Expression and activity profiling of the steroidogenic enzymes of glucocorticoid biosynthesis and the fdx1 co-factors in zebrafish.

The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing ferredoxin co-factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase-PCR and whole-mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a translational model with respect to understanding the roles of the genes for glucocorticoid biosynthesis and fdx co-factors during embryonic development and stress, as well as in brain homeostasis and function.

The Zebrafish as Model for Deciphering the Regulatory Architecture of Vertebrate Genomes.

Despite enormous progress to map cis-regulatory modules (CRMs), like enhancers and promoters in genomes, elucidation of the regulatory landscape of the developing embryo remains a challenge. The zebrafish embryo with its experimental virtues has a great potential to contribute to this endeavor. However, so far progress remained behind expectation. We discuss here available methods and their limitations and how the zebrafish embryo could contribute in the future to unravel the wiring of the vertebrate genome.

Distribution of cannabinoid receptor 1 in the CNS of zebrafish.

The cannabinoid receptor 1 (Cb1) mediates the psychoactive effect of marijuana. In mammals, there is abundant evidence advocating the importance of cannabinoid signaling; activation of Cb1 exerts diverse functions, chiefly by its ability to modulate neurotransmission. Thus, much attention has been devoted to understand its role in health and disease and to evaluate its therapeutic potential. Here, we have cloned zebrafish cb1 and investigated its expression in developing and adult zebrafish brain. Sequence analysis showed that there is a high degree of conservation, especially in residues demonstrated to be critical for function in mammals. In situ hybridization revealed that zebrafish cb1 appears first in the preoptic area at 24 hours post-fertilization. Subsequently, transcripts are detected in the dorsal telencephalon, hypothalamus, pretectum and torus longitudinalis. A similar pattern of expression is recapitulated in the adult brain. While cb1 is intensively stained in the medial zone of the dorsal telencephalon, expression elsewhere is weak by comparison. In particular, localization of cb1 in the telencephalic periventricular matrix is suggestive of the involvement of Cb1 in neurogenesis, bearing strong resemblance in terms of expression and function to the proliferative mammalian hippocampal formation. In addition, a gradient-like expression of cb1 is detected in the torus longitudinalis, a teleost specific neural tissue. In relation to dopaminergic neurons in the diencephalic posterior tuberculum (considered to be the teleostean homologue of the mammalian midbrain dopaminergic system), both cb1 and tyrosine hydroxylase-expressing cells occupy non-overlapping domains. However there is evidence that they are co-localized in the caudal zone of the hypothalamus, implying a direct modulation of dopamine release in this particular region. Collectively, our data indicate the propensity of zebrafish cb1 to participate in multiple neurological processes.

Expression of brain subtype creatine kinase in the zebrafish embryo.

Creatine kinases (CK) play crucial roles in intracellular energy transfer. We have isolated a cDNA from zebrafish embryos, which encodes a CK highly related to the mammalian brain subtype creatine kinase (BCK). The bck mRNA is expressed maternally in the zebrafish embryo and transcripts are distributed uniformly in blastula and gastrula stages. Expression becomes restricted to the prechordal plate and the nervous system during subsequent somitogenesis stages. bck transcripts are abundant in primary neurons in the developing central nervous system of the 1-day-old embryo. While some bck expression persists in the hindbrain, expression vanishes in the spinal cord of the 2-day-old embryo. In summary, the expression pattern of bck is highly dynamic and suggests a role for bck during gastrulation and neuronal differentiation.

Identification of nodal signaling targets by array analysis of induced complex probes.

Nodal signaling controls germ layer formation, left-right asymmetry, and patterning of the brain in the vertebrate embryo. Cellular responses to Nodal signals are complex and include changes in gene expression, cell morphology, and migratory behavior. Only little is known about the genes regulated by Nodal signaling. We designed a subtractive screening strategy by using a constitutively active Nodal receptor to identify putative target genes of Nodal signals in the early gastrula of zebrafish embryos. By quantitative analysis of macro-array hybridizations, 132 genes corresponding to 1.4% of genes on the entire macro-array were identified, which were enriched in the Nodal-induced probe pool. These genes encode components of signal transduction pathways, transcription regulators, proteins involved in protein metabolism but also cytoskeletal components and metabolic enzymes, suggesting dramatic changes of cell physiology in gastrula cells in response to Nodal signals.

Molecular cloning and characterization of dystrophin and Dp71, two products of the Duchenne Muscular Dystrophy gene, in zebrafish.

Dystrophin, the protein responsible for Duchenne Muscular Dystrophy (DMD), plays a critical role in the maintenance of the muscle membrane integrity. There are several forms of dystrophin derived from the DMD gene by alternative promoter usage. In addition to full-length dystrophin (Dp427), four shorter transcripts have been identified: Dp260, Dp140, Dp116 and Dp71. The functional role played by the different products of the DMD gene is not yet determined. To get insight into the function of dystrophin and related products, we have investigated the presence of dystrophin in zebrafish. This choice takes advantage of large-scale mutagenesis screens in zebrafish, which have led to the identification of several mutants with motility defects. The identification and characterization of the genes affected by these mutations is likely to provide relevant information for the understanding of the molecular mechanisms of muscle development and function. Two cDNA clones encoding the homologues of dystrophin and Dp71 in zebrafish were identified and characterized. Both transcripts exhibit a high degree of sequence homology with the dystrophin and Dp71 proteins described in higher vertebrates. In addition, three alternative spliced transcripts that occur at the C-terminal end of the zebrafish DMD gene have been identified. These transcripts exhibit different patterns of tissue expression. We have also determined the chromosomal localization of dystrophin on the radiation hybrid map of the zebrafish genome. Our results indicate that the dystrophin gene is localized to linkage group one. Altogether, these results give new insights on the physiological role played by dystrophin and related proteins, and provide new tools for the identification of mutated genes associated with muscle defects in zebrafish.

Identification and expression of zebrafish Iroquois homeobox gene irx1.

Iroquois homeoproteins are prepatterning factors that positively regulate proneural genes and control neurogenesis. We have identified a zebrafish Iroquois gene, irx1, which is highly homologous to Xenopus Xiro1, Gallus c-Irx1 and mouse Irx1. Expression of irx1 was initially detected at the bud stage. By 16 h post-fertilization (hpf), irx1 expression was exclusively limited to the prospective midbrain and hindbrain. By 24 hpf, irx1 expression was clearly detected in the acousticovestibual ganglia, tectum, tegmentum, cerebellum and rhombomere 1 but not in rhombomere 2 or mid-hindbrain boundary.

A crucial component of the endoderm formation pathway, CASANOVA, is encoded by a novel sox-related gene.

casanova (cas) mutant zebrafish embryos lack endoderm and develop cardia bifida. In a substractive screen for Nodal-responsive genes, we isolated an HMG box-containing gene, 10J3, which is expressed in the endoderm. The cas phenotype is rescued by overexpression of 10J3 and can be mimicked by 10J3-directed morpholinos. Furthermore, we identified a mutation within 10J3 coding sequence that cosegregates with the cas phenotype, clearly demonstrating that cas is encoded by 10J3. Epistasis experiments are consistent with an instructive role for cas in endoderm formation downstream of Nodal signals and upstream of sox17. In the absence of cas activity, endoderm progenitors differentiate into mesodermal derivatives. Thus, cas is an HMG box-containing gene involved in the fate decision between endoderm and mesoderm that acts downstream of Nodal signals.

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.

TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish.

General transcription factors TFIIA, B, D, E, F, H, and RNA polymerase II (Pol II) are required for accurate initiation of Pol II transcription. The TATA binding protein (TBP), a subunit of TFIID, is responsible for recognition of the TATA box, a core element shared by a category of class II promoters [1]. Recently, novel TBP-like factors (TLFs) have been described in metazoan organisms [2]. In spite of the numerous in vitro studies describing the general role of TBP in RNA polymerase II (Pol lI) transcription initiation, the precise function of TBP and the newly described TLF is poorly understood in vivo. We inhibited TBP and TLF function in zebrafish embryos to study the role of these factors during zygotic transcription. A dominant-negative variant of TLF mRNA and a TBP morpholino antisense oligo was used to block either TLF or TBP function. Both TBP- or TLF-blocked embryos developed normally until the midblastula stage; however, they then failed to gastrulate. Several zygotic regulatory genes were downregulated by a block in either TBP or TLF function, while others were differentially affected. These results suggest that TBP is not universally required for Pol II transcription in vertebrates and that there is a differential requirement for TBP and TLF during early embryogenesis.

Expression of the anti-dorsalizing morphogenetic protein gene in the zebrafish embryo.

The BMP3 related anti-dorsalizing morphogenetic protein (ADMP) has been proposed to function in the organizer of chick and Xenopus embryos. We report here the cloning and expression pattern of a zebrafish admp gene. The gene is expressed in involuting cells of the embryonic shield, but not in the non-involuting forerunner cells. During gastrulation, admp transcripts are detected in the posterior prechordal plate, in the notochord primordium and in cells of the dorsal blastoderm margin. Expression is also detectable in the neuroectoderm overlying the posterior prechordal plate. Expression persists in the tail bud until the end of somitogenesis while expression in other areas disappears during early somitogenesis stages.

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS.

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.

Phenotypic effects in Xenopus and zebrafish suggest that one-eyed pinhead functions as antagonist of BMP signalling.

Zebrafish one-eyed pinhead (oep) is essential for embryonic axis and dorsal midline formation by promoting Nodal signalling and is thought to act as a permissive factor. Here we describe that oep elicits profound phenotypic effects when overexpressed in Xenopus and zebrafish. In Xenopus, wild-type oep inhibits mesoderm induction, disrupts axis formation and neuralizes animal caps. A secreted Oep dorsoanteriorizes and neuralizes Xenopus embryos indicative of BMP inhibition. In zebrafish, misexpression of smad1 in oep mutant embryos also reveals an interaction of oep with BMP signalling. Furthermore, the phenotypic effect of nodal overexpression can be rescued by coexpression of oep both in Xenopus and zebrafish. Taken together, our results support an interaction between oep and nodal but they suggest also (1) that the role of oep in Nodal signalling may include negative as well as positive regulation, (2) that oep is able to function in an active fashion and (3) that oep exerts a regulatory effect on the BMP signalling pathway.

Zebrafish developmental genetics and central nervous system development.

The central nervous system (CNS) is the most complex tissue of vertebrates. Recently, the zebrafish has emerged as a powerful genetic system for studying early development, and large-scale mutagenic screens for embryonic patterning defects have been accomplished. Mutants isolated in these screens are proving helpful in unravelling the molecular hierarchies involved in the development of the CNS. We review here recent studies in zebrafish which shed light on the genetic pathways involved in induction and regionalization of the CNS.

Characterization of zebrafish smad1, smad2 and smad5: the amino-terminus of smad1 and smad5 is required for specific function in the embryo.

Members of the TGFbeta superfamily of signalling molecules play important roles in mesendoderm induction and dorsoventral patterning of the vertebrate embryo. We cloned three intracellular mediators of TGFbeta signalling, smad1, 2 and 5, from the zebrafish. The three smad genes are expressed ubiquitously at the onset of gastrulation. The pattern of expression becomes progressively restricted during somitogenesis suggesting that at later stages not only the distribution of the TGFbeta signal but also that of the intracellular smad signal transducer determine the regionally restricted effects of TGFbeta signalling. Forced expression of smad1 leads to an expansion of blood cells resembling the phenotype of moderately ventralized zebrafish mutants. In contrast to Smad1, neither Smad2 nor Smad5 caused a detectable effect when expressed as full-length molecules suggesting that these latter two Smads are more dependent on activation by the cognate TGFbeta ligands. N-terminal truncated Smad2 dorsalized embryos, in agreement with a role downstream of dorsalizing TGFbeta members such as Nodals. In contrast to the C-terminal MH2 domain of Smad2, the C-terminal region of Smad1 and Smad5 lead to pleiotropic effects in embryos giving rize to both dorsalized and ventralized characteristics in injected embryos. Analysis of truncated zebrafish Smad1 in Xenopus embryos supports the notion that the C-terminal domain of smad1 is both a hypomorph and antimorph which can act as activator or inhibitor depending on the region of expression in the embryo. These results indicate a specific function of the MH1 domain of Smad1 and 5 for activity of the molecules.

Intronic enhancers control expression of zebrafish sonic hedgehog in floor plate and notochord.

The signalling molecule Sonic hedgehog (Shh) controls a wide range of differentiation processes during vertebrate development. Numerous studies have suggested that the absolute levels as well as correct spatial and temporal expression of shh are critical for its function. To investigate the regulation of shh expression, we have studied the mechanism controlling its spatial expression in the zebrafish. We employed an enhancer screening strategy in zebrafish embryos based on co-injection of putative enhancer sequences with a reporter construct and analysis of mosaic expression in accumulated expression maps. Enhancers were identified in intron 1 and 2 that mediate floor plate and notochord expression. These enhancers also drive notochord and floor plate expression in the mouse embryo strongly suggesting that the mechanisms controlling shh expression in the midline are conserved between zebrafish and mouse. Functional analysis in the zebrafish embryo revealed that the intronic enhancers have a complex organisation. Two activator regions, ar-A and ar-C, were identified in intron 1 and 2, respectively, which mediate mostly notochord and floor plate expression. In contrast, another activating region, ar-B, in intron 1 drives expression in the floor plate. Deletion fine mapping of ar-C delineated three regions of 40 bp to be essential for activity. These regions do not contain binding sites for HNF3beta, the winged helix transcription factor previously implicated in the regulation of shh expression, indicating the presence of novel regulatory mechanisms. A T-box transcription factor-binding site was found in a functionally important region that forms specific complexes with protein extracts from wild-type but not from notochord-deficient mutant embryos.

Ethanol impairs migration of the prechordal plate in the zebrafish embryo.

Exposure of vertebrate embryos to ethanol causes cyclopia, but little is known about the underlying mechanisms of this effect. Here we show that cyclopia can be induced in the zebrafish by a short ethanol treatment during early gastrula stages and is accompanied by loss of gene expression characteristic of the ventral aspects of the fore- and midbrain. Interestingly, defects in the expression of ventral brain markers are linked to impaired migration of the prechordal plate mesoderm indicating that the correct position of the prechordal plate mesoderm under the anterior neural plate in the zebrafish embryo is required for specification of the anterior neural midline. Ethanol-induced cyclopia does not, however, impair the induction of anterior neuroectodermal structures in general. Finally, as genes like goosecoid and islet-1 are expressed in prechordal plate cells in a temporal pattern similar to control embryos despite the ectopic position of expressing cells, it appears that regulation of prechordal plate-specific gene expression is largely independent of the final position of the prechordal plate.

Conversion of zebrafish blastomeres to an endodermal fate by TGF-beta-related signaling.

The endoderm contributes cells to the gut, and participates in the induction and patterning of the vertebrate head and heart. The mechanisms controlling the formation of endoderm are poorly understood. Commitment of endoderm cells occurs at the onset of gastrulation and requires cell interactions; studies in vitro have implicated transforming growth factor Beta (TGF-beta)-related molecules in this process. TARAM-A is a zebrafish receptor kinase that is related to the type I subunit of the TGF-beta receptor, and is expressed in presumptive endomesodermal cells at gastrulation. We provide here evidence for its involvement in endoderm formation in vivo. Activation of TARAM-A was found to drive blastomeres towards an endodermal fate. The induced endoderm behaved ad endogenous endoderm during gastrulation: it migrated in contact with the yolk and expressed endoderm-specific markers. Loss-of-function mutations in the zebrafish one-eyed-pinhead (OEP) gene lead to defects in heart formation, defects of the ventral central nervous system (CNS) and cyclopia. Mutant embryos also lack endoderm and anterior mesoderm. Endoderm formation in oep mutant embryos was found to be restored by the activation of the TARAM-A signaling pathway. Cardiac and ocular defects, but not midline CNS structures, were rescued non-autonomously, demonstrating that endoderm may provide signals that can pattern the eye anlage, and which are distinct form those specifying the ventral midline of the CNS.