Genomewide expression analysis in zebrafish mind bomb alleles with pancreas defects of different severity identifies putative Notch responsive genes - PubMed (original) (raw)
Genomewide expression analysis in zebrafish mind bomb alleles with pancreas defects of different severity identifies putative Notch responsive genes
Ashok Hegde et al. PLoS One. 2008.
Abstract
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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Expression pattern of pancreas-specific genes at 96 hpf in mib mutants and their wild type (wt) siblings.
The RNA probes used for in situ hybridization are (A and A′–D′) elastaseA, (E and E′–H′) trypsin, (I and I′–L′) somatostatin and (M and M′–P′) insulin. The genotypes are (A, A′, E, E′, I, I′, M and M′) wt, (B′, F′, J′ and N′) mibtfi91, (C′, G′, K′ and O′) mibm132 and (D′, H′, L′ and P′) mibta52b. A′, E′, I′ and M′ are cropped from A, E, I and M, respectively. All panels are lateral views and anterior to the right.
Figure 2. _elastaseA_-GFP, _insulin_-GFP and _pdx1_-GFP expression in embryos of mib mutant alleles and their wt siblings.
Expression of _elastaseA_-GFP in (A) wt, (B) mibtfi91, (C) mibm132 and (D) mibta52b embryos was analyzed at 96 hpf. Expression of _insulin_-GFP in (E and E′) wt, (F′) mibtfi91, (G′) mibm132 and (H′) mibta52b embryos was analyzed at 72 hpf. Expression of _pdx1_-GFP in (I and I′) wt, (J′) mibtfi91, (K′) mibm132 and (L′) mibta52b embryos was analyzed at 72 hpf. Panels A–D are lateral views and the rest are dorsolateral views, oriented anterior to the right.
Figure 3. Venn diagrams show numbers of genes belonging to different groups.
Differentially expressed genes specific to each mib mutant allele, common between two mutant alleles and common to all three mutant alleles are (A) up-regulated and (B) down-regulated at 72 hpf; and (C) up-regulated and (D) down-regulated at 48 hpf; and genes specific to each time point, common between two time points and common to all three time points are (E) up-regulated and (F) down-regulated in mibta52b mutants. Gene set for the analysis of 72-hpf data was selected based on the criteria, q = 0.0 and score(d)>4.0; and for the analysis of 48-hpf data and the three time-point data for mibta52b mutants were both selected based on the criterion, q = 0.0.
Figure 4. Gene expression profile of 91 differentially expressed (q = 0.0, score(d)>4.0) genes (Genbank ID) common to all three mib mutant alleles at 72 hpf.
At least two biological repeats and two technical repeats were carried out for each mutant allele. Each horizontal strip represents expression of a single gene. Color in each cell reflects the expression level of corresponding gene in the respective sample. The up-regulation is shown in red and the down-regulation in green. Grey cells indicate the missing values. The median log2 ratio values for differential expression ranged between −4.07 and 3.32. Note: The gene symbols are mentioned instead of Genbank IDs, where the gene symbols corresponding to the Genbank IDs are available in the Zebrafish Chip Annotation Database. Three Genbank IDs, BI840762, BI839927 and BG306150, encode the same gene, gpm6aa.
Figure 5. Pictorial representation of significantly up-regulated or down-regulated genes (q = 0.0, score(d)>4.0) categorized based on known/related biological functions as assigned in the zebrafish gene ontology database (Unigene Build 85).
Genes in each functional group were searched by using specific or related key words appropriate for that function in the lists of differentially regulated genes for each allele, common 91 genes in all three mib mutant alleles, and the genes specific to each mutant allele. Absolute number of genes in each category is shown next to the corresponding pie sector. (A–D) Pie charts show numbers of genes belonging to different functional categories. (E–H) Pie charts show numbers of genes (excluding the genes without any known function) in different categories.
Figure 6. Significantly up-regulated and down-regulated genes analyzed by IPA (see Materials and Methods) for (A) enrichment of functional groups and (B) enrichment of canonical pathways, shown in histograms.
Figure 7. Validation of expression profile of differentially expressed genes with fluorescent double in situ hybridization.
Expression of down-regulated genes: (A and B) isl1, (C and D) cad and (E and F) wu:fb59c09 and up-regulated genes: (G and H) spon1b and (I and J) isl3 in (B, D, F, H and J) mibta52b mutants embryos and (A, C, E, G and I) wild-type embryos. Left panels show respective gene expression in green; middle panels show insulin (endocrine) or trypsin (exocrine) expression in red; right panels show merged pictures. Expression of wu:fb59c09 is shown in 4-dpf embryos, and the rest are in 3-dpf embryos. All embryos are anterior to the left and ventral views.
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