In the early zebrafish embryo, a ventral to dorsal gradient of bone morphogenetic protein (BMP) activity is established, which is essential for the specification of cell fates along this axis. To visualise and mechanistically determine how this BMP activity gradient forms, we have used a transgenic zebrafish line that expresses monomeric red fluorescent protein (mRFP) under the control of well-characterised BMP responsive elements. We demonstrate that mRFP expression in this line faithfully reports BMP and GDF signalling at both early and late stages of development. Taking advantage of the unstable nature of mRFP transcripts, we use in situ hybridisation to reveal the dynamic spatio-temporal pattern of BMP activity and establish the timing and sequence of events that lead to the formation of the BMP activity gradient. We show that the BMP transcriptional activity gradient is established between 30% and 40% epiboly stages and that it is preceded by graded mRNA expression of the BMP ligands. Both Dharma and FGF signalling contribute to graded bmp transcription during these early stages and it is subsequently maintained through autocrine BMP signalling. We show that BMP2B protein is also expressed in a gradient as early as blastula stages, but do not find any evidence of diffusion of this BMP to generate the BMP transcriptional activity gradient. Thus, in contrast to diffusion/transport-based models of BMP gradient formation in Drosophila, our results indicate that the establishment of the BMP activity gradient in early zebrafish embryos is determined by graded expression of the BMP ligands.

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.

The liver and pancreas arise from common endodermal progenitors. How these distinct cell fates are specified is poorly understood. Here we describe prostaglandin E2 (PGE2) as a regulator of endodermal fate specification during development. Modulating PGE2 activity has opposing effects on liver versus pancreas specification in zebrafish embryos as well as mouse endodermal progenitors. The PGE2 synthetic enzyme cox2a and receptor ep2a are patterned such that cells closest to PGE2 synthesis acquire a liver fate, whereas more distant cells acquire a pancreas fate. PGE2 interacts with the bmp2b pathway to regulate fate specification. At later stages of development, PGE2 acting via the ep4a receptor promotes outgrowth of both the liver and pancreas. PGE2 remains important for adult organ growth, as it modulates liver regeneration. This work provides in vivo evidence that PGE2 may act as a morphogen to regulate cell-fate decisions and outgrowth of the embryonic endodermal anlagen.

Glucocorticoid receptor (GR) signaling is thought to play a key role in embryogenesis, but its specific developmental effects remain unclear. Cortisol is the primary ligand for GR activation in teleosts, and in zebrafish (Danio rerio), the prehatch embryo content of this steroid is of maternal origin. Using early zebrafish developmental stages, we tested the hypothesis that GR signaling is critical for embryo growth and hatching. In zebrafish, maternal GR mRNA is degraded quickly, followed by zygotic synthesis of the receptor. GR protein is widely expressed throughout early development, and we were able to knockdown this protein using morpholino oligonucleotides. This led to a more than 70% reduction in mRNA abundance of matrix metalloproteinase-13 (mmp13), a glucocorticoid-responsive gene. The GR morphants displayed delayed somitogenesis, defects in somite and tail morphogenesis, reduced embryo size, and rarely survived after hatch. This correlated with altered expression of myogenic markers, including myogenin, myostatin, and muscle-specific myosin heavy chain and troponin genes. A key finding was a 70-90% reduction in the mRNA abundance of bone morphogenetic proteins (BMP), including bmp2a, bmp2b, and bmp4 in GR morphants. Bioinformatics analysis confirmed multiple putative glucocorticoid response elements upstream of these BMP genes. GR morphants displayed reduced expression of BMP-modulated genes, including eve1 and pax3. Zebrafish GR mRNA injection rescued the GR morphant phenotype and reversed the disrupted expression of BMP and myogenic genes. Our results for the first time indicate that GR signaling is essential for zebrafish muscle development, and we hypothesize a role for BMP morphogens in this process.

The zebrafish has the potential to regenerate many of its tissues. In this study, we examined caudal fin regeneration in zebrafish that received repeated injuries (fin amputation) at different ages. In zebrafish that received repeated injuries, the potential for caudal fin regeneration, such as tissue growth and the expression of regeneration marker genes (msxb, fgf20a, bmp2b), did not decline in comparison to zebrafish that received only one amputation surgery. The process of initial fin regeneration (e.g., tissue outgrowth and the expression of regeneration marker genes at 7 days post-amputation) did not seem to correlate with age. However, slight differences in fin outgrowth were observed between young and old animals when examined in the late regeneration stages (e.g., 20 and 30 days post-amputation). Together, the data suggest that zebrafish has unlimited regenerative potential in the injured caudal fin.

The dorsal-ventral axis of vertebrate embryos is thought to be specified by a gradient of bone morphogenetic protein (BMP) activity, which, in part, arises through the interaction of dorsally expressed antagonists Chordin and Noggin with the ventralizing BMPs. The zebrafish mercedes(tm305), ogon(m60), and short tail(b180) mutations produce ventralized phenotypes, including expanded bmp2b/4 expression domains. We find that the three mutations are allelic and that the locus they define, renamed ogon (ogo), maps to linkage group 25. The ogo(m60) and ogo(b180) mutations are deficiencies and thus represent null alleles, whereas the ENU-induced allele ogo(tm305) retains partial function. Aspects of the ogo(m60) and ogo(tm305) mutant phenotypes are fully suppressed by overexpression of BMP antagonists. Moreover, swirl(tc300), a null mutation in bmp2b, is epistatic to ogo(m60) mutation, providing further evidence that ogo normally functions in a BMP-dependent manner. Embryonic patterning is highly sensitive to maternal and zygotic ogo gene dosage, especially when the level of zygotic chordin activity is also reduced. However, elimination of the zygotic activity of both genes does not result in a completely ventralized embryo. Thus, while ogo and chordin are required to limit activity of BMPs, additional mechanisms must exist to block these ventralizing signals. We have ruled out zebrafish noggin homologues as candidates for the ogo gene, including a newly identified gene, nog1, which is specifically expressed in the gastrula organizer. The results suggest that ogo encodes an as yet unidentified dorsalizing factor that mediates dorsoventral patterning by directly or indirectly antagonizing BMP activity.

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

We analyzed the interactions between mutations in antagonistic BMP pathway signaling components to examine the roles that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7, lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, we found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, we observed a mutual suppression of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, we found that the absence of Ogon or Chordin antagonism did not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly we observed a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype, when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead we observed an apparent decrease in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly orthologue of chordin, short gastrulation, our paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity.

The zebrafish bozozok (boz) gene encoding a homeodomain protein (also named Dharma/Nieuwkoid) is required during blastula stages for the formation of a complete Spemann-Mangold gastrula organizer and subsequent development of axial mesoderm and anterior neural structures. Expression of bozin the dorsal yolk syncytial layer (YSL) and overlying marginal blastomeres is activated by beta-catenin. Bozozok itself acts as a transcriptional repressor, and promotes organizer formation by directly inhibiting expression of the bmp2b (swirl) gene and by negatively regulating Wnt signaling by an unknown mechanism. boz cooperates with the Nodal-related secreted factors, Cyclops and Squint, in organizer formation. The incomplete organizer in boz mutants is deficient in expression of a number of factors such as Chordin that antagonize Bone morphogenetic proteins (Bmps), and Dickkopf 1, a Wnt antagonist. Conversely, the dorsal blastoderm of boz mutants exhibits ectopic expression of genes normally excluded from the dorsal midline such as wnt8 or tbx6. boz specifies the formation of anterior neuroectoderm by regulating Bmp and Wnt pathways in a fashion consistent with Nieuwkoop's two-step neural patterning model. boz promotes neural induction by limiting the anti-neuralizing activity of Bmp morphogens. In addition, by negative regulation of Wnt signaling, boz limits posteriorization of neuroectoderm. bozozok chordino double mutants exhibit a synergistic loss of head and trunk. This synthetic phenotype is due to dramatically increased Bmp signaling and consequent massive accumulation of cells in the tailbud at the expense of dorso-anterior structures. Therefore, boz and din act in overlapping pathways that provide the main mechanism to limit Bmp signaling in the zebrafish gastrula and allow for head and trunk development. Notably, Bozozok appears to function by repressing transcription of target genes such as swr (bmp2b) gene, and as such is the earliest acting repressor that the nascent dorsal axis is using to antagonize ventral influences.

The Nieuwkoop center provides signals essential for the establishment of the dorsal gastrula organizer in vertebrates. Activation of beta-catenin is one of the events in the Nieuwkoop center that lead to activation of dorsal-specific genes during blastula and early gastrula stages. Zebrafish bozozok (boz) mutant embryos have severe defects in axial mesoderm and anterior neuroectoderm. The boz gene is activated in the organizer in response to beta-catenin signaling, and Boz protein has been demonstrated to contribute to organizer formation by repression of ventralizing genes, including bmp2b, vega1, and vega2. Here, we investigate the timing and molecular mechanism by which boz expression is activated in the organizer. We demonstrate that boz is already expressed before midblastula transition (MBT). We further identify high-affinity binding sites for Tcf/Lef1 within the boz promoter region. These sites, together with the finding that beta-catenin induces boz expression, indicate that transcription of boz may be activated directly by beta-catenin/Lef1. We hypothesize that pre-MBT activation of boz may be important to build up a sufficiently strong antagonizing activity against zygotic ventralizing genes activated immediately post-MBT. Thus, the early onset of boz expression may be crucial for organizer establishment in the presence of ubiquitous maternal activators of ventralizing genes.

It was shown in Xenopus and chick that Spemann's organizer activity is regulated through the negative action of Anti-Dorsalizing Morphogenetic Protein (ADMP). We report the characterization and functional properties of admp in zebrafish. admp expression profile is consistent with a role in the organizer, including the tail organizer. We studied admp function through overexpression experiments, with the use of a dominant-negative form (TR-ADMP) and of an antisense morpholino-modified oligonucleotide. Our results indicate that the ADMP pathway causes the restriction of anterior and axial fates and that ADMP, BMP2b, and BMP7 pathways have distinct actions but cooperate in establishing proper dorso-ventral regionalization. This is shown by partial rescue of the dorsalized mutant snailhouse and of the ventralized mutant chordino, upon admp and tr-admp RNA injection, respectively. Moreover, ADMP and BMP7 probably form heterodimers as shown by the ability of TR-ADMP and BMP7 to antagonize each other. We observed that a MYC-tagged ADMP was secreted and detected in the extracellular space, suggesting that admp could act at a distance. Simultaneous local inhibition of bmp function at the blastoderm margin and impairment of ADMP secretion led to the induction of secondary head structures, confirming that the two pathways cooperatively regulate organizer formation and activity.

IGFBP3 is a multi-functional protein that has IGF-dependent and IGF-independent actions in cultured cells. Here we show that the IGF binding domain (IBD), nuclear localization signal (NLS) and transactivation domain (TA) are conserved and functional in zebrafish Igfbp3. The in vivo roles of these domains were investigated by expression of Igfbp3 and its mutants in zebrafish embryos. Igfbp3, and its NLS and TA mutants had equally strong dorsalizing effects. Human IGFBP3 had similar dorsalizing effects in zebrafish embryos. The activities of IBD and IBD+NLS mutants were lower, but they still caused dorsalization. Thus, the IGF-independent action of Igfbp3 is not related to NLS or TA in this in vivo model. We next tested the hypothesis that Igfbp3 exerts its IGF-independent action by affecting Bmp signaling. Co-expression of Igfbp3 with Bmp2b abolished Bmp2b-induced gene expression and inhibited its ventralizing activity. Biochemical assays and in vitro experiments revealed that IGFBP3 bound BMP2 and inhibited BMP2-induced Smad signaling in cultured human cells. In vivo expression of Igfbp3 increased chordin expression in zebrafish embryos by alleviating the negative regulation of Bmp2. The elevated level of Chordin acted together with Igfbp3 to inhibit the actions of Bmp2. Knockdown of Igfbp3 enhanced the ventralized phenotype caused by chordin knockdown. These results suggest that Igfbp3 exerts its IGF-independent actions by antagonizing Bmp signaling and that this mechanism is conserved.

Endocardial and myocardial progenitors originate in distinct regions of the anterior lateral plate mesoderm and migrate to the midline where they coalesce to form the cardiac tube. Endocardial progenitors acquire a molecular identity distinct from other vascular endothelial cells and initiate expression of specific genes such as nfatc1. Yet the molecular pathways and tissue interactions involved in establishing endocardial identity are poorly understood. The endocardium develops in tight association with cardiomyocytes. To test for a potential role of the myocardium in endocardial morphogenesis, we used two different zebrafish models deficient in cardiomyocytes: the hand2 mutant and a myocardial-specific genetic ablation method. We show that in hand2 mutants endocardial progenitors migrate to the midline but fail to assemble into a cardiac cone and do not express markers of differentiated endocardium. Endocardial differentiation defects were rescued by myocardial but not endocardial-specific expression of hand2. In metronidazole-treated myl7:nitroreductase embryos, myocardial cells were targeted for apoptosis, which resulted in the loss of endocardial nfatc1 expression. However, endocardial cells were present and retained expression of general vascular endothelial markers. We further identified bone morphogenetic protein (BMP) as a candidate myocardium-derived signal required for endocardial differentiation. Chemical and genetic inhibition of BMP signaling at the tailbud stage resulted in severe inhibition of endocardial differentiation while there was little effect on myocardial development. Heat-shock-induced bmp2b expression rescued endocardial nfatc1 expression in hand2 mutants and in myocardium-depleted embryos. Our results indicate that the myocardium is crucial for endocardial morphogenesis and differentiation, and identify BMP as a signal involved in endocardial differentiation.

Timing and pattern of expression of ten candidate segmentation genes or gene pairs were reviewed or examined in developing median fins of late-stage zebrafish, Danio rerio. We found a general correspondence in timing and pattern of expression between zebrafish fin radial segmentation and tetrapod joint development, suggesting that molecular mechanisms underlying radial segmentation have been conserved over 400 million years of evolution. Gene co-expression during segmentation (5.5-6.5 mm SL) is similar between tetrapods and zebrafish: bmp2b, bmp4, chordin, and gdf5 in interradial mesenchyme and ZS; bapx1, col2a1, noggin3, and sox9a in chondrocytes. Surprisingly, wnt9a is not expressed in the developing median fins, though wnt9b is detected. In contrast to all other candidate segmentation genes we examined, bapx1 is not expressed in the caudal fin, which does not segment. Together, these data suggest a scenario of gene interactions underlying radial segmentation based on the patterns and timing of candidate gene expression.

BACKGROUND

The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.

RESULTS

We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/-)) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.

CONCLUSIONS

Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

Bone morphogenetic protein (Bmp) signaling has been shown to play important roles in tooth development at virtually all stages from initiation to hard tissue formation. The specific ligands involved in these processes have not been directly tested by loss-of-function experiments, however. We used morpholino antisense oligonucleotides and mutant analysis in the zebrafish to reduce or eliminate the function of bmp2b and bmp4, two ligands known to be expressed in zebrafish teeth and whose mammalian orthologs are thought to play important roles in tooth development. Surprisingly, we found that elimination of function of these two genes singly and in combination did not prevent the formation of mature, attached teeth. The mostly likely explanation for this result is functional redundancy with other Bmp ligands, which may differ between the zebrafish and the mouse.

The vertebrate Vox/Vent family of transcription factors plays a crucial role in the establishment of the dorsoventral (DV) axis, by repressing organizer genes such as bozozok/dharma, goosecoid, and chordino. In Danio rerio (zebrafish), members of the vox/vent gene family (vox/vega1, vent/vega2, and ved) are thought to share expression patterns and functional properties. Bringing novel insights in the differential activity of the zebrafish vox/vent genes, we propose a critical role for the ved gene in DV patterning of vertebrate embryos. ved is not only expressed as a maternal gene, but it also appears to function as a repressor of dorsal factors involved in organizer formation. At early- and mid-gastrula stage, ved appears to be finely controlled by antagonist crosstalks in a complex regulatory network, involving gradients of bone morphogenetic protein (BMP) activity, dorsal factors, and vox/vent family members. We show that ved transcripts are ventrally restricted by BMP factors such as bmp2b, bmp7, smad5, and alk8, and by dorsal factors (chd and gsc). Alteration of ved expression in both vox and vent deletion mutants and vox and vent mRNAs-injected embryos, suggests that vox and vent function downstream of BMP signaling to negatively regulate ved expression. This inhibitory role is emphasized by a vox and vent redundant activity, compared with single gene effects.

BACKGROUND

Heparan sulfate (HS) is an ubiquitous component of the extracellular matrix that binds and modulates the activity of growth factors, cytokines and proteases. Animals with defective HS biosynthesis display major developmental abnormalities however the processes that are affected remain to be defined. D-glucuronyl-C5-epimerase (Glce) is a key HS chain modifying enzyme that catalyses the conversion of glucuronic acid into iduronic acid, a biosynthetic step that enhances HS biological activity. In this study the role of Glce during early zebrafish development has been investigated.

RESULTS

Two Glce-like proteins (Glce-A and -B) are expressed in zebrafish at all times. They are the products of two distinct genes that, based on chromosomal mapping, are both orthologues of the same single human gene. Transcripts for both proteins were detected in fertilized zebrafish embryos prior to the onset of zygotic transcription indicating their maternal origin. At later developmental stages the epimerases are expressed widely throughout gastrulation and then become restricted to the hindbrain at 24 h post-fertilization. By monitoring the expression of well characterized marker genes during gastrulation, we have found that misexpression of Glce causes a dose-dependent expansion of the ventral structures, whereas protein knockdown using targeted antisense morpholino oligonucleotides promotes axis dorsalization. The ventralizing activity of Bmp2b is enhanced by Glce overexpression whereas Glce knockdown impairs Bmp2b activity.

CONCLUSIONS

Glce activity is an important determinant of of dorso-ventral axis formation and patterning in zebrafish. In particular Glce acts during gastrulation by affecting Bmp-mediated cell specification. The results obtained further corroborate the concept that HS encodes information that affect morphogenesis during early vertebrate development.

It is widely accepted that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils is cardiotoxic. Although PAHs toxic endpoints show strong correlation with the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, and is thought to be a potent inducer of cytochrome P4501A, the action mechanism of PAHs on vertebrate cardiovascular development and disease is unclear. Herein, we address the cardiac developmental effects of exposure to the weak AhR agonist pyrene on the early life-stages of zebrafish. Embryos were exposed to 0, 0.05, 0.5, 5, and 50 nmol/L pyrene up to 72h post-fertilization (hpf). Pyrene-treated embryos showed dose-dependent heart abnormalities, such as pericardial edema and cardiac looping defects. Changes in AhR1a, AhR1b, AhR2, and Cyp1A expression were assessed by real-time RT-PCR. The results showed that low-level pyrene failed to alter these genes expression. However, the homeodomain transcription factor Nkx2.5, which plays an essential role in the development of the cardiovascular system, was down-regulated in a dose-dependent manner by pyrene exposure. The bone morphogenetic protein 2b (Bmp2b), which has been identified as the upstream gene of Nkx2.5, also was inhibited in a dose-dependent manner after treatment with pyrene. Taken together, these data indicated that embryonic exposure of zebrafish to low-level environmental pyrene disrupt normal cardiac development and alter expression of defective cardiac differentiation related genes.

Bone morphogenetic proteins (Bmps) control dorsoventral patterning of vertebrate embryos through the establishment of a ventrodorsal gradient of the activated downstream cytoplasmic effectors Smad1/5/8. Some Bmp ligands are expressed in the ventral and lateral regions and in the organizer during gastrulation of the embryo, but it remains unclear how organizer-derived Bmps contribute to total Bmp ligand levels and to the establishment of the correct phospho-Smad1/5/8 gradient along the ventrodorsal axis. Here we demonstrate that interference with organizer-specific Bmp2b signalling in zebrafish embryos alters the phospho-Smad1/5/8 gradient throughout the ventrodorsal axis, elevates the levels of the Bmp antagonist Chordin and dorsalizes the embryos. Moreover, we show that organizer-derived Bmp2b represses chordin transcription in the organizer and contributes to the control of the Chordin gradient. Combining these experimental results with simulations of Bmp's reaction-diffusion dynamics, our data indicate that organizer-produced Bmp2b is required for the establishment and maintenance of a Bmp activity gradient and for appropriate embryonic dorsoventral patterning during gastrulation.

The Nieuwkoop center is the earliest signaling center during dorsal-ventral pattern formation in amphibian embryos and has been implied to function in induction of the Spemann-Mangold organizer. In zebrafish, Nieuwkoop-center-like activity resides in the dorsal yolk syncytial layer (YSL) at the interface of the vegetal yolk cell and the blastoderm. hex homologs are expressed in the anterior endomesoderm in frogs (Xhex), the anterior visceral endoderm in mice, and the dorsal YSL in zebrafish (hhex). Here, we investigate the control of hhex expression in the YSL. We demonstrate that bozozok (boz) is absolutely required for early hhex expression, while overexpression of boz causes ectopic hhex expression. Activation of Wnt/beta-catenin signaling by LiCl induces hhex expression in wild-type YSL but not in boz mutant embryos, revealing that boz activity is required downstream of Wnt/beta-catenin signaling for hhex expression. Further, we show that the boz-mediated induction of hhex is independent of the Boz-mediated repression of bmp2b. Our data reveal that repressive effects of both Vega1 and Vega2 may be responsible for the exclusion of hhex expression from the ventral and lateral parts of the YSL. In summary, zebrafish hhex appears to be activated by Wnt/beta-catenin in the dorsal YSL, where Boz acts in a permissive way to limit repression of hhex by Vega1 and Vega2.

Using the fluorescent dyes calcein and alcian blue, we stained the F3 generation of chemically (ENU) mutagenized zebrafish embryos and larvae, and screened for mutants with defects in bone development. We identified a mutant line, bone calcification slow (bcs), which showed delayed axial vertebra calcification during development. Before 4-5 days post-fertilization (dpf), the bcs embryos did not display obvious abnormalities in bone development (i.e., normal number, size and shape of cartilage and vertebrae). At 5-6 dpf, when vertebrae calcification starts, bcs embryos began to show defects. At 7 dpf, for example, in most of the bcs embryos examined, calcein staining revealed no signals of vertebrae mineralization, whereas during the same developmental stages, 2-14 mineralized vertebrae were observed in wild-type animals. Decreases in the number of calcified vertebrae were also observed in bcs mutants when examined at 9 and 11 dpf, respectively. Interestingly, by 13 dpf the defects in bcs mutants were no longer evident. There were no significant differences in the number of calcified vertebrae between wild-type and mutant animals. We examined the expression of bone development marker genes (e.g., Sox9b, Bmp2b, and Cyp26b1, which play important roles in bone formation and calcification). In mutant fish, we observed slight increases in Sox9b expression, no alterations in Bmp2b expression, but significant increases in Cyp26b1 expression. Together, the data suggest that bcs delays axial skeletal calcification, but does not affect bone formation and maturation.

Recent studies on the morphogenesis of the fins of Danio rerio (zebrafish) during development and regeneration suggest that a number of inductive signals involved in the process are similar to some of those that affect bone and cartilage differentiation in mammals and humans. Akimenko et al. (2002) has shown that bone morphogenetic protein-2b (BMP2b) is involved in the induction of dermal bone differentiation during fin regeneration. Many other groups have also shown that molecules from the transforming growth factor-beta superfamily (TGFb), including BMP2, are effective in promoting chondrogenesis and osteogenesis in vivo in higher vertebrates, including humans. In the present study, we review the state of the art of this topic by a comparative analysis of skeletal tissue development, regeneration and renewal processes in tetrapods, and fin regeneration in fishes. A general conclusion of this study states that lepidotrichia is a special skeletal tissue different to cartilage, bone, enamel, or dentine in fishes, according to its extracellular matrix (ECM) composition. However, the empirical analysis of inducing signals of skeletal tissues in fishes and tetrapods suggests that lepidotrichia is different to any responding features with main skeletal tissues. A number of new inductive molecules are arising from fin development and regeneration studies that might establish an empirical basis for further molecular approaches to mammal skeletal tissues differentiation. Despite the tissue dissimilarity, this empirical evidence might finally lead to clinical applications to skeletal disorders in humans.