Genomewide Expression Analysis in Zebrafish mind bomb Alleles with Pancreas Defects of Different Severity Identifies Putative Notch Responsive Genes

Abstract

BackgroundNotch 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 FindingsWe 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 mibta52b followed by mibm132 and then mibtfi91, 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/SignificanceThis 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.

Spontaneous Seizures and Altered Gene Expression in GABA Signaling Pathways in amind bombMutant Zebrafish

“…5C), a region where GABAergic interneurons are thought to originate (Mione et al, 2008). Interestingly, GAD1 expression was also found to be decreased by microarray and real-time qPCR analysis of mib ta52b mutants (Hegde et al, 2008). We also analyzed the Danio rerio homolog for the α1 subunit of the GABA-A receptor (Fig.…”

“…In particular, CNS expression patterns for hairy-related genes ( her4, her5, her6, her8a, her9 and hes5 ) and proneural factors ( asc1b, atoh2a, ngn1, neurod ) identified as down-regulated in three different microarray analyses of mib mutants (Table 2; Hegde et al, 2008; Hwang et al, 2009) warrant further analysis. Grossly abnormal development and early fatality suggest important roles for these genes in establishing the mind bomb phenotype.…”

“…Consistent with a lateral inhibition neurogenic phenotype, late-differentiating secondary islet GFP-expressing motor neurons and gata3 -expressing interneurons are decreased in the mib hindbrain (Bingham et al 2003). Genome-wide analysis of mind bomb mutants was recently performed to identify Notch responsive genes required for pancreatic (Hegde et al, 2008) or mesodermic (Hwang et al, 2009) development. However, identification of differentially regulated central nervous system (CNS) genes was not discussed and little is presently known about the functional or behavioral consequences of a defect in E3 ubiquitin ligase activity in mib hi904 mutants.…”

The Notch signalling system: recent insights into the complexity of a conserved pathway

“…Thus, exploring signal integration or crosstalk is of fundamental importance, as identifying points of integration may unveil rules that govern morphogenesis and, ultimately, pathogenesis. The means to study integration in a systematic way have only begun to emerge through the various genomic approaches that we now have at our disposal 48,66–69 . Transcriptome analyses, which have now been enhanced by chromatin immunoprecipitation followed by microarrays (ChIP–chip) or ChIP followed by high-throughput sequencing (ChIP–seq) 48,70 methods, have been instrumental in improving our ability to identify both transcriptional targets of Notch signalling, as well as genes that direct Notch function.…”

Transcriptome analysis of the zebrafish mind bomb mutant

“…Recently, genome-wide expression analysis was done by Hegde et al (2008). Authors provided a global gene expression proWle in mib mutants which could be used as genomic resources for transcriptome analysis and functional study of novel transcripts as was performed using a diVerent method.…”