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.     

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.     

Histone deacetylase 1 regulates retinal neurogenesis in zebrafish by suppressing Wnt and Notch signaling pathways

In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.     

Notch signalling synchronizes the zebrafish segmentation clock but is not needed to create somite boundaries

Somite segmentation depends on a gene expression oscillator or clock in the posterior presomitic mesoderm (PSM) and on read-out machinery in the anterior PSM to convert the pattern of clock phases into a somite pattern. Notch pathway mutations disrupt somitogenesis, and previous studies have suggested that Notch signalling is required both for the oscillations and for the read-out mechanism. By blocking or overactivating the Notch pathway abruptly at different times, we show that Notch signalling has no essential function in the anterior PSM and is required only in the posterior PSM, where it keeps the oscillations of neighbouring cells synchronized. Using a GFP reporter for the oscillator gene her1, we measure the influence of Notch signalling on her1 expression and show by mathematical modelling that this is sufficient for synchronization. Our model, in which intracellular oscillations are generated by delayed autoinhibition of her1 and her7 and synchronized by Notch signalling, explains the observations fully, showing that there are no grounds to invoke any additional role for the Notch pathway in the patterning of somite boundaries in zebrafish.     

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.     

Three novel Notch genes in zebrafish: implications for vertebrate Notch gene evolution and function

 Notch genes encode transmembrane receptors that interact with numerous signal transduction pathways and are essential for animal development. To facilitate analysis of vertebrate Notch gene function, we isolated cDNA fragments of three novel Notch genes from zebrafish (Danio rerio), Notch1b, Notch5 and Notch6. Notch1b is a second zebrafish Notch1 gene. From analysis of the Notch1b sequence we argue that the various vertebrate Notch gene subfamilies encode receptors with different signalling specificities. Notch5 and Notch6 represent novel vertebrate Notch gene subfamilies. Remarkably, Notch1b lacks expression in presomitic mesoderm, Notch5 is expressed in a metameric pattern within the presomitic mesoderm whilst Notch6 expression is excluded from the nervous system. The expression patterns of these genes suggest important roles in gastrulation, somitogenesis, tail bud extension, myogenesis, heart development and neurogenesis. We discuss the implications of our observations for Notch gene evolution and function. Received: 20 January 1997 / Accepted: 12 February 1997     

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD

Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.     

Potential Role of Notch Signalling in CD34+ Chronic Myeloid Leukaemia Cells: Cross-Talk between Notch and BCR-ABL

Notch signalling is critical for haemopoietic stem cell (HSC) self-renewal and survival. The role of Notch signalling has been reported recently in chronic myeloid leukaemia (CML) – a stem cell disease characterized by BCR-ABL tyrosine kinase activation. Therefore, we studied the relationship between BCR-ABL and Notch signalling and assessed the expression patterns of Notch and its downstream target Hes1 in CD34⁺ stem and progenitor cells from chronic-phase CML patients and bone marrow (BM) from normal subjects (NBM). We found significant upregulation (p<0.05) of Notch1, Notch2 and Hes1 on the most primitive CD34⁺Thy⁺ subset of CML CD34⁺ cells suggesting that active Notch signalling in CML primitive progenitors. In addition, Notch1 was also expressed in distinct lymphoid and myeloid progenitors within the CD34⁺ population of primary CML cells. To further delineate the possible role and interactions of Notch with BCR-ABL in CD34⁺ primary cells from chronic-phase CML, we used P-crkl detection as a surrogate assay of BCR-ABL tyrosine kinase activity. Our data revealed that Imatinib (IM) induced BCR-ABL inhibition results in significant (p<0.05) upregulation of Notch activity, assessed by Hes1 expression. Similarly, inhibition of Notch leads to hyperactivation of BCR-ABL. This antagonistic relationship between Notch and BCR-ABL signalling was confirmed in K562 and ALL-SIL cell lines. In K562, we further validated this antagonistic relationship by inhibiting histone deacetylase (HDAC) - an effector pathway of Hes1, using valproic acid (VPA) - a HDAC inhibitor. Finally, we also confirmed the potential antagonism between Notch and BCR/ABL in In Vivo, using publically available GSE-database, by analysing gene expression profile of paired samples from chronic-phase CML patients pre- and post-Imatinib therapy. Thus, we have demonstrated an antagonistic relationship between Notch and BCR-ABL in CML. A combined inhibition of Notch and BCR-ABL may therefore provide superior clinical response over tyrosine-kinase inhibitor monotherapy by targeting both quiescent leukaemic stem cells and differentiated leukaemic cells and hence must be explored.     

Notch Is a Critical Component of the Mouse Somitogenesis Oscillator and Is Essential for the Formation of the Somites

Segmentation of the vertebrate body axis is initiated through somitogenesis, whereby epithelial somites bud off in pairs periodically from the rostral end of the unsegmented presomitic mesoderm (PSM). The periodicity of somitogenesis is governed by a molecular oscillator that drives periodic waves of clock gene expression caudo-rostrally through the PSM with a periodicity that matches somite formation. To date the clock genes comprise components of the Notch, Wnt, and FGF pathways. The literature contains controversial reports as to the absolute role(s) of Notch signalling during the process of somite formation. Recent data in the zebrafish have suggested that the only role of Notch signalling is to synchronise clock gene oscillations across the PSM and that somite formation can continue in the absence of Notch activity. However, it is not clear in the mouse if an FGF/Wnt-based oscillator is sufficient to generate segmented structures, such as the somites, in the absence of all Notch activity. We have investigated the requirement for Notch signalling in the mouse somitogenesis clock by analysing embryos carrying a mutation in different components of the Notch pathway, such as Lunatic fringe (Lfng), Hes7, Rbpj, and presenilin1/presenilin2 (Psen1/Psen2), and by pharmacological blocking of the Notch pathway. In contrast to the fish studies, we show that mouse embryos lacking all Notch activity do not show oscillatory activity, as evidenced by the absence of waves of clock gene expression across the PSM, and they do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for the formation of a segmented body axis.     

The Mammalian Orthologs of Drosophila Lgd,CC2D1A and CC2D1B,Function in the Endocytic Pathway,but Their Individual Loss of Function Does Not Affect Notch Signalling

CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT-III mediated process in metazoans.     

RA and FGF Signalling Are Required in the Zebrafish Otic Vesicle to Pattern and Maintain Ventral Otic Identities

During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.     

Changes in histone acetylation during oocyte meiotic maturation in the diabetic mouse

Although there is considerable evidence that diabetes can adversely affect meiosis in mammalian oocytes, acetylation status of oocytes in a diabetic environment remains unclear. The objective was to determine acetylation or deacetylation patterns (based on immunostaining) of H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16 sites at various stages during meiosis in murine oocytes from control and diabetic mice. According to quantitative real time polymerase chain reaction (qPCR), mean ± SEM relative expression of Gcn5 (1.70 ± 0.14 at metaphase [M]I and 1.27 ± 0.01 at MII, respectively), Ep300 (1.74 ± 0.04 at MI and 1.80 ± 0.001 at MII), and Pcaf (2.01 ± 0.03 at MI and 1.41 ± 0.18 at MII) mRNA in oocytes from diabetic mice were higher than those from controls (P < 0.05), whereas there was no difference (P > 0.05) during the germinal vesicle (GV) stage between the two groups (1.23 ± 0.04 for Gcn5, 0.82 ± 0.06 for Ep300, and 0.80 ± 0.07 for Pcaf). Conversely, relative mRNA expression concentrations of Hdac1, Hdac2, Hdac3, Sirt1 and Sirt2 during the germinal vesicle stage were lower in oocytes of diabetic mice (0.24 ± 0.03 for Hdac1, 0.11 ± 0.001 for Hdac2, 0.31 ± 0.03 for Hdac3, 0.28 ± 0.02 for Sirt1, and 0.55 ± 0.02 for Sirt2; P < 0.05). Similarly, the expression concentrations of these genes at the MI stage were lower in oocytes from diabetic mice (0.79 ± 0.12 for Hdac1, 0.72 ± 0.001 for Hdac2, 0.02 ± 0.001 for Sirt1, and 0.84 ± 0.08 for Sirt2; P < 0.05). Their expression concentrations at the MII stage were also lower in oocytes from diabetic mice (0.46 ± 0.03 for Hdac1, 0.93 ± 0.01 for Hdac2, 0.56 ± 0.01 for Hdac3, 0.01 ± 0.002 for Sirt1, and 0.84 ± 0.04 for Sirt2; P < 0.05). At the MI stage, however, there was no difference in the expression of Hdac3 between the two groups of oocytes (0.96 ± 0.03; P > 0.05). Taken together, diabetes altered the intracellular histone modification system, which may have contributed to changes in histone acetylation, and may be involved in the compromised maturation rate of oocytes in diabetic humans.     

Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models

Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b⁺, ascl1⁺, elavl3⁻ cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.     

SOX1 links the function of neural patterning and Notch signalling in the ventral spinal cord during the neuron-glial fate switch

During neural development the transition from neurogenesis to gliogenesis, known as the neuron-glial (Ν/G) fate switch, requires the coordinated function of patterning factors, pro-glial factors and Notch signalling. How this process is coordinated in the embryonic spinal cord is poorly understood. Here, we demonstrate that during the N/G fate switch in the ventral spinal cord (vSC) SOX1 links the function of neural patterning and Notch signalling. We show that, SOX1 expression in the vSC is regulated by PAX6, NKX2.2 and Notch signalling in a domain-specific manner. We further show that SOX1 regulates the expression of Hes1 and that loss of Sox1 leads to enhanced production of oligodendrocyte precursors from the pMN. Finally, we show that Notch signalling functions upstream of SOX1 during this fate switch and is independently required for the acquisition of the glial fate perse by regulating Nuclear Factor I A expression in a PAX6/SOX1/HES1/HES5-independent manner. These data integrate functional roles of neural patterning factors, Notch signalling and SOX1 during gliogenesis.