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PubMed1995

Differential expression of fork head genes during early Xenopus and zebrafish development.

Intense efforts have been devoted to the identification of genes that are causatively involved in pattern-forming events of invertebrates and vertebrates. Several gene families involved in this process have been identified. Here we focus on the Xenopus fork head domain gene family. One of its members, XFKH1/Pintallavis/XFD1, has been shown previously to be involved in axial formation, and the expression patterns of the other family members discussed below suggest that they too play a major role in the initial steps of patterning and axial organization. In this report, we describe four Xenopus fork head genes (XFKH3, 4, 5, and 6) and analyze the distribution of their transcripts during early development. XFKH3 is expressed in developing somites but not notochord, XFKH4 in forebrain, anterior retina, and neural crest cells, and XFKH5 in a subset of epidermal cells and the neural floor plate. Finally, transcripts of XFKH6 are seen in neural crest-derived cranial ganglia. In addition, we show that at least some of the zebrafish fork head genes might serve a comparable function. Zebrafish zf-FKH1 has a similar expression pattern as Xenopus XFKH1/Pintallavis/XFD1. It is transcribed in the notochord and neural floor plate. The polster or "pillow" also shows very high levels of zf-FKH1 mRNA.

Amino Acid Sequence↗
PubMed2001

Tight transcriptional control of the ETS domain factors Erm and Pea3 by Fgf signaling during early zebrafish development.

Several molecules of the Fibroblast growth factor family have been implicated in the development of the vertebrate brain, but the effectors of these molecules remain largely unknown. Here we study Erm and Pea3, two ETS domain transcription factors, and show that their expression correlates closely with the domains of fgf8 and fgf3 expression. In situ hybridization analysis in wild-type and acerebellar (ace) mutant embryos defective for fgf8 demonstrates a requirement of Fgf8 for normal expression levels of erm and pea3 transcripts in and close to various domains of Fgf8 action, including the prospective midbrain-hindbrain region, the somites, the neural crest, the forebrain, and developing eyes. Morpholino-oligomer-assisted gene knock-down experiments targeted against fgf8 and fgf3 suggest that Fgf3 and Fgf8 are co-regulators of these genes in the early forebrain anlage. Furthermore, inhibition of Fgf signaling by overexpression of sprouty4 or application of the Fgf inhibitor SU5402 leads to a loss of all erm and pea3 expression domains. Conversely, ectopically provided fgf3 mRNA or implanted beads coated with Fgf8 elicit ectopic transcription of erm and pea3. Both activation and loss of transcripts can be observed within short time frames. We conclude that both the transcriptional onset and maintenance of these factors are tightly coupled to Fgf signaling and propose that erm and pea3 transcription is a direct readout of cells to Fgf levels. Given the knowledge that has accumulated on the posttranslational control of ETS domain factors and their combinatorial interactions with other transcription factors, we suggest that the close coupling of erm and pea3 transcription to Fgf signaling might serve to integrate Fgf signaling with other signals to establish refined patterns in embryonic development.

Animals↗
PubMed2004

Expression and mapping of duplicate neuropilin-1 and neuropilin-2 genes in developing zebrafish.

Previously, we described the isolation and characterization of the first zebrafish neuropilin gene, which we now call nrp1a, and found its protein to be a mediator of vascular endothelial growth factor (VEGF)-dependent angiogenesis [Proc. Natl Acad. Sci. USA 99 (2002) 10470]. Subsequently, we have isolated three other full-length neuropilin genes (nrp1b, nrp2a, and nrp2b) and find that they map to independent zebrafish linkage groups. The nrp1s and nrp2s had differential spatio-temporal gene expression profiles with nrp1a being most prominent in the gut, brain, retina, hypochord, motorneurons, fin bud and mandibular cartilage, nrp1b in the brain, dorsal aorta, melanophores, ventral fin, and heart, nrp2a in the brain, retina, heart, and caudal vessels, and nrp2b in the brain, retina, gut, fin bud, melanophores, heart, and caudal vessels. In addition, we have identified an alternatively-spliced transcript of the nrp1b gene (denoted as nrp1b(s)) which is predicted to encode a soluble form of Nrp1b, containing only the a, b, and c extracellular domains. Transcript expression of nrp1b(s) was different from full-length nrp1b transcript, with prominence in the brain, developing mouth, heart, and fin bud. The NRP1s were tested for VEGF-binding ability. Both 125 kDa Nrp1a and 145 kDa Nrp1b bound 125I-labelled VEGFA165. In summary, two nrp1 and two nrp2 genes, with expression patterns similar to higher vertebrates, have been isolated from zebrafish.

Alternative Splicing↗
PubMed2004

Microarray gene expression profiling during the segmentation phase of zebrafish development.

We analyzed 15,512 unique transcripts from wild-type Danio rerio using a long oligonucleotide microarray containing >16,000 65-mers probes. Total RNA was isolated from staged embryos at 2 h intervals over a 24-h period. On average, at any given time point, 27% of the probe set detected corresponding transcripts in embryonic RNA. There were two predominant patterns in the nearly 4000 genes that changed expression in at least one time point during the first 24 hpf. At 12 hpf, we detected 420 up-regulated and 386 down-regulated genes. By 24 hpf, the number of up- and down-regulated genes had increased to 954 and 766, respectively. While the majority of these genes maintained their new level of expression for the duration of the time course, we identified five genes with phasic regulation over the 24-h time course. Two of these genes, germ cell nuclear factor and mesogenin, have been identified as being expressed during gastrulation (5 1/4 to 10 h postfertilization) and subsequently repressed. A cluster containing 36 distinct ribosomal proteins was up-regulated at 12 h, indicating a capability for de novo protein synthesis during and after this stage. Twenty-three muscle-specific genes were up-regulated late during the initial 24 hpf, corresponding to the development and differentiation of the somites.

Animals↗
PubMed2000

Expression of Otx homeodomain proteins induces cell aggregation in developing zebrafish embryos.

In the zebrafish embryo, cells fated to give rise to the rostral brain move in a concerted fashion and retain tissue coherence during morphogenesis. We demonstrate here that Otx proteins have a dramatic effect on cell-cell interactions when expressed ectopically in the zebrafish embryo. Injection of zebrafish Otx1 or Drosophila otd RNAs into a single cell at the 16-cell stage results in aggregation of descendants of the injected cell. The Otx/Otd homeodomain is necessary for aggregation and appears to be sufficient for the effect when substituted for the homeodomain of an unrelated homeodomain protein. When cells containing injected zOtx1 RNA are limited to the area that is normally fated to become the anterior brain and neural retina, the induced aggregates contribute to anterior brain and retina tissues. In many other embryonic regions, which do not express endogenous zOtx1, the aggregates appear to be incompatible with normal development and do not integrate into developing tissues. By using an activatable Otx1-glutocorticoid receptor fusion protein that results in the stimulation of cell association, we demonstrate that cell aggregates can form as a result of Otx1 activity even after gastrulation is completed. Time-lapse analysis of cell movements show that cell aggregation occurs with only a slight inhibition of the rate of convergence. These results suggest that promotion of cell adhesion or mediation of cell repulsion may be one of the normal functions of the Otx proteins in the establishment of the anterior brain.

Animals↗
PubMed2003

Cadherin-mediated differential cell adhesion controls slow muscle cell migration in the developing zebrafish myotome.

Slow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. Here we examine the cellular and molecular basis for slow-twitch muscle cell migration. We show that slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, which correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development.

Animals↗
PubMed1994

Structure and distribution of N-cadherin in developing zebrafish embryos: morphogenetic effects of ectopic over-expression.

N-cadherin cDNA was cloned from a zebrafish embryonic cDNA library. Analysis of the deduced amino acid sequence of this molecule (ZN-cadherin) revealed a high degree of homology to N-cadherins of other species, except that its pre-sequence is considerably shorter. Nevertheless, following transfection into chinese hamster ovary (CHO) cells, the expressed protein was functionally active, namely participated in calcium-dependent intercellular interactions. Moreover, ectopic over-expression of ZN-cadherin, following mRNA microinjection into 2-4 cell embryos, caused microaggregation and uneven segregation of deep cells, resulting in distorted embryos. Developmental Northern and Western blot analyses indicated that both the mRNA and the protein first appear at gastrulation. In-situ hybridization showed that ZN-cadherin mRNA was initially present in all deep cells, and later became restricted to various epithelial and neural tissues. Whole-mount immunostaining indicated that while ZN-cadherin was already present at 50% epiboly, it became associated with cell junctions only 4-5 h later. In developing somites ZN-cadherin expression was prominent but transient. High levels of the protein were detected in epithelial somites and its expression was apparently down regulated concomitantly with the onset of myogenesis.

Amino Acid Sequence↗
PubMed2006

Epithelial mitochondria-rich cells and associated innervation in adult and developing zebrafish.

Studies of ion regulation by mitochondria-rich cells (MRCs) of transport epithelia in fish have revealed many processes by which ion homeostasis is achieved. However, the control of these mechanisms and, particularly, the extent of nervous system involvement are not completely understood. We characterized the potential innervation of MRCs in various gill and extrabranchial tissues involved in ion transport in the model vertebrate the zebrafish. Confocal and conventional microscopy of whole-mount preparations were combined with immunofluorescence techniques to label MRCs with antibodies against a subunit of the enzyme Na(+)/K(+)-ATPase and nerve fibers with a zebrafish neuronal marker, zn-12. MRCs of the gill filaments were identified by their morphology and migration out to the lamellae in response to ion-poor water acclimation. Gill MRCs were intimately associated with nerve fibers originating from outside the filaments. MRCs of the opercular epithelium resembled those of the gill and were also located adjacent to nerve fibers. Mitochondria-rich "pseudobranch cells" were identified in the pseudobranch by immunofluorescence and labeling of dissociated cells with the mitochondrial marker DASPEI. Pseudobranch MRCs resembled gill MRCs and received innervation from a dense network of nerve fibers. In larvae, MRCs were distributed across the surface of the skin. These cells were situated among a dense network of varicose nerve fibers, and some MRCs of the skin displayed extensive cytoplasmic processes. Evidence is presented suggestive of widespread association of MRCs with the nervous system in transport epithelia and the neural control of MRC-mediated ion regulation in teleost fish.

Animals↗
PubMed2002

Characterization of myelination in the developing zebrafish.

Myelination, the process by which glial cells ensheath and electrically insulate axons, has been investigated intensely. Nevertheless, knowledge of how myelination is regulated or how myelinating cells communicate with neurons is still incomplete. As a prelude to genetic analyses of these processes, we have identified zebrafish orthologues of genes encoding major myelin proteins and have characterized myelination in the larval zebrafish. Expression of genes corresponding to proteolipid protein (PLP/DM20), myelin protein zero (P0), and myelin basic protein (MBP) is detected at 2 days postfertilization (dpf), first in the ventral hindbrain, close to the midline. During the next 8 days, expression spreads rostrally to the midbrain and optic nerve, and caudally to the spinal cord. DM20 is expressed in the CNS only, while MBP transcripts are detected both in the CNS and in Schwann cells of the lateral line, cranial nerves, and spinal motor nerves. Unlike its closest homologue, trout IP1, zebrafish P0 transcripts were restricted to the CNS. Ultrastructurally, the expression of myelin genes correlated well with myelination, although myelination showed a temporal lag. Myelinated axons were first detected at 4 dpf in the ventral hindbrain, where they were loosely wrapped by processes of glia cells. By 7 dpf, bundles of heavily myelinated axons were observed in the same region. Axons in the lateral line and optic nerves were also surrounded by compact myelin. Conservation in gene expression patterns and the early appearance of myelinated axons, support using the zebrafish to dissect the process of myelination by a genetic approach.

Amino Acid Sequence↗
PubMed2000

Heart and gut chiralities are controlled independently from initial heart position in the developing zebrafish.

A fundamental problem in developmental biology is how left-right (LR) asymmetry is generated, both on the whole organism level and at the level of an individual organ or structure. To investigate the relationship of organ sidedness to organ chirality, we examined 12 zebrafish mutants for initial heart tube position and later heart looping direction (chirality). Anomalous initial heart position was found in seven mutants, which also demonstrated loss of normal LR asymmetry in lateral plate mesoderm (LPM) antivin/lefty-1 and Pitx2 expression. Those with a relatively normal notochord (cyc(b16), din, and spt) displayed a predictive correlation between initial heart position and heart chirality, whereas initial heart position and heart chirality were independently randomized in those with a defective notochord (flh, boz, ntl, and mom). The predictability of heart chirality in spt, din, and b16 embryos, even in the absence of normal antivin/lefty-1 and Pitx2 expression, strongly suggests that heart chirality is controlled by a process distinct from that which controls appropriate left-sided LPM expression of antivin-Pitx2 signaling pathway molecules. In addition, there was correlation of initial heart position with gut chirality (and also between heart chirality and gut chirality) in the first class of mutants with normal notochord, but not in the second class, which appears to model human heterotaxy syndrome.

Animals↗
PubMed1999

Dicistronic Gene Expression in Developing Zebrafish.

Internal ribosome entry sites (IRESs) allow ribosomal access to messenger RNA without a requirement for cap recognition and subsequent scanning to an initiator AUG. Hence, IRESs have been adapted into dicistronic vectors for the expression of more than one gene from a single mRNA. Dicistronic vectors have been used for many applications in mammalian tissue culture and transgenesis. However, whether the IRESs from mammalian viruses function without temporal or spatial restrictions in nonmammalian organisms like zebra fish (Danio rerio) is unknown. Therefore, we have examined the expression capabilities of the encephalomyocarditis virus (EMCV) IRES during zebrafish embryogenesis. We determined that the EMCV IRES was sufficient to permit detectable expression of several second cistron reporters during zebrafish embryogenesis, including luciferase and green fluorescent protein. This suggests that our dicistronic vectors are suitable for general use in any vertebrate system, from fish to humans.

Journal Article↗
PubMed2004

Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio).

This study investigated whether gonadal sex differentiation of zebrafish (Danio rerio) is susceptible to compounds that interfere with cytochrome P450 aromatase (P450arom). Treatment of zebrafish during the period of gonadal differentiation with either the non-steroidal aromatase inhibitor fadrozole or 17alpha-methyltestosterone (MT) changed gonad morphological differentiation and altered the pattern of P450arom gene (CYP19) expression. Application of fadrozole (500 microg/g of food) between days 35 and 71 post-fertilisation (pf) resulted in 100% masculinisation, i.e. the gonads of all individuals examined ( n = 40) showed testicular morphology. At the same time, fadrozole treatment suppressed gonadal CYP19A (gonad-derived CYP19 gene) mRNA expression, as assessed by means of semi-quantitative RT-PCR. After termination of fadrozole treatment at day 71 pf and subsequent rearing of zebrafish under control conditions until 161 days pf, the gonads of all individuals still displayed testicular morphology. Gonadal CYP19A expression, however, showed a dimorphic pattern, with 14 out of 22 individuals having low CYP19A mRNA levels similar to those found in testes of control fish, while eight fish showed high, ovary-like levels of gonadal CYP19A mRNA. MT treatment (10 microg/l) during the period of gonadal differentiation (days 35 to 71 pf) resulted in phenological feminisation, i.e., all fish examined (n = 28) showed an ovarian gonadal morphology. While gonadal CYP19A expression was suppressed compared to control fish, cerebral CYP19B (brain-derived CYP19 gene) mRNA expression was increased in 71-day-old MT-treated fish. The results from this study provide evidence that exogenous compounds interfering with the P450arom system are able to disrupt, even persistently, gonadal sex differentiation of the protogynic zebrafish.

Animals↗
PubMed2003

In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina.

Two-photon excitation microscopy was used to reconstruct cell divisions in living zebrafish embryonic retinas. Contrary to proposed models for vertebrate asymmetric divisions, no apico-basal cell divisions take place in the zebrafish retina during the generation of postmitotic neurons. However, a surprising shift in the orientation of cell division from central-peripheral to circumferential occurs within the plane of the ventricular surface. In the sonic you (syu) and lakritz (lak) mutants, the shift from central-peripheral to circumferential divisions is absent or delayed, correlating with the delay in neuronal differentiation and neurogenesis in these mutants. The reconstructions here show that mitotic cells always remain in contact with the opposite basal surface by means of a thin basal process that can be inherited asymmetrically.

Animals↗
PubMed1998

Restricted expression of the homeobox gene prox 1 in developing zebrafish.

Prox 1 is a vertebrate homeobox gene which is homologous to the Drosophila transcription factor, prospero. We have isolated a prox 1 cDNA from zebrafish, which encodes a protein that has 82%, 84% and 83% amino acid identity with chicken, mouse and human Prox 1, respectively. Antibodies raised against human Prox 1 cross-react with zebrafish Prox 1 and are used here to determine the expression patterns of Prox 1 during zebrafish embryogenesis by whole-mount immunohistochemistry. In the 10-somite embryo, Prox 1 is expressed over the prospective lens placode and over a broad region of epithelium extending from the eye to the otic vesicle. As embryogenesis proceeds, Prox 1 expression in the eye lens becomes intense, and is detected in maturing muscle pioneer cells and superficial muscle cells. In the CNS, Prox 1 is expressed in a stripe along the forebrain-midbrain boundary, in a segmented pattern in the ventral hindbrain, and in selected cells of the ventral spinal cord. Additional sites of Prox 1 expression include the lateral line primordium, the trigeminal ganglia, the otic vesicle and occasional endodermal cells.

Amino Acid Sequence↗
PubMed2000

Anteroposterior patterning is required within segments for somite boundary formation in developing zebrafish.

Somite formation involves the establishment of a segmental prepattern in the presomitic mesoderm, anteroposterior patterning of each segmental primordium and formation of boundaries between adjacent segments. How these events are co-ordinated remains uncertain. In this study, analysis of expression of zebrafish mesp-a reveals that each segment acquires anteroposterior regionalisation when located in the anterior presomitic mesoderm. Thus anteroposterior patterning is occurring after the establishment of a segmental prepattern in the paraxial mesoderm and prior to somite boundary formation. Zebrafish fss(-), bea(-), des(-) and aei(-) embryos all fail to form somites, yet we demonstrate that a segmental prepattern is established in the presomitic mesoderm of all these mutants and hox gene expression shows that overall anteroposterior patterning of the mesoderm is also normal. However, analysis of various molecular markers reveals that anteroposterior regionalisation within each segment is disturbed in the mutants. In fss(-), there is a loss of anterior segment markers, such that all segments appear posteriorized, whereas in bea(-), des(-) and aei(-), anterior and posterior markers are expressed throughout each segment. Since somite formation is disrupted in these mutants, correct anteroposterior patterning within segments may be a prerequisite for somite boundary formation. In support of this hypothesis, we show that it is possible to rescue boundary formation in fss(-) through the ectopic expression of EphA4, an anterior segment marker, in the paraxial mesoderm. These observations indicate that a key consequence of the anteroposterior regionalisation of segments may be the induction of Eph and ephrin expression at segment interfaces and that Eph/ephrin signalling subsequently contributes to the formation of somite boundaries.

Amino Acid Sequence↗