of the skeletal system and other structures such as the internal organs without mentioning ‐ or doing it in a very brief way ‐ the muscular system. Moreover, within the few published works that did focus on the ontogeny of human muscles, most of them were restricted to a single muscle, a muscle group, a region, or a specific period of time (e.g. before 7 weeks of gestation). This makes the study of muscle anomalies, as well as of variations, very difficult due to the lack of good comparative models about normal development. We recently dissected 17 phenotypically ‘normal’ human newborns, fetuses and embryos between 7 and 39 weeks of development, and compared our results with the scarce literature available on the normal development of the muscular system in humans. Importantly, in the individuals dissected by us there was an indirect relationship between the ages of the fetuses and the number of seemingly fused (i.e. undifferentiated) muscles. That is, muscle anlagen in the early embryonic development often give rise to several muscles even after 7 weeks of gestation. This would contradict what is often described in the literature that by end of week 7 of gestation, all muscles resemble those of the adults (i.e. are differentiated). In addition, the number of the variations (i.e. phenotypes that deviate from the norm, within the ‘normal’ human population) per upper limbs dissected is significantly higher than the number of variations per lower limb studied, a pattern that is strikingly similar to that often reported in the literature about birth defects. Therefore, these and other results support Alberch's (1989) ill‐named “logic of monsters” theory that predicts a parallel between the variant phenotypes of the ‘normal’ populations and the defective phenotypes of genetically ‘abnormal’ individual due to the strong internal constraints that limit the number of possible outcomes. Furthermore, we present, for the first time, detailed data about the development of each head and limb human muscle from late embryonic stages to newborn configuration, and discuss the broader implications of these new data within a broad comparative and developmental framework.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Parvins Are Required for Endothelial Cell–Cell Junctions and Cell Polarity During Embryonic Blood Vessel Formation

During vascular development, integrin-mediated signaling regulates the formation and stabilization of cell-cell junctions, which are required for endothelial cell (EC) apical-basal polarity and proper deposition of the vascular basement membrane. Parvins are actin-binding proteins that facilitate the interaction of integrins with the actin cytoskeleton. The endothelium expresses 2 parvin isoforms: α-pv (α-parvin) and β-pv (β-parvin). Recently, we have shown that α-pv is critical for vessel growth and vessel stability at late embryonic developmental stages. The role of parvins during early embryonic development is unknown. To investigate the role of endothelial parvins in the developing vasculature, we generated mice with ECs lacking both parvin isoforms by deleting α-pv in ECs in global β-pv-/- mice (α-pvΔEC;β-pv-/- mice). Here, we show that α-pvΔEC;β-pv-/- mice die around embryonic day 11.5 and exhibit hemorrhages, immature capillary beds, and severe vascular defects in the central nervous system, including reduced vessel branching, increased vessel diameter, and balloon-like hemorrhagic clusters of ECs. Vessels in α-pvΔEC;β-pv-/- embryos display disorganized cell-cell junctions, impaired endothelial apical-basal polarity, and discontinuous basement membranes. These vascular defects are accompanied by defective pericyte-vessel interaction. Our results show that parvins are critical for the organization of endothelial cell-cell junctions, the establishment of endothelial apical-basal polarity, and the integrity of the basement membrane.

Chicken immune response following in ovo delivery of bacterial flagellin

In ovo immunization of chicken embryos with live vaccines is an effective strategy to protect chickens against several viral pathogens. We investigated the immune response of chicken embryos to purified recombinant protein. In ovo delivery of Salmonella flagellin to 18-day old embryonated eggs resulted in elevated pro-inflammatory chIL-6 and chIL-8 (CXCL8-CXCLi2) cytokine transcript levels in the intestine but not in the spleen at 24 h post-injection. Analysis of the chicken Toll-like receptor (TLR) repertoire in 19-day old embryos revealed gene transcripts in intestinal and spleen tissue for most chicken TLRs, including TLR5 which recognizes Salmonella flagellin (FliC). The in ovo administration of FliC did not alter TLR transcript levels, except for an increase in intestinal chTLR15 expression. Measurement of the antibody response in sera collected at day 11 and day 21 post-hatch demonstrated high titers of FliC-specific antibodies for the animals immunized at the late-embryonic stage in contrast to the mock-treated controls. The successful in ovo immunization with purified bacterial antigen indicates that the immune system of the chicken embryo is sufficiently mature to yield a strong humoral immune response after single exposure to purified protein. This finding strengthens the basis for the development of in ovo protein-based subunit vaccines.

Course-, dose-, and stage-dependent toxic effects of prenatal dexamethasone exposure on fetal articular cartilage development

Dexamethasone, a synthetic long-acting glucocorticoid, is routinely used for treating mothers at risk for preterm delivery. However, intrauterine overexposure to glucocorticoids induces low birth weight and cartilage dysplasia in offspring. Also, the “critical window” and safe dose of this treatment are largely unknown. This study investigated the course-, dose-, and stage-dependent toxic effects and the possible mechanisms of prenatal dexamethasone exposure (PDE) on fetal development and articular cartilage development. Pregnant mice (C57BL/6) received subcutaneous injection of dexamethasone (0.8 mg/kg d) once on gestational day (GD) 15 or once a day from GD 15 to 17, or received various doses of dexamethasone (0, 0.2, 0.8, and 1.2 mg/kg d) on GD 15–17, or received dexamethasone (0.8 mg/kg d) at early stage (GD 12–14) or late stage of pregnancy (GD 15–17). Offspring’s knee joints were harvested at birth for morphological analyses and detection of gene expression. Repeated PDE significantly suppressed fetal and articular cartilage development, which were characterized by decreased body weight and body length, coarse articular cartilage surfaces, and reduced gene and protein expression of Col2a1 and aggrecan. For those newborns treated with repeated PDE at different doses, the toxic effects on fetal and articular cartilage development were observed at doses of 0.8 and 1.2 mg/kg d, whereas no obvious toxic effects were observed at the dose of 0.2 mg/kg d. Moreover, PDE at 0.8 mg/kg d during the early embryonic stage induced stronger toxic effects on fetal and articular cartilage development, compared with PDE during the late embryonic stage. Detection of gene expression showed that the TGFβ signaling pathway in the articular cartilage was down-regulated after PDE. Taken together, PDE induces fetal developmental toxicity and articular cartilage developmental toxicity in a course-, dose-, and stage-dependent manner.

Morphological changes of the optic lobe from late embryonic to adult stages in oval squids Sepioteuthis lessoniana.

The optic lobe is the largest brain area within the central nervous system of cephalopods and it plays important roles in the processing of visual information, the regulation of body patterning, and locomotive behavior. The oval squid Sepioteuthis lessoniana has relatively large optic lobes that are responsible for visual communication via dynamic body patterning. It has been observed that the visual behaviors of oval squids change as the animals mature, yet little is known about how the structure of the optic lobes changes during development. The aim of the present study was to characterize the ontogenetic changes in neural organization of the optic lobes of S. lessoniana from late embryonic stage to adulthood. Magnetic resonance imaging and micro-CT scans were acquired to reconstruct the 3D-structure of the optic lobes and examine the external morphology at different developmental stages. In addition, optic lobe slices with nuclear staining were used to reveal changes in the internal morphology throughout development. As oval squids mature, the proportion of the brain making up the optic lobes increases continuously, and the optic lobes appear to have a prominent dent on the ventrolateral side. Inside the optic lobe, the cortex and the medulla expand steadily from the late embryonic stage to adulthood, but the cell islands in the tangential zone of the optic lobe decrease continuously in parallel. Interestingly, the size of the nuclei of cells within the medulla of the optic lobe increases throughout development. These findings suggest that the optic lobe undergoes continuous external morphological change and internal neural reorganization throughout the oval squid's development. These morphological changes in the optic lobe are likely to be responsible for changes in the visuomotor behavior of oval squids from hatching to adulthood.

Reproductive biology of Macrobrachium amazonicum (Heller, 1862) (Decapoda: Palaemonidae) in a reservoir situated in Minas Gerais State, southeastern Brazil

The palaemonid shrimp Macrobrachium amazonicum is widely distributed in coastal rivers, estuaries and inland waters of South America. Despite this, little is known regarding its biology in the natural environment. Furthermore, this species is economically important because it is the main freshwater shrimp commercially exploited by artisanal fisheries in northern and northeastern Brazil, besides showing great potential for aquaculture. Considering this, the present study aimed to analyze aspects of the reproductive biology of M. amazonicum . A total of 251 females were analyzed, 102 of these being ovigerous. The carapace length ranged from 6.03 to 10.06 mm (average ± standard deviation: 7.52 ± 0.84 mm). Reproduction was continuous with a gradual increase from April to June 2012. No significant relationship between frequency of ovigerous females and environmental variables was observed. The fecundity showed a positive linear relation with the carapace length and the number of eggs varied from 33 to 389 (average ± standard deviation: 203 ± 78.06 eggs/female). A dominance of eggs at an early stage was observed in most months. There was an increase in the size/volume of eggs from early to late embryonic stage. Ovigerous females produced eggs at the same development stage, with the same size and volume regardless of their body size. The results demonstrated higher similarity of this population to other continental M. amazonicum populations than from estuarine ones, showing the environment influence in the life-history of individuals.

Hes5 regulates the transition timing of neurogenesis and gliogenesis in mammalian neocortical development.

During mammalian neocortical development, neural stem/progenitor cells (NSCs) sequentially give rise to deep layer neurons and superficial layer neurons through mid- to late-embryonic stages, shifting to gliogenic phase at perinatal stages. Previously, we found that the Hes genes inhibit neuronal differentiation and maintain NSCs. Here, we generated transgenic mice that overexpress Hes5 in NSCs of the central nervous system, and found that the transition timing from deep to superficial layer neurogenesis was shifted earlier, while gliogenesis precociously occurred in the developing neocortex of Hes5-overexpressing mice. By contrast, the transition from deep to superficial layer neurogenesis and the onset of gliogenesis were delayed in Hes5 knockout (KO) mice. We found that the Hmga genes (Hmga1/2) were downregulated in the neocortical regions of Hes5-overexpressing brain, whereas they were upregulated in the Hes5 KO brain. Furthermore, we found that Hes5 expression led to suppression of Hmga1/2 promoter activity. These results suggest that Hes5 regulates the transition timing between phases for specification of neocortical neurons and between neurogenesis and gliogenesis, accompanied by alteration in the expression levels of Hgma genes, in mammalian neocortical development.

Neural Organization of the Optic Lobe Changes Steadily from Late Embryonic Stage to Adulthood in Cuttlefish Sepia pharaonis.

The optic lobe is the largest structure in the cuttlefish brain. While the general morphology of the optic lobe in adult cuttlefish has been well described, the 3D structure and ontogenetic development of its neural organization have not been characterized. To correlate observed behavioral changes within the brain structure along the development of this animal, optic lobes from the late embryonic stage to adulthood were examined systematically in the present study. The MRI scan revealed that the so called “cell islands” in the medulla of the cephalopod's optic lobe (Young, 1962, 1974) are in fact a contiguous tree-like structure. Quantification of the neural organizational development of optic lobes showed that structural features of the cortex and radial column zone were established earlier than those of the tangential zone during embryonic and post-hatching stages. Within the cell islands, the density of nuclei was decreased while the size of nuclei was increased during the development. Furthermore, the visual processing area in the optic lobe showed a significant variation in lateralization during embryonic and juvenile stages. Our observation of a continuous increase in neural fibers and nucleus size in the tangential zone of the optic lobe from late embryonic stage to adulthood indicates that the neural organization of the optic lobe is modified along the development of cuttlefish. These findings thus support that the ontogenetic change of the optic lobe is responsible for their continuously increased complexity in body patterning and visuomotor behaviors.

Dusky works upstream of Four-jointed and Forked in wing morphogenesis in Tribolium castaneum.

Dusky (dy) is required for cytoskeletal reorganization during wing morphogenesis in Drosophila melanogaster, but which genes participate together with dy for wing morphogenesis has remained unclear. In Tribolium castaneum, dy is highly expressed at the late embryonic stage. Tissue-specific expression analysis indicated high expression levels of dy in the epidermis, head and fat body of late-stage larvae. RNA interference (RNAi) targeting dy significantly decreased adult wing size and caused improper folding of the elytra. Meanwhile, dy knockdown reduced the transcription of four-jointed (fj) and forked (f). Our results show that fj RNAi reduces adult wing size and that silencing f results in abnormal wing folding in T.castaneum. Interestingly, knocking down fj and f simultaneously phenocopies dy RNAi, suggesting that dy probably acts upstream of fj and f to regulate wing morphogenesis in T.castaneum.