Distinct roles of Shh and Fgf signaling in regulating cell proliferation during zebrafish pectoral fin development.

BACKGROUND: Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh) and Fibroblast Growth Factor (Fgf) families of secreted signaling proteins. Sonic hedgehog (Shh) directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb. RESULTS: We used the genetic and pharmacological amenability of the zebrafish model system to dissect the relative importance of Shh and Fgf signaling in regulating proliferation during development of the pectoral fin buds. In zebrafish mutants disrupting the shh gene, proliferation in the pectoral fin buds is initially normal, but later is strongly reduced. Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants. Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both. In contrast, even short periods of pharmacological inhibition of Fgf signaling lead to strong disruption of proliferation in the fin buds, without affecting Shh signaling. To directly test the ability of Fgf signaling to regulate proliferation in the absence of Shh signaling, we implanted beads soaked with Fgf protein into shh mutant fin buds. We find that Fgf-soaked beads rescue proliferation in the pectoral find buds of shh mutants, indicating that Fgf signaling is sufficient to direct proliferation in zebrafish fin buds in the absence of Shh. CONCLUSION: Previous studies have shown that both Shh and Fgf signaling are crucial for outgrowth of the vertebrate limb. The results presented here show that the role of Shh in this process is indirect, and is mediated by its effect on Fgf signaling. By contrast, the activity of the Fgf pathway affects proliferation directly and independently of its effect on Shh. These results show that Fgf signaling is of primary importance in directing outgrowth of the limb bud, and clarify the role of the Shh-Fgf feedback loop in regulating proliferation.

Mutation of zebrafish caf-1b results in S phase arrest, defective differentiation, and p53-mediated apoptosis during organogenesis.

The cell cycle of multicellular organisms must be tightly coordinated with organogenesis and differentiation. Experiments done in vitro have identified chromatin assembly factor 1 (CAF-1) as a protein complex promoting chromatin assembly during DNA replication, but the in vivo role of CAF-1 in multicellular animals is still poorly understood. Here we describe the characterization of a zebrafish mutant disrupting CAF-1b activity, and show that it leads to defective cell cycle progression and differentiation in several organs, including the retina, optic tectum, pectoral fins, and head skeleton. Retinal precursor cells mutant for caf-1b arrest in S phase and undergo p53-mediated apoptosis. While p53 deficiency is able to rescue apoptosis in caf-1b mutants, it fails to rescue differentiation, indicating that CAF-1 activity is essential for differentiation in these organs. In addition, we also show that regulation of caf-1b expression in the retina depends on a group of genes that regulate the switch from proliferation to differentiation.

Reciprocal endoderm-mesoderm interactions mediated by fgf24 and fgf10 govern pancreas development.

In amniotes, the pancreatic mesenchyme plays a crucial role in pancreatic epithelium growth, notably through the secretion of fibroblast growth factors. However, the factors involved in the formation of the pancreatic mesenchyme are still largely unknown. In this study, we characterize, in zebrafish embryos, the pancreatic lateral plate mesoderm, which is located adjacent to the ventral pancreatic bud and is essential for its specification and growth. We firstly show that the endoderm, by expressing the fgf24 gene at early stages, triggers the patterning of the pancreatic lateral plate mesoderm. Based on the expression of isl1, fgf10 and meis genes, this tissue is analogous to the murine pancreatic mesenchyme. Secondly, Fgf10 acts redundantly with Fgf24 in the pancreatic lateral plate mesoderm and they are both required to specify the ventral pancreas. Our results unveil sequential signaling between the endoderm and mesoderm that is critical for the specification and growth of the ventral pancreas, and explain why the zebrafish ventral pancreatic bud generates the whole exocrine tissue.

Glucocorticoids play a key role in circadian cell cycle rhythms.

Clock output pathways play a pivotal role by relaying timing information from the circadian clock to a diversity of physiological systems. Both cell-autonomous and systemic mechanisms have been implicated as clock outputs; however, the relative importance and interplay between these mechanisms are poorly understood. The cell cycle represents a highly conserved regulatory target of the circadian timing system. Previously, we have demonstrated that in zebrafish, the circadian clock has the capacity to generate daily rhythms of S phase by a cell-autonomous mechanism in vitro. Here, by studying a panel of zebrafish mutants, we reveal that the pituitary-adrenal axis also plays an essential role in establishing these rhythms in the whole animal. Mutants with a reduction or a complete absence of corticotrope pituitary cells show attenuated cell-proliferation rhythms, whereas expression of circadian clock genes is not affected. We show that the corticotrope deficiency is associated with reduced cortisol levels, implicating glucocorticoids as a component of a systemic signaling pathway required for circadian cell cycle rhythmicity. Strikingly, high-amplitude rhythms can be rescued by exposing mutant larvae to a tonic concentration of a glucocorticoid agonist. Our work suggests that cell-autonomous clock mechanisms are not sufficient to establish circadian cell cycle rhythms at the whole-animal level. Instead, they act in concert with a systemic signaling environment of which glucocorticoids are an essential part.

Fgf10 regulates hepatopancreatic ductal system patterning and differentiation.

During organogenesis, the foregut endoderm gives rise to the many different cell types that comprise the hepatopancreatic system, including hepatic, pancreatic and gallbladder cells, as well as the epithelial cells of the hepatopancreatic ductal system that connects these organs together and with the intestine. However, the mechanisms responsible for demarcating ducts versus organs are poorly understood. Here, we show that Fgf10 signaling from the adjacent mesenchyme is responsible for refining the boundaries between the hepatopancreatic duct and organs. In zebrafish fgf10 mutants, the hepatopancreatic ductal epithelium is severely dysmorphic, and cells of the hepatopancreatic ductal system and adjacent intestine misdifferentiate toward hepatic and pancreatic fates. Furthermore, Fgf10 also functions to prevent the differentiation of the proximal pancreas and liver into hepatic and pancreatic cells, respectively. These data shed light onto how the multipotent cells of the foregut endoderm, and subsequently those of the hepatopancreatic duct, are directed toward different organ fates.

Prdm1 acts downstream of a sequential RA, Wnt and Fgf signaling cascade during zebrafish forelimb induction.

Vertebrate limb induction is triggered in the lateral plate mesoderm (LPM) by a cascade of signaling events originating in the axial mesoderm. While it is known that Fgf, Wnt and retinoic acid (RA) signals are involved in this cascade, their precise regulatory hierarchy has not been determined in any species. tbx5 is the earliest gene expressed in the limb bud mesenchyme. Recently, another transcription factor, Prdm1, has been shown to be crucial for zebrafish forelimb development. Here, we show that Prdm1 is downstream of RA, Wnt2b and Tbx5 activity. We find that RA activity, but not Fgf signaling, is necessary for wnt2b expression. Fgf signaling is required for prdm1 expression in the fin bud, but is not necessary for the initiation of tbx5 expression. We propose a model in which RA signaling from the somitic mesoderm leads to activation of wnt2b expression in the intermediate mesoderm, which then signals to the LPM to trigger tbx5 expression. tbx5 is required for Fgf signaling in the limb bud leading to activation of prdm1 expression, which in turn is required for downstream activation of fgf10 expression.

Zebrafish Trap230/Med12 is required as a coactivator for Sox9-dependent neural crest, cartilage and ear development.

The vertebrate Sox9 transcription factor directs the development of neural crest, otic placodes, cartilage and bone. In zebrafish, there are two Sox9 orthologs, Sox9a and Sox9b, which together perform the functions of the single-copy tetrapod Sox9. In a large-scale genetic screen, we have identified a novel zebrafish mutant that strongly resembles the Sox9a/Sox9b double mutant phenotype. We show that this mutation disrupts the zebrafish Trap230/Med12 ortholog, a member of the Mediator complex. Mediator is a coactivator complex transducing the interaction of DNA-binding transcription factors with RNA polymerase II, and our results reveal a critical function of the Trap230 subunit as a coactivator for Sox9.

HSPG synthesis by zebrafish Ext2 and Extl3 is required for Fgf10 signalling during limb development.

Heparan sulphate proteoglycans (HSPGs) are known to be crucial for signalling by the secreted Wnt, Hedgehog, Bmp and Fgf proteins during invertebrate development. However, relatively little is known about their effect on developmental signalling in vertebrates. Here, we report the analysis of daedalus, a novel zebrafish pectoral fin mutant. Positional cloning identified fgf10 as the gene disrupted in daedalus. We find that fgf10 mutants strongly resemble zebrafish ext2 and extl3 mutants, which encode glycosyltransferases required for heparan sulphate biosynthesis. This suggests that HSPGs are crucial for Fgf10 signalling during limb development. Consistent with this proposal, we observe a strong genetic interaction between fgf10 and extl3 mutants. Furthermore, application of Fgf10 protein can rescue target gene activation in fgf10, but not in ext2 or extl3 mutants. By contrast, application of Fgf4 protein can activate target genes in both ext2 and extl3 mutants, indicating that ext2 and extl3 are differentially required for Fgf10, but not Fgf4, signalling during limb development. This reveals an unexpected specificity of HSPGs in regulating distinct vertebrate Fgfs.

Hedgehogs as negative regulators of the cell cycle.

During the development of multicellular animals, cell proliferation must be precisely controlled, as deregulated proliferation can lead to overgrowth and cancer. In addition, proliferation must be tightly integrated with pattern formation and differentiation to generate the required number of cells in the right organs, and at the right time. All major signaling pathways employed during embryogenesis have been implicated in cell cycle regulation, indicating that no single pathway has been dedicated to this task. Also, the precise role of a particular signaling pathway in regulating proliferation is highly dependent on the cellular context, and may have opposite effects on cell cycle progression in different cells and tissues. The Hedgehog (Hh) family of signaling proteins is known to control both differentiation and proliferation during development. So far, studies addressing the effect of Hh signaling on proliferation have shown it to have a stimulatory effect on cell cycle progression. Here we review several recent studies indicating that Hh signaling can also have the opposite effect, directing cell cycle exit in a number of cell types in vertebrate and in invertebrate embryos.

Shh directs cell-cycle exit by activating p57Kip2 in the zebrafish retina.

The Hedgehog (Hh) family of signalling proteins control both differentiation and proliferation during animal development. Previous studies have shown that Hh signalling has a stimulatory effect on the cell cycle in several organs by controlling core cell-cycle components. Here, we show that Sonic hedgehog (Shh) signalling has the opposite effect in the zebrafish retina, where it leads to cell-cycle exit, and that this is mediated by transcriptional activation of the cyclin kinase inhibitor p57Kip2. The loss of p57Kip2 activity strongly resembles the Shh mutant eye phenotype, and overexpression of p57Kip2 rescues cell-cycle exit in Shh mutants, indicating that p57Kip2 is both necessary and sufficient to mediate Shh-induced cell-cycle exit in the retina. These findings raise the possibility that stimulation of cell-cycle exit through regulation of core cell-cycle components may be part of a general mechanism required for Hh-directed differentiation.

Histone deacetylase 1 is required for cell cycle exit and differentiation in the zebrafish retina.

Histone acetylation is an important epigenetic mechanism for the control of eukaryotic transcription. The histone deacetylase 1 (HDAC1) gene has been implicated in controlling the transcription of core cell cycle regulators, but the in vivo role of HDACs in cell cycle regulation is still poorly understood. Loss of HDAC1 activity causes underproliferation in several contexts during vertebrate development. In contrast, we show here that HDAC1 has the opposite effect in the zebrafish visual system, where loss of HDAC1 activity leads to failure of cells to exit the cell cycle in the retina and in the optic stalk. The effect of HDAC1 on cell cycle exit is cell-autonomous, and loss of HDAC1 in the retina leads to up-regulation of cyclin D and E transcripts. These results demonstrate that the in vivo role of HDAC1 in regulating cell cycle progression is region-specific, as HDAC1 promotes cell cycle exit in the retina but stimulates proliferation in other cellular contexts.

Monorail/Foxa2 regulates floorplate differentiation and specification of oligodendrocytes, serotonergic raphé neurones and cranial motoneurones.

In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphenucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.

Sonic hedgehog, secreted by amacrine cells, acts as a short-range signal to direct differentiation and lamination in the zebrafish retina.

Neurogenesis in the zebrafish retina occurs in several waves of differentiation. The first neurogenic wave generates ganglion cells and depends on hedgehog (hh) signaling activity. Using transgenic zebrafish embryos that express GFP under the control of the sonic hedgehog (shh) promoter, we imaged the differentiation wave in the retina and show that, in addition to the wave in the ganglion cell layer, shh expression also spreads in the inner nuclear layer. This second wave generates amacrine cells expressing shh, and although it overlaps temporally with the first wave, it does not depend on it, as it occurs in the absence of ganglion cells. We also show that differentiation of cell types found in the inner and outer nuclear layers, as well as lamination of the retina, depends on shh. By performing mosaic analysis, we demonstrate that Shh directs these events as a short-range signal within the neural retina.

The role of hedgehog signaling in the development of the zebrafish visual system.

The vertebrate visual system is a region of the nervous system that is characterized by relative simplicity, and its development has hence been studied intensively, to serve as a paradigm for the rest of the central nervous system. The zebrafish model organism offers an impressive array of tools to dissect this process experimentally, and in recent years has helped to significantly deepen our understanding of the development of the visual system. A number of these studies have focused on the role of the Hedgehog family of secreted signaling molecules in eye development, and this is the main topic of this review. Hedgehog signaling plays an important role in all major steps of visual system development, starting with the regionalization of the eye primordium into proximal and distal territories, continuing with the control of cellular differentiation in the retina, and ending with the guidance of axonal projections from the retina to the optic centers of the brain.

The zebrafish fgf24 mutant identifies an additional level of Fgf signaling involved in vertebrate forelimb initiation.

The development of vertebrate limb buds is triggered in the lateral plate mesoderm by a cascade of genes, including members of the Fgf and Wnt families, as well as the transcription factor tbx5. Fgf8, which is expressed in the intermediate mesoderm, is thought to initiate forelimb formation by activating wnt2b, which then induces the expression of tbx5 in the adjacent lateral plate mesoderm. Tbx5, in turn, is required for the activation of fgf10, which relays the limb inducing signal to the overlying ectoderm. We show that the zebrafish fgf24 gene, which belongs to the Fgf8/17/18 subfamily of Fgf ligands, acts downstream of tbx5 to activate fgf10 expression in the lateral plate mesoderm. We also show that fgf24 activity is necessary for the migration of tbx5-expressing cells to the fin bud, and for the activation of shh, but not hand2, expression in the posterior fin bud.