Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.     

Conditional inactivation of Tgfbr2 in cranial neural crest causes cleft palate and calvaria defects

Cleft palate and skull malformations represent some of the most frequent congenital birth defects in the human population. Previous studies have shown that TGFbeta signaling regulates the fate of the medial edge epithelium during palatal fusion and postnatal cranial suture closure during skull development. It is not understood, however, what the functional significance of TGFbeta signaling is in regulating the fate of cranial neural crest (CNC) cells during craniofacial development. We show that mice with Tgfbr2 conditional gene ablation in the CNC have complete cleft secondary palate, calvaria agenesis, and other skull defects with complete phenotype penetrance. Significantly, disruption of the TGFbeta signaling does not adversely affect CNC migration. Cleft palate in Tgfbr2 mutant mice results from a cell proliferation defect within the CNC-derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Our data suggests that TGFbeta IIR plays a crucial, cell-autonomous role in regulating the fate of CNC cells during palatogenesis. During skull development, disruption of TGFbeta signaling in the CNC severely impairs cell proliferation in the dura mater, consequently resulting in calvaria agenesis. We provide in vivo evidence that TGFbeta signaling within the CNC-derived dura mater provides essential inductive instruction for both the CNC- and mesoderm-derived calvarial bone development. This study demonstrates that TGFbeta IIR plays an essential role in the development of the CNC and provides a model for the study of abnormal CNC development.     

Valproic acid silencing of ascl1b/Ascl1 results in the failure of serotonergic differentiation in a zebrafish model of fetal valproate syndrome

Fetal valproate syndrome (FVS) is caused by in utero exposure to the drug sodium valproate. Valproate is used worldwide for the treatment of epilepsy, as a mood stabiliser and for its pain-relieving properties. In addition to birth defects, FVS is associated with an increased risk of autism spectrum disorder (ASD), which is characterised by abnormal behaviours. Valproate perturbs multiple biochemical pathways and alters gene expression through its inhibition of histone deacetylases. Which, if any, of these mechanisms is relevant to the genesis of its behavioural side effects is unclear. Neuroanatomical changes associated with FVS have been reported and, among these, altered serotonergic neuronal differentiation is a consistent finding. Altered serotonin homeostasis is also associated with autism. Here we have used a chemical-genetics approach to investigate the underlying molecular defect in a zebrafish FVS model. Valproate causes the selective failure of zebrafish central serotonin expression. It does so by downregulating the proneural gene ascl1b, an ortholog of mammalian Ascl1, which is a known determinant of serotonergic identity in the mammalian brainstem. ascl1b is sufficient to rescue serotonin expression in valproate-treated embryos. Chemical and genetic blockade of the histone deacetylase Hdac1 downregulates ascl1b, consistent with the Hdac1-mediated silencing of ascl1b expression by valproate. Moreover, tonic Notch signalling is crucial for ascl1b repression by valproate. Concomitant blockade of Notch signalling restores ascl1b expression and serotonin expression in both valproate-exposed and hdac1 mutant embryos. Together, these data provide a molecular explanation for serotonergic defects in FVS and highlight an epigenetic mechanism for genome-environment interaction in disease.KEY WORDS: Serotonin, Fetal valproate syndrome, Zebrafish, Notch, Proneural gene, Hdac1     

Patterning of palatal rugae through sequential addition reveals an anterior/posterior boundary in palatal development

Background

The development of the secondary palate has been a main topic in craniofacial research, as its failure results in cleft palate, one of the most common birth defects in human. Nevertheless, palatal rugae (or rugae palatinae), which are transversal ridges developing on the secondary palate, received little attention. However, rugae could be useful as landmarks to monitor anterior/posterior (A/P) palatal growth, and they provide a simple model of mesenchymal-epithelial structures arranged in a serial pattern.

Results

We first determined in which order the nine mouse rugae appear during development. Our results revealed a reiterative process, which is coupled with A/P growth of palatal shelves, and by which rugae 3 to 7b are sequentially interposed, in the increasing distance between the second most anterior ruga, ruga 2, and the two most posterior rugae, rugae 8 and 9. We characterized the steps of ruga interposition in detail, showing that a new ruga forms from an active zone of high proliferation rate, next to the last formed ruga. Then, by analyzing the polymorphism of wild type and Eda^Ta mutant mice, we suggest that activation-inhibition mechanisms may be involved in positioning new rugae, like for other skin appendages. Finally, we show that the ruga in front of which new rugae form, i.e. ruga 8 in mouse, coincides with an A/P gene expression boundary in the palatal shelves (Shox2/Meox2-Tbx22). This coincidence is significant, since we also found it in hamster, despite differences in the adult ruga pattern of these two species.

Conclusion

We showed that palatal rugae are sequentially added to the growing palate, in an interposition process that appears to be dependent on activation-inhibition mechanisms and reveals a new developmental boundary in the growing palate. Further studies on rugae may help to shed light on both the development and evolution of structures arranged in regular patterns. Moreover, rugae will undoubtedly be powerful tools to further study the anteroposterior regionalization of the growing palate.     

A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development

Background

Development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. Despite decades of research our knowledge of the formation and maintenance of the blood-brain (BBB) and blood-retinal (BRB) barrier is very limited. We have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin D-binding protein fused with enhanced green fluorescent protein (DBP-EGFP) in blood plasma, as an endogenous tracer.

Results

The temporal establishment of the BBB and BRB was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. We also examined the expression of claudin-5, a component of tight junctions during the first 4 days of development. We observed that the BBB of zebrafish starts to develop by 3 dpf, with expression of claudin-5 in the central arteries preceding it at 2 dpf. The hyaloid vasculature in the zebrafish retina develops a barrier function at 3 dpf, which endows the zebrafish with unique advantages for studying the BRB.

Conclusion

Zebrafish embryos develop BBB and BRB function simultaneously by 3 dpf, which is regulated by tight junction proteins. The Tg(l-fabp:DBP-EGFP) zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model.     

The mych gene is required for neural crest survival during zebrafish development

Background

Among Myc family genes, c-Myc is known to have a role in neural crest specification in Xenopus and in craniofacial development in the mouse. There is no information on the function of other Myc genes in neural crest development, or about any developmental role of zebrafish Myc genes.

Principal Findings

We isolated the zebrafish mych (myc homologue) gene. Knockdown of mych leads to severe defects in craniofacial development and in certain other tissues including the eye. These phenotypes appear to be caused by cell death in the neural crest and in the eye field in the anterior brain.

Significance

Mych is a novel factor required for neural crest cell survival in zebrafish.     

Population genetics of foxtail millet and its wild ancestor

Background

The size of the vertebrate eye and the retina is likely to be controlled at several stages of embryogenesis by mechanisms that affect cell cycle length as well as cell survival. A mutation in the zebrafish out of sight (out) locus results in a particularly severe reduction of eye size. The goal of this study is to characterize the out ^m233 mutant, and to determine whether mutations in the out gene cause microphthalmia in humans.

Results

In this study, we show that the severe reduction of eye size in the out ^m233 mutant is caused by a mutation in the zebrafish gdf6a gene. Despite the small eye size, the overall retinal architecture appears largely intact, and immunohistochemical studies confirm that all major cell types are present in out ^m233 retinae. Subtle cell fate and patterning changes are present predominantly in amacrine interneurons. Acridine orange and TUNEL staining reveal that the levels of apoptosis are abnormally high in out ^m233 mutant eyes during early neurogenesis. Mutation analysis of the GDF6 gene in 200 patients with microphthalmia revealed amino acid substitutions in four of them. In two patients additional skeletal defects were observed.

Conclusions

This study confirms the essential role of GDF6 in the regulation of vertebrate eye size. The reduced eye size in the zebrafish out ^m233 mutant is likely to be caused by a transient wave of apoptosis at the onset of neurogenesis. Amino acid substitutions in GDF6 were detected in 4 (2%) of 200 patients with microphthalmia. In two patients different skeletal defects were also observed, suggesting pleitrophic effects of GDF6 variants. Parents carrying these variants are asymptomatic, suggesting that GDF6 sequence alterations are likely to contribute to the phenotype, but are not the sole cause of the disease. Variable expressivity and penetrance suggest a complex non-Mendelian inheritance pattern where other genetic factors may influence the outcome of the phenotype.     

Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.     

Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice

Background

The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood.

Results

We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region.

Conclusion

We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.     

Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

Background  

The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC). Genetic studies in zebrafish and mice have established that the Hedgehog (Hh)-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE), which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia [1, 2]. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA) skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1) for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1).     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Histone Deacetylase 2 (HDAC2) Regulates Chromosome Segregation and Kinetochore Function via H4K16 Deacetylation during Oocyte Maturation in Mouse

Changes in histone acetylation occur during oocyte development and maturation, but the role of specific histone deacetylases in these processes is poorly defined. We report here that mice harboring Hdac1 ^−/+/Hdac2 ^−/− or Hdac2 ^−/− oocytes are infertile or sub-fertile, respectively. Depleting maternal HDAC2 results in hyperacetylation of H4K16 as determined by immunocytochemistry—normal deacetylation of other lysine residues of histone H3 or H4 is observed—and defective chromosome condensation and segregation during oocyte maturation occurs in a sub-population of oocytes. The resulting increased incidence of aneuploidy likely accounts for the observed sub-fertility of mice harboring Hdac2 ^−/− oocytes. The infertility of mice harboring Hdac1 ^−/+/Hdac2 ^−/−oocytes is attributed to failure of those few eggs that properly mature to metaphase II to initiate DNA replication following fertilization. The increased amount of acetylated H4K16 likely impairs kinetochore function in oocytes lacking HDAC2 because kinetochores in mutant oocytes are less able to form cold-stable microtubule attachments and less CENP-A is located at the centromere. These results implicate HDAC2 as the major HDAC that regulates global histone acetylation during oocyte development and, furthermore, suggest HDAC2 is largely responsible for the deacetylation of H4K16 during maturation. In addition, the results provide additional support that histone deacetylation that occurs during oocyte maturation is critical for proper chromosome segregation.