Enveloping Layer Cells Research Articles

Cell-type-specific mRNA transcription and degradation kinetics in zebrafish embryogenesis from metabolically labeled single-cell RNA-seq

During embryonic development, pluripotent cells assume specialized identities by adopting particular gene expression profiles. However, systematically dissecting the relative contributions of mRNA transcription and degradation to shaping those profiles remains challenging, especially within embryos with diverse cellular identities. Here, we combine single-cell RNA-Seq and metabolic labeling to capture temporal cellular transcriptomes of zebrafish embryos where newly-transcribed (zygotic) and pre-existing (maternal) mRNA can be distinguished. We introduce kinetic models to quantify mRNA transcription and degradation rates within individual cell types during their specification. These models reveal highly varied regulatory rates across thousands of genes, coordinated transcription and destruction rates for many transcripts, and link differences in degradation to specific sequence elements. They also identify cell-type-specific differences in degradation, namely selective retention of maternal transcripts within primordial germ cells and enveloping layer cells, two of the earliest specified cell types. Our study provides a quantitative approach to study mRNA regulation during a dynamic spatio-temporal response.

Single-Cell Sequencing Reveals Heterogeneity Effects of Bisphenol A on Zebrafish Embryonic Development.

The embryonic period is a sensitive window for bisphenol A (BPA) exposure. However, embryonic development is a highly dynamic process with changing cell populations. The heterogeneity effects of BPA on fish embryo cells during development remain unclear. We applied single-cell RNA sequencing to analyze the impact of BPA exposure on transcriptome heterogeneity of 64 683 cells from zebrafish embryos at 8, 12, and 30 h postfertilization (hpf). Thirty-eight cell populations were identified and gene expression profiles of 16 cell populations were significantly altered by BPA. At 8 hpf, BPA mainly influenced the outer layer cell populations of embryos, such as neural plate border and enveloping layer cells. At 12 and 30 hpf, nervous system formation and heart morphogenesis were disturbed. The altered differential processes of the neural plate border, neural crest, and neuronal cells were found to lead to increased neurogenesis in zebrafish larvae. In the forebrain, midbrain, neurons, and optic cells, pathways related to cell division and DNA replication and repair were altered. Moreover, BPA also changed transforming growth factor (TGF) β signaling and heart tube morphogenesis in heart cells, leading to a decreased heartbeat in zebrafish larvae. Our study provides a comprehensive understanding of BPA toxicity on fish embryonic development at a single-cell level.

Spatiotemporal characterization of dynamic epithelial filopodia during zebrafish epiboly

During zebrafish epiboly, the embryonic cell mass, or blastoderm, spreads to enclose the yolk cell. The blastoderm consists of an outer epithelial sheet, the enveloping layer (EVL), and the underlying deep cell layer (DEL). Studies have provided insights into the mechanisms of EVL and deep cell epiboly, but little is known about the interactions between the two cell layers and what role they may play during epiboly. We used live imaging to examine EVL basal protrusions. We identified them as filopodia based on f-actin content and localization of fluorescently tagged filopodial markers. A spatiotemporal analysis revealed that the largest number of EVL filopodia were present during early epiboly at the animal pole. In functional studies, expression of a constitutively active actin-bundling protein resulted in increased filopodial length and delayed gastrulation. We identified protrusions on the basal surface of EVL cells as filopodia and showed that they are present throughout the EVL during epiboly. The largest number of filopodia was at the animal pole during early epiboly, which is when and where deep cell radial intercalations occur to the greatest extent. These findings suggest that EVL filopodia may function during epiboly to promote deep cell rearrangements during epiboly initiation.

Identification of regulatory elements recapitulating early expression of L-plastin in the zebrafish enveloping layer and embryonic periderm

We have cloned and characterized an intronic fragment of zebrafish lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) that drives expression to the zebrafish enveloping layer (EVL). L-plastin is a calcium-dependent actin-bundling protein belonging to the plastin/fimbrin family of proteins, and is necessary for the proper migration and attachment of several adult cell types, including leukocytes and osteoclasts. However, in zebrafish lcp1 is abundantly expressed much earlier, during differentiation of the EVL. The cells of this epithelial layer migrate collectively, spreading vegetally over the yolk. L-plastin expression persists into the larval periderm, a transient epithelial tissue that forms the first larval skin. This finding establishes that L-plastin is activated in two different embryonic waves, with a distinct regulatory switch between the early EVL and the later leukocyte. To better study L-plastin expressing cells we attempted CRISPR/Cas9 homology-driven recombination (HDR) to insert a self-cleaving peptide (Cre-P2A-EGFP-CAAX) downstream of the native lcp1 promoter. This produced a stable zebrafish line expressing Cre recombinase in EVL nuclei and green fluorescence in EVL cell membranes. In vivo tracking of these labeled cells provided enhanced views of EVL migration behavior, membrane extensions, and mitotic events. Finally, we experimentally dissected key elements of the targeted lcp1 locus, discovering a ∼300 bp intronic sequence sufficient to drive EVL expression. The lcp1: Cre-P2A-EGFP-CAAX zebrafish should be useful for studying enveloping layer specification, gastrulation movements and periderm development in this widely used vertebrate model. In addition, the conserved regulatory sequences we have isolated predict that L-plastin orthologs may have a similar early expression pattern in other vertebrate embryos.

Physical Model for Cell Migration Guided by Elastic Properties of the Substrate

During annual fish epiboly, between the egg yolk and the enveloping layer cells (EVL),there is a group of deep cells that move in contact with EVL layer. It is experimentally observed that these cells migrate toward the EVL borders and move freely on them. Apparently,the phenomenon is due to elastic properties of the EVLs: either a difference in the Young modulus on the border or a stress gradient inside the cell. In this work,a model is developed to elucidate how this kind of migration works and what are sensing the cells. It considers a cell modelled as a circle with four contacts on a thin elastic membrane,representing the EVL layer. Each of the four contacts simulates a random protrusion which elongates their end out of the center of the cell,attach rigidly to the substrate, and contracts without gliding. We study the elastic response of the substrate and its implications in the cell dynamics when it has different stiffness and when it is under internal tensions. In the first case, the substrate has a stiffer strip which simulates the EVL border which in theory has a higher Young modulus. In this case,the results do not show the expected durotaxis effect. In fact, the cell shows a random walk movement added to a slight repulsion generated by the border so that a stiffer strip on a thin plate is not enough to reproduce the phenomenon. In the second case,when the substrate is under internal applied tensions,the results show that in a substrate with circular geometry,if the contractive circumferential stress component increases with the radial distance,the cell migrates and reproduce experimental observations. We conclude that cell migration could be due to differences in internal tensions instead of thickness or stiffness differences.

Wilson cell origin for kupffer's vesicle in the zebrafish

Bilaterally symmetric animals have evolved highly reproducible asymmetries between left and right. In teleosts, Kupffer's vesicle, the structure necessary for the determination of left-right asymmetry, is derived from a group of cells in the gastrula termed the dorsal forerunners. Wilson cells are a ring of marginal enveloping layer cells that are cytoplasmically connected to the yolk cell and thus the last blastomeres to inherit yolk cell cytoplasm. Afterward, they collapse into the yolk to form the yolk syncytial layer. Without exception, forerunner cells are the progeny of dorsal Wilson cells. At the beginning of gastrulation, these Wilson cell progeny ingress beneath the enveloping layer, transform into Kupffer's vesicle, and eventually become tail notochord and muscle. Before ingressing, the forerunner precursor cells express endodermal promoting genes and require high-levels of Nodal signaling. Despite a derived function of the enveloping layer as an epithelium covering the entire embryo, its dorsal margin retains many behaviors of what might be expected of the dorsal superficial layers of the ancestral fish embryo, including an early program of endodermal development, cell ingression, and an eventual contribution of cells to caudal notochord and muscle, as well as the control of laterality. Developmental Dynamics 247:1057-1069, 2018. © 2018 Wiley Periodicals, Inc.

Flotillins control zebrafish epiboly through their role in cadherin-mediated cell-cell adhesion.

Zebrafish gastrulation and particularly epiboly that involves coordinated movements of several cell layers is a dynamic process for which regulators remain to be identified. We show here that Flotillin 1 and 2, ubiquitous and highly conserved proteins, are required for epiboly. Flotillins knockdown compromised embryo survival, strongly delayed epiboly and impaired deep cell radial intercalation and directed collective migration without affecting enveloping layer cell movement. At the molecular level, we identified that Flotillins are required for the formation of E-cadherin-mediated cell-cell junctions. These results provide the first in vivo evidence that Flotillins regulate E-cadherin-mediated cell-cell junctions to allow epiboly progression.

Cloning and characterization of ifitm1 and ifitm3 expression during early zebrafish development.

The family of interferon-inducible transmembrane proteins (IFITMs) plays a crucial role in inhibiting proliferation, promoting homotypic cell adhesion and mediating germ cell development. In the present study, the full-length cDNAs of zebrafish ifitm1 (744 bp) and ifitm3 (702 bp) were obtained by rapid amplification of cDNA ends (RACE). Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ifitm1 mRNA was expressed in the ovary, testis, brain, muscle, liver and kidney, while ifitm3 mRNA was only detected in the ovary. Based on in situ hybridization, ifitm1 mRNA was found to be strongly expressed in the ooplasm from stage I to stage II and ifitm3 mRNA was also strongly expressed in the ooplasm from stage I to stage II, furthermore ifitm3 expression ultimately localized to the cortex region beneath the plasma membrane of stage IV oocytes. During development, ifitm1 expression was initially detected in the enveloping layer cells and deep layer cells of shield stage embryos. Then, throughout the segmentation phase (10.25-24 hours post-fertilization (hpf)), ifitm1 expression was mainly detected in the head, trunk and tail regions. Unlike ifitm1, ifitm3 expression was initially detected in sphere stage embryos and was then broadly expressed throughout the embryo from the 70% epiboly stage to 24 hpf. Interestingly, ifitm3 was also expressed in primordial germ cells (PGCs) from the bud stage to 24 hpf. This expression analysis indicates that zebrafish ifitm1 may play a critical role in early organogenesis and may perform immune or hematopoietic functions and ifitm3 might be necessary for PGC migration and the formation of female germ cells.

Pou5f1 protein expression and posttranslational modification during early zebrafish development

Pou5f1/Oct4 is a transcription factor essential for maintenance of pluripotency in mammals and for control of blastula and gastrula stage gene regulatory networks in zebrafish. Information on Pou5f1 protein distribution was before this study not available for zebrafish. Therefore, we generated polyclonal antibodies that selectively recognize Pou5f1 and analyzed its protein distribution and modification during development. Pou5f1 protein is present in unfertilized oocytes, and persists in all embryonic and enveloping layer cell nuclei until the end of gastrulation, but is absent from yolk syncytial nuclei. Pou5f1 is subject to multiple developmentally regulated phosphorylations, with the higher phosphorylated forms prevailing in the oocyte and during late gastrulation. The developmental protein profile correlates with the stages during which deep cells are not committed to a specific germ layer. The posttranslational modification by phosphorylation opens the possibility that Pou5f1 may be subject to temporal or region specific modulation of its activity or stability by embryonic signaling mechanisms.

Ethanol exposure disrupts extraembryonic microtubule cytoskeleton and embryonic blastomere cell adhesion, producing epiboly and gastrulation defects

SummaryFetal alcohol spectrum disorder (FASD) occurs when pregnant mothers consume alcohol, causing embryonic ethanol exposure and characteristic birth defects that include craniofacial, neural and cardiac defects. Gastrulation is a particularly sensitive developmental stage for teratogen exposure, and zebrafish is an outstanding model to study gastrulation and FASD. Epiboly (spreading blastomere cells over the yolk cell), prechordal plate migration and convergence/extension cell movements are sensitive to early ethanol exposure. Here, experiments are presented that characterize mechanisms of ethanol toxicity on epiboly and gastrulation. Epiboly mechanisms include blastomere radial intercalation cell movements and yolk cell microtubule cytoskeleton pulling the embryo to the vegetal pole. Both of these processes were disrupted by ethanol exposure. Ethanol effects on cell migration also indicated that cell adhesion was affected, which was confirmed by cell aggregation assays. E-cadherin cell adhesion molecule expression was not affected by ethanol exposure, but E-cadherin distribution, which controls epiboly and gastrulation, was changed. E-cadherin was redistributed into cytoplasmic aggregates in blastomeres and dramatically redistributed in the extraembryonic yolk cell. Gene expression microarray analysis was used to identify potential causative factors for early development defects, and expression of the cell adhesion molecule protocadherin-18a (pcdh18a), which controls epiboly, was significantly reduced in ethanol exposed embryos. Injecting pcdh18a synthetic mRNA in ethanol treated embryos partially rescued epiboly cell movements, including enveloping layer cell shape changes. Together, data show that epiboly and gastrulation defects induced by ethanol are multifactorial, and include yolk cell (extraembryonic tissue) microtubule cytoskeleton disruption and blastomere adhesion defects, in part caused by reduced pcdh18a expression.

Zeb1 Regulates E-cadherin and Epcam (Epithelial Cell Adhesion Molecule) Expression to Control Cell Behavior in Early Zebrafish Development

The ZEB1 transcription factor is best known as an inducer of epithelial-mesenchymal transitions (EMT) in cancer metastasis, acting through transcriptional repression of CDH1 (encoding E-cadherin) and the EMT-suppressing microRNA-200s (miR-200s). Here we analyze roles of the ZEB1 zebrafish orthologs, Zeb1a and Zeb1b, and of miR-200s in control of cell adhesion and morphogenesis during gastrulation and segmentation stages. Loss and gain of function analyses revealed that Zeb1 represses cdh1 expression to fine-tune adhesiveness of migrating deep blastodermal cells. Furthermore, Zeb1 acts as a repressor of epcam in the deep cells of the blastoderm and may contribute to control of epithelial integrity of enveloping layer cells, the outermost cells of the blastoderm. We found a similar ZEB1-dependent repression of EPCAM expression in human pancreatic and breast cancer cell lines, mediated through direct binding of ZEB1 to the EPCAM promoter. Thus, Zeb1 proteins employ several evolutionary conserved mechanisms to regulate cell-cell adhesion during development and cancer.

Necessary role for intracellular Ca2+ transients in initiating the apical-basolateral thinning of enveloping layer cells during the early blastula period of zebrafish development

During the early blastula period of zebrafish embryos, the outermost blastomeres begin to undergo a significant thinning in the apical/basolateral dimension to form the first distinct cellular domain of the embryo, the enveloping layer (EVL). During this shape transformation, only the EVL-precursor cells generate a coincidental series of highly restricted Ca(2+) transients. To investigate the role of these localized Ca(2+) transients in this shape-change process, embryos were treated with a Ca(2+) chelator (5,5'-difluoro BAPTA AM; DFB), or the Ca(2+) ionophore (A23187), to downregulate and upregulate the transients, respectively, while the shape-change of the forming EVL cells was measured. DFB was shown to significantly slow, and A23187 to significantly facilitate the shape change of the forming EVL cells. In addition, to investigate the possible involvement of the phosphoinositide and Wnt/Ca(2+) signaling pathways in the Ca(2+) transient generation and/or shape-change processes, embryos were treated with antagonists (thapsigargin, 2-APB and U73122) or an agonist (Wnt-5A) of these pathways. Wnt-5A upregulated the EVL-restricted Ca(2+) transients and facilitated the change in shape of the EVL cells, while 2-APB downregulated the Ca(2+) transients and significantly slowed the cell shape-change process. Furthermore, thapsigargin and U73122 also both inhibited the EVL cell shape-change. We hypothesize, therefore, that the highly localized and coincidental Ca(2+) transients play a necessary role in initiating the shape-change of the EVL cells.

Structural components and morphogenetic mechanics of the zebrafish yolk extension, a developmental module

The yolk extension (YE) appears to be a novel developmental module that has been inserted into the phylotypic period of teleostean development, specifically in the order Cypriniformes. The zebrafish YE informs the study of morphogenetic movements reshaping ventral tissues because (1) this trait is easily visible, so disruptions are easy to score; (2) its ontogenesis occurs quickly; and (3) the yolk cell isolates the tissues elongating the ventrum from the rest of the embryo, serving as a three-dimensional in vivo "tissue culture." We determined that three histological compartments comprise the structural components of the YE: (1) the internal yolk cell; (2) the mesendodermal mantle external to the yolk cell; and (3) the external embryonic integument, consisting of an embryonic epidermis plus enveloping layer cells. These structural components interact with one another in a hierarchical manner, resulting in the morphogenesis of the elongated and tubular embryonic zebrafish ventrum as the cylindrical YE forms. Time-lapse videomicroscopy and experimental manipulation show that the yolk mass is a cohesive, viscoelastic foam, which resists compression. Moreover, as the mesodermal mantle participates in tubulation of the posterior trunk, Kupffer's Vesicle, the organ of laterality in teleosts, separates from the posterior pole of the yolk syncytial layer. Additionally, the embryonic integument becomes contractile over the posterior yolk cell, constricting the yolk mass to form the YE. These findings constitute an initial assessment of the morphogenetic mechanics underlying formation of the YE developmental module in zebrafish.

Poky/chuk/ikk1 is required for differentiation of the zebrafish embryonic epidermis

An epidermis surrounds all vertebrates, forming a water barrier between the external environment and the internal space of the organism. In the zebrafish, the embryonic epidermis consists of an outer enveloping layer (EVL) and an inner basal layer that have distinct embryonic origins. Differentiation of the EVL requires the maternal effect gene poky/ikk1 in EVL cells prior to establishment of the basal layer. This requirement is transient and maternal Ikk1 is sufficient to allow establishment of the EVL and formation of normal skin in adults. Similar to the requirement for Ikk1 in mouse epidermis, EVL cells in poky mutants fail to exit the cell cycle or express specific markers of differentiation. In spite of the similarity in phenotype, the molecular requirement for Ikk1 is different between mouse and zebrafish. Unlike the mouse, EVL differentiation requires functioning Poky/Ikk1 kinase activity but does not require the HLH domain. Previous work suggested that the EVL was a transient embryonic structure, and that maturation of the epidermis required replacement of the EVL with cells from the basal layer. We show here that the EVL is not lost during embryogenesis but persists to larval stages. Our results show that while the requirement for poky/ikk1 is conserved, the differences in molecular activity indicate that diversification of an epithelial differentiation program has allowed at least two developmental modes of establishing a multilayered epidermis in vertebrates.

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers.

Identification of common and unique modifiers of zebrafish midline bifurcation and cyclopia

Loss of the zebrafish Nodal-related protein Squint causes a spectrum of phenotypes including cyclopia and midline bifurcations (MB). Here we examine MBs and their relation to cyclopia in maternal-zygotic squint (MZsqt) mutants. There is a concordance of MB with cyclopia in MZsqt embryos. Heat treatment and depletion of Hsp90a are “common” risk factors, each of which increases the incidence of both phenotypes. Midline identity is specified on both sides of MBs, and deep-layer cells are initially lacking within bifurcations, whereas enveloping layer cells are intact. Bifurcations do not appear until the completion of gastrulation and are preceded by gaps in the expression of wnt5b, an essential regulator of dorsal convergence. The incidence of early MBs and wnt5b expression defects in heated MZsqt embryos is high, but there is also substantial recovery. Wnt5b depletion increases the incidence of MB, but not cyclopia, and as such Wnt5b is a “unique” risk factor for MB. Reciprocally, depletion of Wnt11 or Hsp90b increases cyclopia only. In summary, we find that MB arises after gastrulation in regions that fail to express wnt5b, and we show that two complex dysmorphologies – MB and cyclopia – can be promoted by either common or unique risk factors.

Cell adhesion and the actin cytoskeleton of the enveloping layer in the zebrafish embryo during epiboly

As the zebrafish embryo undergoes gastrulation and epiboly, the cells of the enveloping layer (EVL) expand, covering the entire yolk cell. During the epiboly process, the EVL cells move as a coherent layer, remaining tightly attached to each other and to the underlying yolk syncytial layer (YSL). In view of the central role of the actin cytoskeleton, in both cell motility and cell-cell adhesion, we have labeled these cells in situ with fluorescent phalloidin and anti-actin antibodies. We show that, throughout their migration, the EVL cells retain a conspicuous cortical actin cytoskeletal belt coinciding with cell surface cadherins. At the margins approaching the YSL, the EVL cells extend, from their apicolateral domains, actin-rich filopodial protrusions devoid of detectable cadherin. We have studied the role of the actin cytoskeleton in the maintenance of EVL cohesion during epiboly. Cytochalasin treatment of embryos induces EVL dissociation accompanied by general detachment of the rest of the embryonic cells. In the dissociating EVL cells, the cortical actin belt undergoes fragmentation with the formation of actin aggregates; cadherins, on the other hand, remain evenly distributed at the junctional cell surface. Removal of Ca2+ by ethyleneglycolbis (amino-ethyl-ether)-tetraacetic acid (EGTA) treatment also induces cell dissociation without visible disruption of the cortical actin belt. The protein kinase inhibitor (1-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H-7), which blocks acto-myosin contractility and disrupts actin cables in cultured cells, also potentiates cytochalasin-induced dissociation and promotes the projection of numerous actin-rich lamellipodial extensions. The fact that EVL cells produce microspike-like structures towards the YSL and are capable of lamellipodial activity lend further support to the suggestion (R.W. Keller and J.P. Trinkaus. 1987. Dev. Biol. 120: 12-24) that the EVL cells are not passively mobilized on the expanding YSL but actively participate in epiboly.

Developmental Activation of the Capability to Undergo Checkpoint-Induced Apoptosis in the Early Zebrafish Embryo

In this study, we demonstrate the developmental activation, in the zebrafish embryo, of a surveillance mechanism which triggers apoptosis to remove damaged cells. We determine the time course of activation of this mechanism by exposing embryos to camptothecin, an agent which specifically inhibits topoisomerase I within the DNA replication complex and which, as a consequence of this inhibition, also produces strand breaks in the genomic DNA. In response to an early (pre-gastrula) treatment with camptothecin, apoptosis is induced at a time corresponding approximately to mid-gastrula stage in controls. This apoptotic response to a block of DNA replication can also be induced by early (pre-MBT) treatment with the DNA synthesis inhibitors hydroxyurea and aphidicolin. After camptothecin treatment, a high proportion of cells in two of the embryo's three mitotic domains (the enveloping and deep cell layers), but not in the remaining domain (the yolk syncytial layer), undergoes apoptosis in a cell-autonomous fashion. The first step in this response is an arrest of the proliferation of all deep- and enveloping-layer cells. These cells continue to increase in nuclear volume and to synthesize DNA. Eventually they become apoptotic, by a stereotypic pathway which involves cell membrane blebbing, “margination” and fragmentation of nuclei, and cleavage of the genomic DNA to produce a nucleosomal ladder. Fragmentation of nuclei can be blocked by the caspase-1,4,5 inhibitor Ac-YVAD-CHO, but not by the caspase-2,3,7[,1] inhibitor Ac-DEVD-CHO. This suggests a functional requirement for caspase-4 or caspase-5 in the apoptotic response to camptothecin. Recently,Xenopushas been shown to display a developmental activation of the capability for stress- or damaged-induced apoptosis at early gastrula stage.En masse,our experiments suggest that the apoptotic responses in zebrafish andXenopusare fundamentally similar. Thus, as for mammals, embryos of the lower vertebrates exhibit the activation of surveillance mechanisms, early in development, to produce the selective apoptosis of damaged cells.

Spatially distinct domains of cell behavior in the zebrafish organizer region

To determine the sequence of cell behaviors that is involved in the morphogenesis of the zebrafish organizer region, we have examined the dorsal marginal zone of vitally stained zebrafish embryos using time-lapse confocal microscopy. During the late-blastula stage, the zebrafish dorsal marginal zone segregates into several cellular domains, including a group of noninvoluting, highly endocytic marginal (NEM) cells. The NEM cell cluster, which lies in a superficial location of the dorsal marginal zone, is composed of both enveloping layer cells and one or two layers of underlying deep cells. The longitudinal position of this cellular domain accurately predicts the site of embryonic shield formation and occupies a homologous location to the organizer epithelium in Xenopus laevis. At the onset of gastrulation, deep cells underneath the superficial NEM cell domain undergo involution to form the nascent hypoblast of the embryonic shield. Deep cells within the NEM cell cluster, however, do not involute during early shield formation, but instead move in front of the blastoderm margin to form a loose mass of cells called forerunner cells. Forerunner cells coalesce into a wedge-shaped mass during late gastrulation and eventually become overlapped by the converging lateral lips of the germ ring. During early zebrafish tail elongation, most forerunner cells are incorporated into the epithelial lining of Kupffer's vesicle, a transient teleostean organ rudiment long thought to be an evolutionary vestige of the neurenteric canal. Owing to the location of NEM cells at the dorsal margin of blastula-stage embryos, as well as their early segregation from other deep cells, we hypothesized that NEM cells are specified by an early-acting dorsalizing signal. To test this possibility, we briefly treated early-blastula stage embryos with LiCl, an agent known to produce hyperdorsalized zebrafish embryos with varying degrees of expanded organizer tissue. In Li(+)-treated embryos, NEM cells appear either within expanded spatial domains or in ectopic locations, primarily within the marginal zone of the blastoderm. These results suggest that NEM cells represent a specific cell type that is specified by an early dorsal patterning pathway.

Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2.

We have used zebrafish embryos to dissect the promoter activity of a gene with a complex expression pattern during embryogenesis. GATA-2 is a transcription factor required for hematopoiesis and is dynamically expressed in hematopoietic tissues and in the central nervous system. Using constructs containing zebrafish GATA-2 genomic flanking sequences and the green fluorescent protein (GFP) reporter gene, we demonstrate that distinct regulatory domains are required for hematopoietic, enveloping layer (EVL), and neuronal expression of GATA-2. During gastrulation, GFP expression is confined to the ventral ectoderm and lateral mesoderm and is lacking in the dorsal shield. Cells derived from the regions expressing GFP give rise to hematopoietic progenitors, EVL cells, and neurons. Deletion analysis of the 7.3-kb GATA-2 promoter region revealed that a 1.1-kb DNA sequence is critical for expression of GATA-2 in neurons. Fine mapping revealed that a 31-bp region is required for neuron enhancer activity, and mutagenesis showed that the DNA motif CCCTCCT is essential for GATA-2 promoter activity in the central nervous system of zebrafish. Our use of zebrafish embryos can be exploited as a whole animal system for the dissection of any developmentally regulated vertebrate promoter.