Table 1.
Top 20 genes with one peak of expression during chicken somitogenesis.
Times in minutes, assuming a 90mn periodicity, are provided for each peak. Accuracy has been estimated using a Monte Carlo method. All experimentally validated cyclic genes were detected using our algorithm (see [15]).
Fig 1.
Phase conservation between mouse and chicken.
Axin2, Wnt-related cyclic gene oscillates opposite phase to Hes1 (Notch-related gene) suggesting a phase conservation between mouse and chicken. Raf1, an Fgf-related gene previously shown to have two peaks of expression during mouse somitogenesis showed the same pattern of expression in chicken. Phase of gene expression varies from 0 to 1 and corresponds to 120 and 90 minutes periodicity for mouse and chicken respectively.
Table 2.
Top 20 genes with one peak of expression during zebrafish somitogenesis.
Times in minutes, assuming a 30mn periodicity, are provided for each peak. Accuracy has been estimated using a Monte Carlo method. All experimentally validated cyclic genes were detected using our algorithm (see [15]).
Fig 2.
Hes5 cyclic expression is conserved between mouse, chicken and zebrafish.
The expression pattern of Hes5 is shown as periodic during mouse, chicken and zebrafish somitogenesis. Moreover, the oscillations are in phase in all three species as depicted by the expression profile. Phase of gene expression varies from 0 to 1 and corresponds to 120, 90, and 30 minutes periodicity of mouse, chicken, and zebrafish respectively.
Fig 3.
The timing of the significant genes involved in the chicken and zebrafish somitogenesis.
Position of a gene symbol on the plots reflects time of peak timing (angle; clockwise) and the mean expression level (genes with high expression level are closer to the center). The timing is based on 90 and 30 minutes periodicity for the chicken and zebrafish somite cycle respectively. Genes are color-coded according to their known pathway association with green for Notch, brown for Fgf, and purple for Wnt. Genes in blue have never been reported as cyclic but are regulated during chicken and zebrafish somitogenesis. Also, a comparison with Fig 3 of [15] suggests that notable similarities exist between the temporal organization of the pathways involved in the somite clock in chick and mouse, with certain aspects (as the phase of expression of Notch genes) conserved also in the zebrafish.
Fig 4.
Gene Ontology analysis of the candidate cyclic genes with one peak of expression during mouse, chicken, and zebrafish somitogenesis.
Shown are the top three GO terms for each species. The analysis was done using DAVID, an online set of tools for functional analysis of co-regulated genes. Only GO terms containing at least 5 of the input genes and a q-value (Benjamini corrected p-value) < 0.05 were selected.
Table 3.
Published cyclic genes of mouse, chicken and zebrafish somitogenesis used for motifs enrichment.
We considered cyclic genes in the same pathway as co-regulated and used their sequences for cis-regulatory elements finding. Moreover, as somitogenesis is better described in mouse that any other species, mouse sequences are used for de novo motif finding and the subsequent motifs were used for enrichment analysis in chicken and zebrafish.
Fig 5.
De novo motif discovery led to 20 statistically significant motifs overrepresented in the promoter regions of mouse cyclic genes.
We found 10 motifs (named N1-N10) in the promoter region of Notch cyclic genes, 5 motifs for Wnt cyclic genes (W1-W5) and Fgf cyclic genes (F1-F5). The motifs were ruled significant after passing all steps described in the Methods section.
Fig 6.
Several TFs may cooperatively regulate the expression of cyclic genes during mouse somitogenesis.
A GC rich regulatory motif denoted as A1 in the text was found to be overrepresented in the promoter of nearly ¾ of reported cyclic genes during chicken somitogenesis. The average position of the motif is around 300 bps upstream of the transcription start site for mouse genes. When tested for enrichment in the database of experimentally validated TFBSs, it appears this regulatory motif may contain the binding sites of several couple of TFs acting cooperatively to regulate the expression of corresponding genes. The first and second possibilities (panels A and B) is the TF E2F1 binding upstream of EGR1 and ZNF263 respectively and the others possibilities (panels C and D) involve the TF PLAG1 binding upstream of RREB1 and EGR2.
Table 4.
De novo identified motifs and their conservation between pathways and species.
We tested the reference motif list for pathway conservation and between species conservation. For each motif, we computed the enrichment in known TFBSs databases and reported the corresponding significant TFs. It appears that several TFs may be acting cooperatively to regulate the expression of cyclic genes. We also noticed motif conservation between mouse and chicken as opposed to zebrafish where any of the discovered motifs could be found in the promoters of known cyclic genes. The conservation between species is given in terms of percentage of reported cyclic genes of the species with the corresponding motif in their promoter region.
Table 5.
Distribution of G-quadruplex structures in the promoter regions of mouse, chicken, zebrafish, and human genes.
We computed the distribution of G4 in the promoter (2 kbps upstream of the TSS) of mouse, chicken, zebrafish, and human genes and compared to that of corresponding cyclic genes. As cyclic genes in human are not well defined, we used homologs of mouse cyclic genes for comparison purposes. G4 is significantly represented in the promoter of mouse cyclic genes. Indeed 85% of known mouse cyclic genes contain the G4 structures, suggesting a possible role during mouse somitogenesis. The p-value is computed using Fisher exact test.
Table 6.
Motif enrichment in the lists of candidate cyclic genes from mouse, chicken, and zebrafish datasets.
We have compiled the lists of candidate cyclic genes with one and two peak of expression during mouse, chicken, and zebrafish somitogenesis and tested them against the reference of regulatory motifs. For each species, we reported the number of genes containing the corresponding motif in their promoter as well as the genes with the highest E-values. These genes represent leading cyclic candidate genes. As expected, none of the motifs discovered in mouse was found to be overrepresented in the promoter of zebrafish candidate cyclic genes.
Table 7.
Parameters describing the wave deceleration and the geometry of the system for collected data in mouse, chicken and zebrafish somitogenesis.
The parameters α and d are computed assuming that the cycle phase φ is random at the time when the embryos are sacrificed [15].
Fig 7.
Relationship between the phase of gene expression and position along the PSM of the chicken, mouse and zebrafish embryos.
The waves start as moving fast for small values of x (most posterior part of the PSM), and slow down as the wave progresses toward larger values of x. The four snapshots depict a sequence of time-points, corresponding to different phases of the somite cycle.