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Ac-SYN is the core protein of a cell surface proteoglycan of the sea urchin Anthocidaris crassispina . To examine the functions of Ac-SYN, embryos were cultured in the presence of affinity-purified antibody against Ac-SYN. At the late pluteus stage, severe inhibition of elongation of the postoral arms was seen in treated embryos compared with control embryos. Blastocoeleic microinjection of the antibody did not affect morphogenesis. The relationship between the number of cells in the postoral arms and the length of the postoral rods was investigated in normal embryos. This showed that postoral arm elongation has two phases: the first phase accompanies the increase in cell numbers while the second does not. The syndecan antibody inhibited the increase in cell numbers in the postoral arms. Furthermore, in the treated embryos, cell numbers continued to increase normally until 31 h post fertilization (hpf), while cell division stopped after 31 hpf. These results suggest that Ac-SYN participates in postoral arm formation via cell division in sea urchin embryos. [ABSTRACT FROM AUTHOR]

The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription–polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae. [ABSTRACT FROM AUTHOR]

The SM30α gene encodes a protein in the embryonic endoskeleton of the sea urchin Strongylocentrotus purpuratus, and is specifically expressed in the skeletogenic primary mesenchyme cell lineage. To clarify the mechanism for the differentiation of this cell lineage, which proceeds rather autonomously in the embryo, regulation of the SM30α gene was investigated previously and it was shown that the distal DNA region upstream of this gene from - 1.6 to - 1.0 kb contained numerous negative regulatory elements that suppressed the ectopic expression of the gene in the gut. Here we study the influence of the proximal region from - 303 to + 104 bp. Analysis of the expression of reporter constructs indicated that a strong positive enhancer element existed in the region from - 142 to - 105 bp. This element worked both in forward and reverse orientations and additively when placed tandemly upstream to the reporter gene. In addition, other weaker positive and negative regulatory sites were also detected throughout the proximal region. Electrophoretic gel mobility shift analyses showed that multiple nuclear proteins were bound to the putative strong enhancer region. One of the proteins binding to this region was present in early blastulae, a time when the SM30 gene was still silent, but it was not in prism embryos actively expressing the gene. The binding region for this blastula-specific protein was narrowed down to the region from - 132 to - 122 bp, which included the consensus binding site for the mammalian proto-oncogene product, Ets. Two possible SpGCF1 binding sites were identified in the vicinity of the enhancer region. This information was used to make a comparison of the general regulatory architecture of genes that contribute to the formation of the skeletal spicule. [ABSTRACT FROM AUTHOR]

We have found that partial resolution of newly replicated nucleosomes can be achieved by rate zonal centrifugation through sucrose density gradients preformed in heavy water. Nucleosome samples were obtained from MH-134SC cells density labeled with 5-iododeoxyuridine in the presence of suitable isotopic precursors. The method is simple and can be performed under conditions that do not destabilize the nucleosome structure. This gave us an exciting opportunity to study the deposition sites of newly synthesized histones. Nucleosomes were obtained from cells pulse-labeled simultaneously with 5-iododeoxyuridine and [³H]lysine for the rate zonal analysis. Proteins in the resulting fractions were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and visualized by silver staining and fluorography. The distribution of newly synthesized H2A and H2B coincided closely with that of bulk nucleosomes. The distribution of newly synthesized H3 and H4 was shifted to the bottom sides of the bulk nucleosome peaks, but not so far as to the putative peaks of newly replicated (dense) nucleosomes. This means that newly synthesized histones are deposited on DNA in disproportionate amounts and that their sites of deposition are not restricted to newly replicated DNA. [ABSTRACT FROM AUTHOR]

The vertebrate telencephalic lobes consist of the pallium (dorsal) and subpallium (ventral). The subpallium gives rise to the basal ganglia, encompassing the pallidum and striatum. The development of this region is believed to depend on Foxg1/Foxg1a functions in both mice and zebrafish. This study aims to elucidate the genetic regulatory network controlled by foxg1a in subpallium development using zebrafish as a model. The expression gradient of foxg1a within the developing telencephalon was examined semi‐quantitatively in initial investigations. Utilizing the CRISPR/Cas9 technique, we subsequently established a foxg1a mutant line and observed the resultant phenotypes. Morphological assessment revealed that foxg1a mutants exhibit a thin telencephalon together with a misshapen preoptic area (POA). Notably, accumulation of apoptotic cells was identified in this region. In mutants at 24 h postfertilization, the expression of pallium markers expanded ventrally, while that of subpallium markers was markedly suppressed. Concurrently, the expression of fgf8a, vax2, and six3b was shifted ventrally, causing anomalous expression in regions typical of POA formation in wild‐type embryos. Consequently, the foxg1a mutation led to expansion of the pallium and disrupted the subpallium and POA. This highlights a pivotal role of foxg1a in directing the dorsoventral patterning of the telencephalon, particularly in subpallium differentiation, mirroring observations in mice. Additionally, reduced expression of neural progenitor maintenance genes was detected in mutants, suggesting the necessity of foxg1a in preserving neural progenitors. Collectively, these findings underscore evolutionarily conserved functions of foxg1 in the development of the subpallium in vertebrate embryos. [ABSTRACT FROM AUTHOR]

Nuclear receptor subfamily 2 group F (Nr2f) proteins are essential for brain development in mice, but little is known about their precise roles and their evolutionary diversification. In the present study, the expression patterns of major nr2f genes (nr2f1a, nr2f1b, and nr2f2) during early brain development were investigated in zebrafish. Comparisons of their expression patterns revealed similar but temporally and spatially distinct patterns after early somite stages in the brain. Frameshift mutations in the three nr2f genes, achieved using the CRISPR/Cas9 method, resulted in a smaller telencephalon and smaller eyes in the nr2f1a mutants; milder forms of those defects were present in the nr2f1b and nr2f2 mutants. Acridine orange staining revealed enhanced cell death in the brain and/or eyes in all nr2f homozygous mutants. The expression of regional markers in the brain did not suggest global defects in brain regionalization; however, shha expression in the preoptic area and hypothalamus, as well as fgf8a expression in the anterior telencephalon, was disturbed in nr2f1a and nr2f1b mutants, potentially leading to a defective telencephalon. Specification of the retina and optic stalk was also significantly affected. The overexpression of nr2f1b by injection of mRNA disrupted the anterior brain at a high dose, and the expression of pax6a in the eyes and fgf8a in the telencephalon at a low dose. The results of these loss‐ and gain‐of‐function approaches showed that nr2f genes regulate the development of the telencephalon and eyes in zebrafish embryos. [ABSTRACT FROM AUTHOR]

Abstract: The roles of the FGF family growth factors and their receptors (FGFRs) in zebrafish embryos were examined using variously modified versions of the four FGFR genes (fgfr1–4). Constitutively active forms of all of the examined FGFRs (ca-FGFRs) caused dorsalization, brain caudalization, and secondary axis formation, indicating that the main FGF signal transduction downstream of the receptor is highly similar among FGFRs. All of the membrane-bound type of dominant-negative FGFRs (mdn-FGFRs) derived from the four fgfr genes, which interfere with endogenous FGFRs, produced posterior truncation, as previously reported in both Xenopus and zebrafish. mdn-FGFR3c had the strongest effects on embryos, progressively disrupting the posterior structure as the dose increased. At the highest dose, only the forebrain was formed. At lower doses, mdn-FGFR3c mainly suppressed the paraxial mesoderm. The co-injection of mRNA for different mdn-FGFRs and FGFs resulted in diverse suppression spectra of the respective FGFRs against FGFs. Only mdn-FGFR3c severely suppressed all of the FGFs examined. We also examined the effects of the secretory type of dominant-negative FGFRs (sdn-FGFRs), which are released from cells and trap FGF ligands. Only sdn-FGFR3c resulted in the characteristic effect of selectively disrupting the isthmic development, as well as the tailbud. The co-injection of the mRNA for sdn-FGFRs and FGFs suggested that sdn-FGFR3c inhibits FGFs of the FGF8 subfamily, which is consistent with its specific effects on development. We discuss the implications of our findings obtained in the present study. [Copyright &y& Elsevier]

Abstract: Fgf8 is expressed in the isthmic region of the developing brain, serving an organizing function in vertebrate embryos. We previously identified S4.2 downstream to the zebrafish fgf8 gene as a regulatory region that drives transcription in the anterior hindbrain. Here, we investigated the mechanism of fgf8 regulation by the S4.2 region during development. Reporter analyses in embryos revealed that S4.2 closely recapitulates fgf8 expression in the anteriormost hindbrain during somitogenesis. This region contains a sequence highly conserved in fgf8 of diverse vertebrates. Further analyses of S4.2 revealed a 342-bp core region composed of three subregions (#2, #3, and #4). Regions #3 and #4 drove expression broadly in the brain from the midbrain to r5 of the hindbrain, whereas a 28-bp sequence in #2 repressed ectopic expression in the midbrain and in r2 to r5. The enhancer function of S4.2 was absent in pax2a mutant embryos, while it was activated ectopically by pax2a misexpression in the hindbrain. We identified two sites in the core region that are bound by Pax2a in vitro and in vivo, the disruption of which abrogated the S4.2 activity. Thus, fgf8 expression in the anteriormost hindbrain involves activation and repression, with Pax2a as a pivotal regulator. [Copyright &y& Elsevier]

βig-h3, which is structurally related to the insect fasciclin I, is assumed to act as a cell adhesion molecule through binding to cell-surface integrins. In this study, we obtained cDNA clones for the zebrafish orthologue of βig-h3 and examined the expression of the gene (βig-h3) in zebrafish embryos using in situ hybridization. Expression is first seen at the bud stage in the presomitic mesoderm. Throughout the somitogenesis stage, βig-h3 is expressed in all the segmented somites, as well as in the presomitic mesoderm (S0 and S-I). High expression is observed in the dorsolateral part of the somite until the mid-somitogenesis stage. At late somitogenesis stages, the βig-h3 expression in the dorsolateral somite fades away, while expression is upregulated in the ventromedial part of the somite that corresponds to the sclerotome. In embryos after completion of somitogenesis and fry after hatching, βig-h3 continues to be expressed in the sclerotome. In addition, new expression starts in the mesenchyme cells in the head, pharyngeal arches, and pectoral fins. In the embryonic brain, expression is observed along the anterior and postoptic commissures, as well as along the optic nerve. [Copyright &y& Elsevier]

Egg activation at fertilization requires the release of Ca2+ from the egg’s endoplasmic reticulum, and recent evidence has indicated that a Src family kinase (SFK) may function in initiating this signaling pathway in echinoderm eggs. Here, we identify and characterize a SFK from the sea urchin Strongylocentrotus purpuratus, SpSFK1. SpSFK1 RNA is present in eggs, and an antibody made against a SpSFK1 peptide recognizes an ∼58-kDa egg membrane-associated protein in eggs of S. purpuratus as well as another sea urchin Lytechinus variegatus. Injection of both species of sea urchin eggs with dominant-interfering Src homology 2 domains of SpSFK1 delays and reduces the release of Ca2+ at fertilization. Injection of an antibody against SpSFK1 into S. purpuratus eggs also causes a small increase in the delay between sperm–egg fusion and Ca2+ release. In contrast, when injected into eggs of L. variegatus, this same antibody has a dramatic stimulatory effect: it causes PLCγ-dependent Ca2+ release like that occurring at fertilization. Correspondingly, in lysates of L. variegatus eggs, but not S. purpuratus eggs, the antibody stimulates SFK activity. Injection of L. variegatus eggs with another antibody that recognizes the L. variegatus egg SFK also causes PLCγ-dependent Ca2+ release like that at fertilization. These results indicate that activation of a Src family kinase present in sea urchin eggs is necessary to cause Ca2+ release at fertilization and is capable of stimulating Ca2+ release in the unfertilized egg via PLCγ, as at fertilization. [Copyright &y& Elsevier]

In vertebrate embryogenesis, elongation of the posterior body is driven by de novo production of the axial and paraxial mesoderm as well as the neural tube at the posterior end. This process is presumed to depend on the stem cell‐like population in the tail bud region, but the details of the gene regulatory network involved are unknown. Previous studies suggested the involvement of pou5f3, an Oct4‐type POU gene in zebrafish, in axial elongation. In the present study, we first found that pou5f3 is expressed mainly in the dorsal region of the tail bud immediately after gastrulation, and that this expression is restricted to the posterior‐most region of the elongating neural tube during somitogenesis. This pou5f3 expression was complementary to the broad expression of sox3 in the neural tube, and formed a sharp boundary with specific expression of tbxta (orthologue of mammalian T/Brachyury) in the tail bud, implicating pou5f3 in the specification of tail bud‐derived cells toward neural differentiation in the spinal cord. When pou5f3 was functionally impaired after gastrulation by induction of a dominant‐interfering pou5f3 mutant gene (en‐pou5f3), trunk and tail elongation were markedly disturbed at distinct positions along the axis depending on the stage. This finding showed involvement of pou5f3 in de novo generation of the body from the tail bud. Conditional functional abrogation also showed that pou5f3 downregulates mesoderm‐forming genes but promotes neural development by activating neurogenesis genes around the tail bud. These results suggest that pou5f3 is involved in formation of the posterior spinal cord. [ABSTRACT FROM AUTHOR]

The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1 , established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs. • A mutation termed kanazutsi was identified that showed novel and broad phenotypes. • Defects were observed mainly in the neural crest-derived and mesodermal tissues. • Downregulation of cytoglobin 1 is probably responsible for the phenotype. • This study revealed the role of oxygen metabolism in aquatic vertebrate embryos. [ABSTRACT FROM AUTHOR]

In the vertebrate body, a metameric structure is present along the anterior–posterior axis. Zebrafish tbx6−/− larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three‐dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6−/− larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two‐colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6−/−. The parapophyses in tbx6−/− juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish. [ABSTRACT FROM AUTHOR]

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4 -related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3 -expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2 , and also soxB1 , but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1 , a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3 , her5 , and her9 , further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1 , but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3 -mediated gene regulatory network in neural development in vertebrate embryos. • An Oct4-type PouV factor Pou5f3 regulates neurogenesis in the neural plate. • The expression domains of pou5f3 correspond to proneural clusters. • pou5f3 expression is regulated by proneural genes and Notch signaling. • Precise expression patterns of neurogenesis genes and soxB1 genes were determined. [ABSTRACT FROM AUTHOR]

Highlights • 4D imaging and analysis were performed to examine cell behavior in the neural tube. • Migratory behavior of cells linked to brain compartmentalization was characterized. • A novel cell migration pattern from the midbrain-hindbrain boundary was elucidated. • gbx2 -expressing cells showed two-step migration, followed by neuronal differentiation. Abstract One of the pivotal events in neural development is compartmentalization, wherein the neural tissue divides into domains and undergoes functional differentiation. For example, midbrain-hindbrain boundary (MHB) formation and subsequent isthmus development are key steps in cerebellar development. Although several regulatory mechanisms are known to underlie this event, little is known about cellular behaviors. In this study, to examine the cellular dynamics around the MHB region, we performed confocal time-lapse imaging in zebrafish embryos to track cell populations in the neural tube via 4D analysis. We used a transgenic line wherein enhanced green fluorescent protein (EGFP) expression is driven by the gastrulation brain homeobox 2 (gbx2) enhancer, which is involved in MHB maintenance. 4D time-lapse imaging of 5–20 h revealed a novel pattern in cell migration: a dynamic ventrocaudally directed migration from the MHB region toward the hindbrain. Furthermore, in the hindbrain region, these EGFP-positive cells altered their shapes and extended the axons. Immunohistochemical analysis and retrograde labeling showed that these cells in the hindbrain were in the process of neuronal differentiation, including reticulospinal neurons. These results revealed the dynamic and two-step behavior and possible fate of the cell population, which are linked to brain compartmentalization, leading to a deeper understanding of brain development and formation of neuronal circuits. [ABSTRACT FROM AUTHOR]

In the zebrafish segmentation clock, hairy/enhancer of split‐related genes her1, her7, and hes6 encodes components of core oscillators. Since the expression of cyclic genes proceeds rapidly in the presomitic mesoderm (PSM), these hairy‐related mRNAs are subject to strict post‐transcriptional regulation. In this study, we demonstrate that inhibition of the CCR4‐NOT deadenylase complex lengthens poly(A) tails of hairy‐related mRNAs and increases the amount of these mRNAs, which is accompanied by defective somite segmentation. In transgenic embryos, we show that EGFPmRNAs with 3′UTRs of hairy‐related genes exhibit turnover similar to endogenous mRNAs. Our results suggest that turnover rates of her1, her7, and hes6 mRNAs are differently regulated by the CCR4‐NOT deadenylase complex possibly through their 3′UTRs in the zebrafish PSM. [ABSTRACT FROM AUTHOR]

Somites sequentially form with a regular interval by the segmentation from the anterior region of the presomitic mesoderm (PSM). The expression of several genes involved in the somite segmentation is switched off at the transition from the anterior PSM to somites. Zebrafish Ripply1, which down-regulates a T-box transcription factor Tbx6, is required for the suppression of segmentation gene expression. However, the functional roles of the Ripply-mediated suppression of segmentation gene expression at the anterior PSM remain elusive. In this study, we generated ripply1 mutants and examined genetic interaction between ripply1 / 2 and tbx6 . Zebrafish ripply1 −/− embryos failed to form the somite boundaries as was observed in knockdown embryos. We found that somite segmentation defects in ripply1 mutants were suppressed by heterozygous mutation of tbx6 or partial translational inhibition of tbx6 by antisense morpholino. We further showed that somite boundaries that were recovered in tbx6 +/− ; ripply1 −/− embryos were dependent on the function of ripply2 , indicating that relative gene dosage between ripply1 / 2 and tbx6 plays a critical role in the somite formation. Interestingly, the expression of segmentation genes such mesp as was still not fully suppressed at the anterior PSM of tbx6 +/− ; ripply1 −/− embryos although the somite formation and rostral-caudal polarity of somites were properly established. Furthermore, impaired myogenesis was observed in the segmented somites in tbx6 +/− ; ripply1 −/− embryos. These results revealed that partial suppression of the segmentation gene expression by Ripply is sufficient to establish the rostral-caudal polarity of somites, and that stronger suppression of the segmentation gene expression by Ripply is required for proper myogenesis in zebrafish embryos. [ABSTRACT FROM AUTHOR]

The enteric nervous system (ENS) is the largest part of the peripheral nervous system in vertebrates. Toward the visualization of the development of the vertebrate ENS, we report our creation of a new transgenic line, Tg( chata : GGFF2 ) which has a 1.5-kb upstream region of the zebrafish choline acetyltransferase a (chata) gene followed by modified green fluorescent protein ( gfp ). During development, GFP + cells were detected in the gut by 60 h post-fertilization (hpf). In the gut of 6- and 12-days post-fertilization (dpf) larvae, an average of 92% of the GFP + cells were positive for the neuronal marker HuC/D, suggesting that GFP marks enteric neurons in this transgenic line. We also observed that 66% of the GFP + cells were choline acetyltransferase (ChAT)-immunopositive at 1.5 months. Thus, GFP is expressed at the larval stages at which ChAT protein expression is not yet detected by immunostaining. We studied the spatiotemporal pattern of neural differentiation in the ENS by live-imaging of this transgenic line. We observed that GFP + or gfp  + cells initially formed a pair of bilateral rows at 60 hpf or 53 hpf, respectively, in the migrating enteric neural crest cells. Most of the GFP + cells did not migrate, and most of the new GFP + cells were added to fill the space among the previously formed GFP + cells. GFP expression reached the anus by 72 hpf. New GFP + cells then also appeared in the dorsal and ventral sides of the initial GFP + rows, resulting in their distribution on the entire gut by 4 dpf. A small number of new GFP + cells were found to move among older GFP + cells just before the cells stopped migration, suggesting that the moving GFP + cells may represent neural precursor cells searching for a place for the final differentiation. Our data suggest that the Tg( chata : GGFF2 ) line could serve as a useful tool for studies of enteric neural differentiation and cell behavior. [ABSTRACT FROM AUTHOR]

Zebrafish pou5f3 (previously named pou2 ), a close homologue of mouse Oct4 , encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3 , which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3 —the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3 , which are frequently coexpressed in early embryos, in the regulation of early development. [ABSTRACT FROM AUTHOR]

It is well established that the gbx2 homeobox gene contributes to the positioning of the midbrain-hindbrain boundary (MHB) governing the development of adjacent brain regions in vertebrate embryos, but the specific aspects of the gene regulatory network regulated by gbx2 during brain development remain unclear. In the present study, we sought to comprehensively identify gbx2 target genes in zebrafish embryos by microarray analysis around the end of gastrulation, when the MHB is established, using transgenic embryos harboring heat-inducible gbx2 . This analysis revealed that a large number of genes were either upregulated or downregulated following gbx2 induction, and the time course of induction differed depending on the genes. The differences in response to gbx2 were found by functional annotation analysis to be related to the functions and structures of the target genes. Among the significantly downregulated genes was her5 , whose expression in the midbrain was precisely complementary to gbx2 expression around the MHB, suggesting that gbx2 expression in the anterior hindbrain restricts her5 expression to the midbrain. Because her5 represses neurogenesis, gbx2 may positively regulate neural development in its expression domain. Indeed, we showed further that gbx2 induction upregulated neural marker expression in the midbrain. Quantitative PCR analysis revealed that gbx2 upregulated the expression of the zebrafish proneural gene ebf2 , whereas it repressed notch1a , which generally represses neurogenesis. Taken together, these results demonstrate that gbx2 not only functions to position the MHB but also regulates neurogenesis in the anterior hindbrain. [ABSTRACT FROM AUTHOR]

Chromatin immunoprecipitation (ChIP) against enhancer-associated marks with massive sequencing is a powerful approach to identify genome-wide distributions of putative enhancers. However, functional in vivo analysis is required to elucidate the activities of predicted enhancers. Using zebrafish embryos, we established a ChIP-Injection method that enables identification of functional enhancers based on their enhancer activities in embryos. Each reporter gene possessing the enhancer-associated genomic region enriched by ChIP was injected into zebrafish embryos to analyze the activity of putative enhancers. By using the ChIP-Injection, we identified 32 distinct putative enhancers that drove specific expression. Additionally, we generated transgenic lines that exhibit distributions of the EGFP signal as was observed in the screening. Furthermore, the expression pattern driven by the identified somite-specific enhancer resembled that of the gene acta2 . The results indicate that ChIP-Injection provides an efficient approach for identification of active enhancers in a potentially wide variety of developmental tissues and stages. [ABSTRACT FROM AUTHOR]

In vertebrates, the periodic formation of somites from the presomitic mesoderm (PSM) is driven by the molecular oscillator known as the segmentation clock. The hairy -related gene, hes6/her13.2, functions as a hub by dimerizing with other oscillators of the segmentation clock in zebrafish embryos. Although hes6 exhibits a posterior–anterior expression gradient in the posterior PSM with a peak at the tailbud, the detailed mechanisms underlying this unique expression pattern have not yet been clarified. By establishing several transgenic lines, we found that the transcriptional regulatory region downstream of hes6 in combination with the hes6 3′UTR recapitulates the endogenous gradient of hes6 mRNA expression. This downstream region, which we termed the PT enhancer, possessed several putative binding sites for the T-box and Ets transcription factors that were required for the regulatory activity. Indeed, the T-box transcription factor (Tbx16) and Ets transcription factor (Pea3) bound specifically to the putative binding sites and regulated the enhancer activity in zebrafish embryos. In addition, the 3′UTR of hes6 is required for recapitulation of the endogenous mRNA expression. Furthermore, the PT enhancer with the 3′UTR of hes6 responded to the inhibition of retinoic acid synthesis and fibroblast growth factor signaling in a manner similar to endogenous hes6 . The results showed that transcriptional regulation by the T-box and Ets transcription factors, combined with the mRNA stability given by the 3′UTR, is responsible for the unique expression gradient of hes6 mRNA in the posterior PSM of zebrafish embryos. [ABSTRACT FROM AUTHOR]

Highlights: [•] Both specification and morphogenesis of the isthmus are regulated by gbx2. [•] Transgenic fish allowing for conditional gbx2 upregulation has been established. [•] Isthmus specification is highly sensitive to gbx2 around the end of epiboly. [•] Functional microdissection of Gbx2 revealed several distinct subdomains. [•] Gbx2 functions as a transcriptional repressor in embryos and P19 cells. [Copyright &y& Elsevier]

Zebrafish pou2, encoding the class V POU transcription factor orthologous to mouse Oct-3/4, has been implicated in different aspects of development, such as dorsoventral patterning, endoderm formation, and brain regionalization, by analyzing pou2 mutant embryos. In the present study, we first conducted overexpression of pou2-modified genes by m RNA injection, and found that pou2 and its active form ( vp- pou2) augmented mesoderm formation and suppressed endoderm specification, whereas repressive pou2 ( en-pou2) affected the formation of the mesoderm and endoderm in a different manner. To avoid complications that might arise from different pou2 functions during the course of development, we used a transgenic line harboring inducible en-pou2 ( HEP), which could function in a dominant-negative manner. We found that suppressing endogenous pou2 by HEP induction at the mid-blastula stage enhanced endoderm development at the expense of mesoderm, whereas the same treatment in the late blastulae promoted mesoderm formation and suppressed the endoderm specification. Further analyses using HEP induction revealed that, from late epiboly to early somitogenesis, pou2 regulated additional developmental aspects, such as brain regionalization, heart development, and tail formation. Our findings suggest that Pou2 functions in multiple aspects of vertebrate development, especially in the binary decision of the mesendoderm to mesoderm and endoderm in different ways depending on the developmental stage. [ABSTRACT FROM AUTHOR]

Abstract: Zebrafish pou2, which encodes a class V POU transcription factor considered to be an orthologue of mouse Oct-3/4, has been implicated by mutant analysis in dorsoventral (DV) patterning, gastrulation, and endoderm formation in early embryos and later in the regionalization of the neural plate. A series of gain-of-function experiments were conducted in the present study to directly reveal the roles pou2 plays in embryogenesis. We first revealed that injecting low-dose wild-type pou2 mRNA ventralizes embryos. Similar overexpression of activated (vp-) pou2 resulted in the same effects, whereas repressive (en-) pou2 caused dorsalization, supporting the previously proposed idea that pou2 is involved in DV patterning and that pou2 is a transcriptional activator. In contrast, high-dose mRNA for pou2 and its modified genes affected convergent extension (CE) movement. We observed similar activities for mouse Oct-3/4, suggesting conservation of the roles of this POU family in vertebrate development. To determine the critical stage for the functions of pou2 in embryos, we established a transgenic (Tg) fish line harboring en-pou2 under regulation of a heat-shock promoter (HEP) and found that the exposure of HEP Tg embryos to heat shock at the midblastula (sphere) stage dorsalized embryos, whereas induction of HEP at the late blastula stage (30–50% epiboly) affected CE movement. The defects due to HEP induction were rescued by introducing wild-type pou2 mRNA before the heat treatments. Collectively, these data demonstrated that pou2 regulates DV patterning and CE movement in zebrafish embryos at the midblastula and late blastula stages, respectively. [Copyright &y& Elsevier]

Abstract: Zebrafish hoxb1b is expressed during epiboly in the posterior neural plate, with its anterior boundary at the prospective r4 region providing a positional cue for hindbrain formation. A similar function and expression is known for Hoxa1 in mice, suggesting a shared regulatory mechanism for hindbrain patterning in vertebrate embryos. To understand the evolution of the regulatory mechanisms of key genes in patterning of the central nervous system, we examined how hoxb1b transcription is regulated in zebrafish embryos and compared the regulatory mechanisms between mammals and teleosts that have undergone an additional genome duplication. By promoter analysis, we found that the expression of the reporter gene recapitulated hoxb1b expression when driven in transgenic embryos by a combination of the upstream 8.0-kb DNA and downstream 4.6-kb DNA. Furthermore, reporter expression expanded anteriorly when transgenic embryos were exposed to retinoic acid (RA) or LiCl, or injected with fgf3/8 mRNA, implicating the flanking DNA examined here in the responsiveness of hoxb1b to posteriorizing signals. We further identified at least two functional RA responsive elements in the downstream DNA that were shown to be major regulators of early hoxb1b expression during gastrulation, while the upstream DNA, which harbors repetitive sequences with apparent similarity to the autoregulatory sequence of mouse Hoxb1, contributed only to later hoxb1b expression, during somitogenesis. Possible implications in vertebrate evolution are discussed based on these findings. [ABSTRACT FROM AUTHOR]

Abstract: In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the regions flanking the gene. [Copyright &y& Elsevier]

Fgf8 is among the members of the fibroblast growth factor (FGF) family that play pivotal roles in vertebrate development. In the present study, the genomic DNA of the zebrafish fgf8 gene was cloned to elucidate the regulatory mechanism behind the temporally and spatially restricted expression of the gene in vertebrate embryos. Structural analysis revealed that the exon–intron organization of fgf8 is highly conserved during vertebrate evolution, from teleosts to mammals. Close inspection of the genomic sequence and reverse transcription–polymerase chain reaction analysis revealed that zebrafish fgf8 encodes two splicing variants, corresponding to Fgf8a and Fgf8b, among the four to seven splicing variants known in mammals. Misexpression of the two variants in zebrafish embryos following mRNA injection showed that both variants have dorsalizing activities on zebrafish embryos, with Fgf8b being more potent. Reporter gene analysis of the transcriptional regulation of zebrafish fgf8 suggested that its complicated expression pattern, which is considered essential for its multiple roles in development, is mediated by combinations of different regulatory regions in the upstream and downstream regions of the gene. Furthermore, comparison of the genomic sequence of fgf8 among different vertebrate species suggests that this regulatory mechanism is conserved during vertebrate evolution. [ABSTRACT FROM AUTHOR]

The EGIP gene for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are structurally related to the epidermal growth factor, is activated at the onset of gastrulation in subdomains of the embryonic ectoderm. We showed in our previous study that the spatial and temporal regulation of EGIP is conducted by the upstream region from −372 to +194, and that there is an enhancer element between −372 and −210. In this study, we introduced into sea urchin embryos PCR-amplified DNA containing differently truncated EGIP flanking region that was ligated to the GFP reporter gene, and examined the transient expression of the reporter gene, showing that both the −270/−238 and −249/−210 regions were essential for the enhancer activity. We further showed that there is another activating element between −65 and −21, and that even the region between −65 and +194 is sufficient for ectoderm-specific expression of the EGIP gene. The electrophoretic mobility shift assay showed that the −270/−210 enhancer region and the proximal −61/+30 region include specific binding sites for nuclear proteins of sea urchin embryos. Besides these unique sites, the presence of multiple binding sites for GCF1-like nuclear proteins have been revealed in the upstream DNA. [Copyright &y& Elsevier]

We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes (fgfr1–4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2–4 is absent. [Copyright &y& Elsevier]

Exogastrula-inducing peptides (EGIPs) were identified in embryos of the sea urchin Anthocidaris crassispina as polypeptides with structural similarity to epidermal growth factor (EGF) that severely affect gastrulation of sea urchin embryos to induce exogastrulation. Here we have obtained genomic clones for the EGIP precursor gene (EGIP) and determined its genomic organization. The EGIP gene spans the length of 9 kb in the genome and is composed of seven exons and six introns. Each of the four EGF motifs in the precursor protein is encoded by a single exon, and all the exon boundaries are in phase 1, suggesting that EGIP have been generated during evolution by duplication of an exon encoding a single ancient EGIP sequence. The 5′-flanking sequence of EGIP from −4372 to +194 revealed the presence of multiple repeat sequences including direct and inverted repeats as well as two clusters of GGGG/CCCC elements. The function of the upstream flanking region of EGIP was examined by introducing the gene constructs into embryos in which different regions from the flanking DNA were placed upstream to the GFP reporter gene. Systematic deletion of the upstream DNA revealed the presence of potent enhancer activity between −372 and −210. [Copyright &y& Elsevier]

Exogastrula-inducing peptides (EGIP) of the sea urchin Anthocidaris crassispina are endogenous peptides related to epidermal growth factor (EGF), which induce exogastrulation in the embryo. Recently, a protein(s) from sea urchin embryos that binds to one of the EGIP, EGIP-D (EGIP-D-binding protein, EBP) was purified. The isolation and characterization of the cDNA clones for two EBP proteins (EBP-α and EBP-β) is reported. The two EBP proteins were highly similar in structure to each other; both possessed putative cell-binding sites and two repeated sequences characteristically seen in the insect neuronal cell adhesion protein, fasciclin I. The EBP showed similarity with other sea urchin proteins HLC-32, Bep1, and Bep4. It has been confirmed that bacterially expressed EBP proteins associate with EGIP-D as does native EBP, suggesting the interaction between EGF-related proteins and fasciclin I-related proteins. An EBP transcript of 1.4 kb was strongly expressed in immature ovaries but not in immature testes. A somewhat lower level of the transcript existed in unfertilized eggs and the amount gradually declined to an almost undetectable level by the pluteus stage. The EBP proteins were present throughout embryonic development at nearly constant levels. Although most of the proteins were distributed rather evenly in the cytoplasm, a small portion was detected on the apical surface of blastomeres and ectodermal cells, showing that EBP are components of the hyaline layer. [ABSTRACT FROM AUTHOR]

By screening a cDNA library and 3′-rapid amplification of cDNA ends, the cDNA for a non-receptor type protein tyrosine kinase from the sea urchin Anthocidaris crassispina was analyzed. The deduced protein (AcSrc1) with the highest identity of about 60% to mammalian Src family kinases shows the characteristic features of the Src family. AcSrc1 mRNA is maternally expressed in unfertilized eggs, while zygotic expression is first detected in blastulae and continues through the pluteus stage. Zygotic mRNA expression, visualized by in situ hybridization, is detected specifically in archenteron at the gastrula stage, while it is restricted in plutei to the midgut and hindgut, suggesting specific roles for AcSrc1 in the formation and/or functions of the digestive tract. Meanwhile, western blot analysis has shown that the AcSrc1 protein is constantly expressed throughout embryogenesis. By immunostaining, it was found that the protein (distributed evenly in the cytoplasm of unfertilized eggs) is translocated to the membrane after fertilization. All through the following development, AcSrc1 was localized to the peripheries of different embryonic cells, although at a relatively low level of localization at the boundaries between adjacent cells. [ABSTRACT FROM AUTHOR]

We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes (fgfr1–4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2–4 is absent. [Copyright &y& Elsevier]