Genomewide expression analysis in zebrafish mind bomb alleles with pancreas defects of different severity identifies putative Notch responsive genes

Background

Notch signaling is an evolutionarily conserved developmental pathway. Zebrafish mind bomb (mib) mutants carry mutations on mib gene, which encodes a RING E3 ligase required for Notch activation via Delta/Jagged ubiquitylation and internalization.

Methodology/Principal Findings

We examined the mib mutants for defects in pancreas development using in situ hybridization and GFP expression analysis of pancreas-specific GFP lines, carried out the global gene expression profile analysis of three different mib mutant alleles and validated the microarray data using real-time PCR and fluorescent double in situ hybridization. Our study showed that the mib mutants have diminished exocrine pancreas and this defect was most severe in mib^ta52b followed by mib^m132 and then mib^tfi91, which is consistent with the compromised Notch activity found in corresponding mib mutant alleles. Global expression profile analysis of mib mutants showed that there is a significant difference in gene expression profile of wt and three mib mutant alleles. There are 91 differentially expressed genes that are common to all three mib alleles. Through detailed analysis of microarray data, we have identified several previously characterized genes and some putative Notch-responsive genes involved in pancreas development. Moreover, results from real-time PCR and fluorescent double in situ hybridization were largely consistent with microarray data.

Conclusions/Significance

This study provides, for the first time, a global gene expression profile in mib mutants generating useful genomic resources and providing an opportunity to identify the function of novel genes involved in Notch signaling and Notch-regulated developmental processes.     

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.     

Development and Notch signaling requirements of the zebrafish choroid plexus

Background

The choroid plexus (CP) is an epithelial and vascular structure in the ventricular system of the brain that is a critical part of the blood-brain barrier. The CP has two primary functions, 1) to produce and regulate components of the cerebral spinal fluid, and 2) to inhibit entry into the brain of exogenous substances. Despite its importance in neurobiology, little is known about how this structure forms.

Methodology and Principal Findings

Here we show that the transposon-mediated enhancer trap zebrafish line Et^Mn16 expresses green fluorescent protein within a population of cells that migrate toward the midline and coalesce to form the definitive CP. We further demonstrate the development of the integral vascular network of the definitive CP. Utilizing pharmacologic pan-notch inhibition and specific morpholino-mediated knockdown, we demonstrate a requirement for Notch signaling in choroid plexus development. We identify three Notch signaling pathway members as mediating this effect, notch1b, deltaA, and deltaD.

Conclusions and Significance

This work is the first to identify the zebrafish choroid plexus and to characterize its epithelial and vasculature integration. This study, in the context of other comparative anatomical studies, strongly indicates a conserved mechanism for development of the CP. Finally, we characterize a requirement for Notch signaling in the developing CP. This establishes the zebrafish CP as an important new system for the determination of key signaling pathways in the formation of this essential component of the vertebrate brain.     

Fibroblast Growth Factor Receptor 2c Signaling Is Required for Intestinal Cell Differentiation in Zebrafish

Background

There are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood.

Methodology/Principal Findings

We used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mib^ta52b mutant that had defective Notch signaling.

Conclusions/Significance

In conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mib^ta52b mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.     

Timely Inhibition of Notch Signaling by DAPT Promotes Cardiac Differentiation of Murine Pluripotent Stem Cells

The Notch signaling pathway plays versatile roles during heart development. However, there is contradictory evidence that Notch pathway either facilitates or impairs cardiomyogenesis in vitro. In this study, we developed iPSCs by reprogramming of murine fibroblasts with GFP expression governed by Oct4 promoter, and identified an effective strategy to enhance cardiac differentiation through timely modulation of Notch signaling. The Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) alone drove the iPSCs to a neuronal fate. After mesoderm induction of embryoid bodies initiated by ascorbic acid (AA), the subsequent treatment of DAPT accelerated the generation of spontaneously beating cardiomyocytes. The timed synergy of AA and DAPT yielded an optimal efficiency of cardiac differentiation. Mechanistic studies showed that Notch pathway plays a biphasic role in cardiomyogenesis. It favors the early–stage cardiac differentiation, but exerts negative effects on the late-stage differentiation. Therefore, DAPT administration at the late stage enforced the inhibition of endogenous Notch activity, thereby enhancing cardiomyogenesis. In parallel, DAPT dramatically augmented the expression of Wnt3a, Wnt11, BMP2, and BMP4. In conclusion, our results highlight a practicable approach to generate cardiomyocytes from iPSCs based on the stage-specific biphasic roles of Notch signaling in cardiomyogenesis.     

The Role of her4 in Inner Ear Development and Its Relationship with Proneural Genes and Notch Signalling

The generation of sensory neurons and hair cells of the inner ear is under tight control. Different members of the Hairy and Enhancer of Split genes (HES) are expressed in the inner ear, their full array of functions still not being disclosed. We have previously shown that zebrafish her9 acts as a patterning gene to restrict otic neurogenesis to an anterior domain. Here, we disclose the role of another her gene, her4, a zebrafish ortholog of Hes5 that is expressed in the neurogenic and sensory domains of the inner ear. The expression of her4 is highly dynamic and spatiotemporally regulated. We demonstrate by loss of function experiments that in the neurogenic domain her4 expression is under the regulation of neurogenin1 (neurog1) and the Notch pathway. Moreover, her4 participates in lateral inhibition during otic neurogenesis since her4 knockdown results in overproduction of the number of neurog1 and deltaB-positive otic neurons. In contrast, during sensorigenesis her4 is initially Notch-independent and induced by atoh1b in a broad prosensory domain. At later stages her4 expression becomes Notch-dependent in the future sensory domains but loss of her4 does not result in hair cell overproduction, suggesting that there other her genes can compensate its function.     

Notch signaling regulates endocrine cell specification in the zebrafish anterior pituitary

The vertebrate pituitary gland is a key endocrine control organ that contains six distinct hormone secreting cell types. In this study, we analyzed the role of direct cell-to-cell Delta-Notch signaling in zebrafish anterior pituitary cell type specification. We demonstrate that initial formation of the anterior pituitary placode is independent of Notch signaling. Later however, loss of Notch signaling in mind bomb (mib) mutant embryos or by DAPT treatment leads to increased numbers of lactotropes and loss of corticotropes in the anterior pars distalis (APD), increased number of thyrotropes and loss of somatotrope cell types in the posterior pars distalis (PPD), and fewer melanotropes in the posterior region of the adenohypophysis, the pars intermedia (PI). Conversely, Notch gain of function leads to the opposite result, loss of lactotrope and thyrotrope cell specification, and an increased number of corticotropes, melanotropes, and gonadotropes in the pituitary. Our results suggest that Notch acts on placodal cells, presumably as a permissive signal, to regulate progenitor cell specification to hormone secreting cell types. We propose that Notch mediated lateral inhibition regulates the relative numbers of specified hormone cell types in the three pituitary subdomains.     

Temporal Notch activation through Notch1a and Notch3 is required for maintaining zebrafish rhombomere boundaries

In vertebrates, hindbrain is subdivided into seven segments termed rhombomeres and the interface between each rhombomere forms the boundary. Similar to the D/V boundary formation in Drosophila, Notch activation has been shown to regulate the segregation of rhombomere boundary cells. Here we further explored the function of Notch signaling in the formation of rhombomere boundaries. By using bodipy ceramide cell-labeling technique, we found that the hindbrain boundary is formed initially in mib mutants but lost after 24 hours post-fertilization (hpf). This phenotype was more severe in mib ^ta52b allele than in mib ^tfi91 allele. Similarly, injection of su(h)-MO led to boundary defects in a dosage-dependent manner. Boundary cells were recovered in mib ^ta52b mutants in the hdac1-deficient background, where neurogenesis is inhibited. Furthermore, boundary cells lost sensitivity to reduced Notch activation from 15 somite stage onwards. We also showed that knockdown of notch3 function in notch1a mutants leads to the loss of rhombomere boundary cells and causes neuronal hyperplasia, indicating that Notch1a and Notch3 play a redundant role in the maintenance of rhombomere boundary.     

Notch2 is required for maintaining sustentacular cell function in the adult mouse main olfactory epithelium

Notch receptors are expressed in neurons and glia in the adult nervous system, but why this expression persists is not well-understood. Here we examine the role of the Notch pathway in the postnatal mouse main olfactory system, and show evidence consistent with a model where Notch2 is required for maintaining sustentacular cell function. In the absence of Notch2, the laminar nature of these glial-like cells is disrupted. Hes1, Hey1, and Six1, which are downstream effectors of the Notch pathway, are down-regulated, and cytochrome P450 and Glutathione S-transferase (GST) expression by sustentacular cells is reduced. Functional levels of GST activity are also reduced. These disruptions are associated with increased olfactory sensory neuron degeneration. Surprisingly, expression of Notch3 is also down-regulated. This suggests the existence of a feedback loop where expression of Notch3 is initially independent of Notch2, but requires Notch2 for maintained expression. While the Notch pathway has previously been shown to be important for promoting gliogenesis during development, this is the first demonstration that the persistent expression of Notch receptors is required for maintaining glial function in adult.     

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.     

hnRNP I Inhibits Notch Signaling and Regulates Intestinal Epithelial Homeostasis in the Zebrafish

Regulated intestinal stem cell proliferation and differentiation are required for normal intestinal homeostasis and repair after injury. The Notch signaling pathway plays fundamental roles in the intestinal epithelium. Despite the fact that Notch signaling maintains intestinal stem cells in a proliferative state and promotes absorptive cell differentiation in most species, it remains largely unclear how Notch signaling itself is precisely controlled during intestinal homeostasis. We characterized the intestinal phenotypes of brom bones, a zebrafish mutant carrying a nonsense mutation in hnRNP I. We found that the brom bones mutant displays a number of intestinal defects, including compromised secretory goblet cell differentiation, hyperproliferation, and enhanced apoptosis. These phenotypes are accompanied by a markedly elevated Notch signaling activity in the intestinal epithelium. When overexpressed, hnRNP I destabilizes the Notch intracellular domain (NICD) and inhibits Notch signaling. This activity of hnRNP I is conserved from zebrafish to human. In addition, our biochemistry experiments demonstrate that the effect of hnRNP I on NICD turnover requires the C-terminal portion of the RAM domain of NICD. Our results demonstrate that hnRNP I is an evolutionarily conserved Notch inhibitor and plays an essential role in intestinal homeostasis.