Differentiation In Zebrafish Research Articles

21-P043 Fibroblast growth factor (Fgf) signaling regulates left–right asymmetry and intestinal cell differentiation in zebrafish

21-P043 Fibroblast growth factor (Fgf) signaling regulates left–right asymmetry and intestinal cell differentiation in zebrafish

Regulation of Neurogenesis by Interkinetic Nuclear Migration through an Apical-Basal Notch Gradient

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.

Six1a is required for the onset of fast muscle differentiation in zebrafish

Vertebrate skeletal muscles arise from two major types of precursor cell populations which differentiate into slow and fast fibers. Six1 homeodomain transcription factor was implicated in myogenesis in mammals, but its role in the development of different types of muscle precursors remained unclear. In zebrafish, there are two close homologs of Six1: six1a (known earlier as six1) and six1b identified in this study. Here we studied the role of six1a whose expression is initiated in the fast muscle precursor region of the forming somite. In the six1a loss-of-function conditions, initiation of myog expression was compromised in fast muscle precursors whereas myod expression appeared unaffected suggestive of six1a requirement for fast muscle differentiation. Expression of myog recovered soon, but differentiation of fast muscle proceeded abnormally. Exclusion of muscle-specific transcripts, myhz1 and tpma, from the dorsal and posterior part of somites demonstrated early abnormalities in fast muscle formation. U-shaped somites, reduced birefringence, and abnormal cell morphology were observed in morphant fast muscle upon terminal differentiation. In contrast, differentiation of slow fibers appeared largely unaffected. We conclude that Six1a plays an essential role at the onset of fast muscle differentiation.

Intestinal Differentiation in Zebrafish Requires Cdx1b, a Functional Equivalent of Mammalian Cdx2

The ParaHox transcription factor Cdx2 is an essential determinant of intestinal phenotype in mammals throughout development, influencing gut function, homeostasis, and epithelial barrier integrity. Cdx2 expression demarcates the zones of intestinal stem cell proliferation in the adult gut, with deregulated expression implicated in intestinal metaplasia and cancer. However, in vivo analysis of these prospective roles has been limited because inactivation of Cdx2 in mice leads to preimplantation embryonic lethality. We used the zebrafish, a valuable model for studying gut development, to generate a system to further understanding of the role of Cdx2 in normal intestinal function and in disease states. We isolated and characterized the zebrafish cdx1b ortholog and analyzed its function by antisense morpholino gene knockdown. We showed that zebrafish Cdx1b replaces the role of Cdx2 in gut development. Evolutionary studies have indicated that the zebrafish cdx2 loci were lost following the genome-wide duplication event that occurred in teleosts. Zebrafish Cdx1b is expressed exclusively in the developing intestine during late embryogenesis and regulates intestinal cell proliferation and terminal differentiation. This work established an in vivo system to explore further the activity of Cdx2 in the gut and its impact on processes such as inflammation and cancer.

Expression profiles for six zebrafish genes during gonadal sex differentiation.

BackgroundThe mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation.ResultsIn the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high in females (fig alpha and cyp19a1a) was segregated in two groups with more than 10 times difference in expression levels. All of the investigated genes showed peaks in expression levels during the time of sex determination and gonadal sex differentiation. Expression of all genes was investigated on cDNA from the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three "male" genes (ar, sox9a and dmrt1) in the investigated period and 81% were high or low expressers of both "female" genes (fig alpha and cyp19a1a). When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish.ConclusionIn zebrafish, the first significant peak in gene expression during the investigated period (2–40 dph) was dmrt1 at 10 dph which indicates involvement of this gene in the early gonadal sex differentiation of males.

Calcitonin receptor-like receptor guides arterial differentiation in zebrafish

AbstractThe calcitonin receptor-like receptor (crlr) is a major endothelial cell receptor for adrenomedullin, a peptide vasodilator involved in cardiovascular development, homeostasis, and disease. Here, we used the zebrafish (Danio rerio) model to characterize the role of crlr in vascular development. Crlr is expressed within somites from the 4- to the 13-somite stage and by arterial progenitors and axial vessels during zebrafish development. Loss of crlr results in profound alterations in vascular development and angiogenesis, including atrophic trunk dorsal aorta and interruption of anterior aortic bifurcation, delay in intersomitic vessel development, and lack of blood circulation. Remarkably, crlr morphants are characterized by the loss of arterial endothelial cell identity in dorsal aorta, as shown by the lack of expression of the arterial markers ephrin-B2a, DeltaC, and notch5. Down-regulation of crlr affects vascular endothelial growth factor (vegf) expression, whereas vegf overexpression is sufficient to rescue arterial differentiation in crlr morphants. Finally, genetic and biochemical evidences indicate that somitic crlr expression is under the control of sonic hedgehog. These data demonstrate that crlr plays a nonredundant role in arterial differentiation, representing a novel element of the sonic hedgehog–vegf-notch signaling cascade that controls arterial/venous fate.

Involvement of DMAP1 in the neural migration and differentiation in zebrafish

Involvement of DMAP1 in the neural migration and differentiation in zebrafish

Zebrafish sex determination and differentiation: Involvement of FTZ-F1 genes

Sex determination is the process deciding the sex of a developing embryo. This is usually determined genetically; however it is a delicate process, which in many cases can be influenced by environmental factors. The mechanisms controlling zebrafish sex determination and differentiation are not known. To date no sex linked genes have been identified in zebrafish and no sex chromosomes have been identified. However, a number of genes, as presented here, have been linked to the process of sex determination or differentiation in zebrafish. The zebrafish FTZ-F1 genes are of central interest as they are involved in regulating interrenal development and thereby steroid biosynthesis, as well as that they show expression patterns congruent with reproductive tissue differentiation and function. Zebrafish can be sex reversed by exposure to estrogens, suggesting that the estrogen levels are crucial during sex differentiation. The Cyp19 gene product aromatase converts testosterone into 17 beta-estradiol, and when inhibited leads to male to female sex reversal. FTZ-F1 genes are strongly linked to steroid biosynthesis and the regulatory region of Cyp19 contains binding sites for FTZ-F1 genes, further linking FTZ-F1 to this process. The role of FTZ-F1 and other candidates for zebrafish sex determination and differentiation is in focus of this review.

Sox4b is a key player of pancreatic α cell differentiation in zebrafish

Pancreas development relies on a network of transcription factors belonging mainly to the Homeodomain and basic Helix–Loop–Helix families. We show in this study that, in zebrafish, sox4, a member of the SRY-like HMG-box (SOX) family, is required for proper endocrine cell differentiation. We found that two genes orthologous to mammalian Sox4 are present in zebrafish and that only one of them, sox4b, is strongly expressed in the pancreatic anlage. Transcripts of sox4b were detected in mid-trunk endoderm from the 5-somite stage, well before the onset of expression of the early pancreatic gene pdx-1. Furthermore, by fluorescent double in situ hybridization, we found that expression of sox4b is mostly restricted to precursors of the endocrine compartment. This expression is not maintained in differentiated cells although transient expression can be detected in α cells and some β cells. That sox4b-expressing cells belong to the endocrine lineage is further illustrated by their absence from the pancreata of slow-muscle-omitted mutant embryos, which specifically lack all early endocrine markers while retaining expression of exocrine markers. The involvement of sox4b in cell differentiation is suggested firstly by its up-regulation in mind bomb mutant embryos displaying accelerated pancreatic cell differentiation. In addition, sox4b knock-down leads to a drastic reduction in glucagon expression, while other pancreatic markers including insulin, somatostatin, and trypsin are not significantly affected. This disruption of α cell differentiation is due to down-regulation of the homeobox arx gene specifically in the pancreas. Taken together, these data demonstrate that, in zebrafish, sox4b is expressed transiently during endocrine cell differentiation and plays a crucial role in the generation of α endocrine cells.

Development without germ cells: The role of the germ line in zebrafish sex differentiation

The progenitors of the gametes, the primordial germ cells (PGCs) are typically specified early in the development in positions, which are distinct from the gonad. These cells then migrate toward the gonad where they differentiate into sperms and eggs. Here, we study the role of the germ cells in somatic development and particularly the role of the germ line in the sex differentiation in zebrafish. To this end, we ablated the germ cells using two independent methods and followed the development of the experimental fish. First, PGCs were ablated by knocking down the function of dead end, a gene important for the survival of this lineage. Second, a method to eliminate the PGCs using the toxin-antitoxin components of the parD bacterial genetic system was used. Specifically, we expressed a bacterial toxin Kid preferentially in the PGCs and at the same time protected somatic cells by uniformly expressing the specific antidote Kis. Our results demonstrate an unexpected role for the germ line in promoting female development because PGC-ablated fish invariably developed as males.

Effect of Exposure to Bisphenol A on the Sex Differentiation in Zebrafish (Danio rerio)

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Intestinal growth and differentiation in zebrafish

Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.

MALE OR FEMALE? CAN THE ENVIRONMENT DECIDE?

![Figure][1] Sexual differentiation in zebrafish occurs after hatching and is a labile process. During early life, gonads are undifferentiated and become either testes or ovaries during sexual differentiation. The balance between circulating levels of male (androgen) and female (oestrogen)

Transgene driving GFP expression from the promoter of the zona pellucida gene zpc is expressed in oocytes and provides an early marker for gonad differentiation in zebrafish.

Although mechanisms of sex differentiation have been studied intensely in mammals, insects, and worms, little is known about this process in lower vertebrates. To establish a marker for female gonad differentiation in zebrafish, we generated a transgenic line in which 412 bp from the promoter and 5' mRNA leader of the female-specific zebrafish zona pellucida gene zpc are fused to the coding region of green fluorescent protein (GFP). The zpc0.5:GFP transgene is expressed exclusively in oocytes, starting from the onset of female-specific differentiation, and closely resembles the expression pattern of the wild-type zpc. Strong GFP expression persists throughout oogenesis and is visible through the body wall of females. We have also characterized a putative upstream factor of zpc, FIGalpha, and show that distribution of FIGalpha RNA is compatible with its postulated role in the regulation of zpc. The zpc0.5:GFP transgenic line described here will be useful for studying oocyte development and the mechanisms that determine sex-specific gene expression in the zebrafish. It is also the first promoter characterized to date to drive stable and efficient expression specifically in the zebrafish female germline.

Extraretinal and retinal hedgehog signaling sequentially regulate retinal differentiation in zebrafish

Hedgehog (Hh) signaling is required for eye development in vertebrates; known roles in the zebrafish include regulation of eye morphogenesis and ganglion cell and photoreceptor differentiation. We employed a temporally selective Hh signaling knockdown strategy, by using antisense morpholino oligonucleotides or the teratogenic alkaloid cyclopamine, in order to dissect the separate roles of Hh signaling arising from specific sources. We also examined the eye phenotype of zebrafish slow muscle-omitted (smu) mutants, which lack a functional smoothened gene, encoding a component of the Hh signal transduction pathway. We find that Hh signaling from extraretinal sources is required for the initiation of retinal differentiation, but this involvement may be independent of the effects of Hh signaling on optic stalk development. We also find that Hh signals from ganglion cells participate in propagating expression of ath5, and we suggest that the effects of Hh signals from the retinal pigmented epithelium on photoreceptor differentiation may be mediated by the transcription factor rx1.

2SY04-5 A novel mutation, s202 affects early endothelial cell differentiation in zebrafish

2SY04-5 A novel mutation, s202 affects early endothelial cell differentiation in zebrafish

Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish

Large numbers of apoptotic early diplotene oocytes were observed during the transition from ovary-like undifferentiated gonadal tissue to testes during sex differentiation in presumptive males of the zebrafish (Danio rerio). The percentage of terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labelling (TUNEL)-positive apoptotic oocytes in the gonads of presumptive males was approximately eight- to 12-fold higher than in genetic all-females. By 29 days post-hatching, all oocytes had disappeared from the gonads of presumptive males. In these males, we also observed apoptotic somatic cells in the ovarian cavity between 23 and 35 days post-hatching. Therefore, the disappearance of oocytes and the decomposition of the ovarian cavity caused by apoptosis during sex differentiation were male-specific events. In genetic all-females, apoptosis in a proportion of early diplotene oocytes was found in the undifferentiated gonads at 15-19 days post-hatching, probably as a result of programmed oocyte loss during ovarian development. These findings suggest that oocyte apoptosis is the mechanism of testicular and ovarian differentiation in zebrafish.

Critical role of biklf in erythroid cell differentiation in zebrafish

Hematopoietic cells arise from ventral mesoderm in different vertebrates, but the mechanisms through which various factors contribute to the hematopoietic processes, including erythrogenesis, remain incompletely understood. The Krüppel-like transcription factor Biklf is preferentially expressed in blood islands throughout zebrafish embryogenesis, marking the region of future erythropoiesis [1]. In this paper, we show that expression of biklf is significantly suppressed in the blood-less mutants vampire and m683 in which primitive hematopoiesis is impaired. Knockdown of biklf using morpholino-based antisense oligonucleotides (biklf-MO) led to a potent reduction in the number of circulating blood cells and deficiency in hemoglobin production. Consistently, we found that the expression of βe3globin is strongly suppressed in biklf-MO-injected embryos, while gata1 expression is partly inhibited at the 10-somite stage. In addition, analysis of reporter constructs driven by the GATA1 and β-globin promoters showed that Biklf can positively regulate both genes. These results indicate that Biklf is required for erythroid cell differentiation in zebrafish.

Bmp2b and Oep Promote Early Myocardial Differentiation through Their Regulation of gata5

Members of both the bone morphogenetic protein (Bmp) and EGF-CFC families have been implicated in vertebrate myocardial development. Zebrafish swirl (swr) encodes Bmp2b, a member of the Bmp family required for patterning the dorsoventral axis. Zebrafish one-eyed pinhead (oep) encodes a maternally and zygotically expressed member of the EGF-CFC family essential for Nodal signaling. Both swr/bmp2b and oep mutants exhibit severe defects in myocardial development. swr/bmp2b mutants exhibit reduced or absent expression of nkx2.5, an early marker of the myocardial precursors. Embryos lacking zygotic oep (Zoep mutants) display cardia bifida and, as we show here, also display reduced or absent nkx2.5 expression. Recently, we have demonstrated that the zinc finger transcription factor Gata5 is an essential regulator of nkx2.5 expression. In this paper, we investigate the relationships between bmp2b, oep, gata5, and nkx2.5. We show that both swr/bmp2b and Zoep mutants exhibit defects in gata5 expression in the myocardial precursors. Forced expression of gata5 in swr/bmp2b and Zoep mutants restores robust nkx2.5 expression. Moreover, overexpression of gata5 in Zoep mutants restores expression of cmlc1, a myocardial sarcomeric gene. These results indicate that both Bmp2b and Oep regulate gata5 expression in the myocardial precursors, and that Gata5 does not require Bmp2b or Oep to promote early myocardial differentiation. We conclude that Bmp2b and Oep function at least partly through Gata5 to regulate nkx2.5 expression and promote myocardial differentiation. We integrate these and other data to propose a pathway of the molecular events regulating early myocardial differentiation in zebrafish.

Dystrophin and Dp71, two products of the DMD gene, show a different pattern of expression during embryonic development in zebrafish.

Dystrophin, the protein defective in Duchenne muscular dystrophy (DMD), plays a critical role in the formation and maintenance of the neuromuscular junction. In addition to dystrophin, activation of internal promoters of the DMD gene leads to the production of several short products. Among these, Dp71, which consists of the C-terminal domain of dystrophin, is the most abundant product of the gene in non-muscle tissues and brain. In this report, we compare the temporal and regional expression patterns of dystrophin and Dp71 at different stages of embryonic development and during retinal differentiation in zebrafish. The Dp71 transcripts are the earliest to be expressed at 9-10 h post-fertilization (hpf) in the axial mesoderm. As development proceeds, intense Dp71 staining is observed in the notochord, the developing brain, the marginal regions of the somites and the eye primordium. At the completion of retinal differentiation, Dp71 is expressed in the ganglion and inner nuclear layers. Transcripts encoding dystrophin have a slightly later onset of expression, 13-14 hpf, and remain restricted to the transverse myosepta through all the developmental stages examined. The complementary patterns of expression of dystrophin and Dp71 suggest that these two proteins exert different functions during embryonic development in zebrafish.