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PLoS One
2010 Dec 02;512:e15459. doi: 10.1371/journal.pone.0015459.
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HES6-1 and HES6-2 function through different mechanisms during neuronal differentiation.
Vilas-Boas F
,
Henrique D
.
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BACKGROUND: Notch signalling plays a central role in the mechanisms regulating neuronal differentiation in the vertebrate nervous system. The transcriptional repressors encoded by Hes genes are the main effectors of this pathway, acting in neural progenitors during the lateral inhibition process to repress proneural genes and inhibit differentiation. However, Hes6 genes seem to behave differently: they are expressed in differentiating neurons and facilitate the activity of proneural genes in promoting neurogenesis. Still, the molecular mechanisms underlying this unique function of Hes6 genes are not yet understood.
METHODOLOGY/PRINCIPAL FINDINGS: Here, we identify two subgroups of Hes6 genes that seem conserved in most vertebrate species and characterize a novel Hes6 gene in chicken: cHes6-1. The embryonic expression pattern of cHes6-1 suggests roles for this gene in the formation of the pancreas, nervous system and in the generation of body asymmetry. We show that cHes6-1 is negatively regulated by Notch signalling in the developing embryonic spinal cord and in pancreatic progenitors, but requires Notch for the observed asymmetric expression at the lateralmesoderm. Functional studies by ectopic expression in the chick embryonic neural tube revealed that cHES6-1 up-regulates the expression of cDelta1 and cHes5 genes, in contrast with overexpression of cHES6-2, which represses the same genes. We show that this activity of cHES6-2 is dependent on its capacity to bind DNA and repress transcription, while cHES6-1 seems to function by sequestering other HES proteins and inhibit their activity as transcriptional repressors.
CONCLUSIONS/SIGNIFICANCE: Our results indicate that the two chick HES6 proteins act at different phases of neuronal differentiation, contributing to the progression of neurogenesis by different mechanisms: while cHES6-2 represses the transcription of Hes genes, cHES6-1 acts later, sequestering HES proteins. Together, the two cHES6 proteins progressively shut down the Notch-mediated progenitor program and ensure that neuronal differentiation can proceed.
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21151987
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Figure 1. HES6 subgroups.(A) Location and orientation of the single mouse Hes6 and the two chick Hes6 genes in chromosomes 1 and 9, respectively. In both genomes, Hes6 genes are present in close proximity to the Period2 gene. (B) Sequence alignments of HES6 proteins from different species. HES6 protein sequences were obtained in Ensemble or NCBI and have the following accession numbers. Homo sapiens: HES6, ENSP00000272937. Mus musculus: HES6, ENSMUSP00000084062. Gallus gallus: HES6-2, ENSGALP00000008850. Xenopus tropicalis: HES6-1, ENSXETP00000013003; HES6-2, NP_001072210. Oryzias latipes: HER13.1, ENSORLP00000020797; HER13.2, ENSORLP00000019320; HER8, ENSORLP00000019320. Danio rerio: HER13.1, ENSDARP00000012990; HER13.2 ENSDARP00000021078; HER8.1, ENSDARP00000010206; HER8.2, ENSDARP00000103093. Accession number of Gallus gallus HES6-1 is unavailable, but can be identified in GenBank (BI393243). Shaded areas represent regions of homology. The HES6 proteins are divided in two subgroups: HES6-1 and HES6-2. The main protein domains are indicated. Amino acid residues that were mutated in our experiments are marked with asterisks. h: human; m: mouse; c: chick; x: Xenopus; meda: medaka; zf: zebrafish.
Figure 2. cHes6-1 expression pattern and response to Notch inhibition.(A-G) Expression of cHes6-1 at stages (A) HH4, (B) HH5, (C) HH6, (D) HH8, (E) HH9 (dorsal view), (F) HH9 (ventral view), (G) HH10. (Gi-Gvi) Sections through the regions marked with the white dashed lines on HH10 embryo in (G). Arrows in (D-F) point to the asymmetric expression of cHes6-1 in the mesoderm lateral to the primitive streak. (H-I) Ventral view of embryos showing expression of (H) cHes6-2 at HH12 and (I) cDelta1 at HH10 in pancreatic progenitors, identified by arrows. (J-N) Expression of cHes6-1 in embryos treated with the Notch signalling inhibitor LY411575. Down-regulation of cHes6-1 can be detected in the mesoderm lateral to the primitive streak (L, dorsal view) and up-regulation of cHes6-1 in pancreatic progenitors (M, ventral view) and neural tube (N, dorsal view), when compared to control embryos treated with PBS (J, dorsal, and K, ventral view). Arrows in (J-N) point to regions of the embryo where cHes6-1 expression if affected by Notch signalling inhibition. ep: epiblast; h: heart; hf: head fold; hn: Hensen's node; hp: head process; mlps: mesoderm lateral to the primitive streak; n: notochord; nf: neural fold; nt: neural tube; p: pancreatic progenitors; sr: sinus rhomboidalis.
Figure 3. Expression of cHes6-1 in differentiating cells of the spinal cord.(A-C) Expression of cHes6-1 (red) in spinal cord sections of (A) E3, (B) E4 and (C) E7 embryos. Nuclei were counterstained with DAPI (blue). (D-Li) cHes6-1 (red) is expressed in post-mitotic cells of spinal cord of E4 embryos, as shown by the absence of BrdU incorporation (30 minute pulse (green)) in these cells (D-Fi). However, cHes6-1-expressing cells are not fully differentiated, as demonstrated by absence of Tuj-1 co-labelling (green) (G-Ii). By contrast, in cranial ganglia cHes6-1 is expressed in Tuj-1+ differentiated neurons (J-Li). (Fi), (Ii), (Li) are magnifications of the selected areas in (F), (I), (L), respectively. Arrows identify cells where cHes6-1 expression coincides with Tuj-1. drg: dorsal root ganglion; n: notochord; nt: neural tube.
Figure 4. cHes6-1 expression relative to expression of various genes involved in neurogenesis.(A-Oi) Double in situ hybridization shows partial overlapping patterns and co-expression between cHes6-1 (green) and the following genes in spinal cord of E4 embryos: (A-Ci) cNeurog1, (D-Fi) cNeurog2, (G-Ii) cNeuroM, (J-Li) cHes6-2 and (M-Oi) cDelta1 (red). (Ci), (Fi), (Ii), (Li), (Oi) are magnifications of the selected areas in (C), (F), (I), (L), (O), respectively. Arrows indicate cells where genes are co-expressed. Horizontal lines in (F) and (Fi) separate the dorsal and ventral domains of the spinal cord. (P) Neuronal differentiation is a step-wise process: differentiating neural progenitors exit the IVZ (inner ventricular zone) and sequentially activate neuronal differentiating genes during migration to the ML (mantle layer). cHes6-1 is transiently expressed in the OVZ (outer ventricular zone), where it is co-expressed with various genes involved in neurogenesis.
Figure 5. Regulation of cHes6-1 expression.(A-Ci) Double in situ hybridization for cHes6-1 (green) and cNotch1 (red), showing no co-expression of the two genes in spinal cord of E4 embryos. (D-Li) cHes6-1 expression in the neural tube decreases after ectopic expression of NICD (D-Fi), and increases after ectopic expression of CSLDN (G-Ii) and NEUROG2 (J-Li). (Ci), (Fi), (Ii), (Li) are magnifications of the selected areas in (C), (F), (I), (L), respectively. Arrows pinpoint electroporated cells (GFP+) with increased expression.
Figure 6. cHES6 overexpression phenotypes.(A-Fi) Overexpression of cHES6-1 causes up-regulation of (A-Ci) cDelta1 and (D-Fi) cHes5-1. (Ci), (Fi) are magnifications of the selected areas in (C) and (F), respectively. Arrows pinpoint electroporated cells (GFP+) with increased expression. (G-J) Whole-mount analysis of cHes5-1 expression in electroporated embryos shows that overexpression of cHES6-2 down-regulates cHes5-1 (I–J), contrasting with the up-regulation caused by cHES6-1 overexpression (G–H).
Figure 7. Phenotypical analysis of overexpressed cHES6 variants.(A) Schematic representation of various modified HES6 proteins overexpressed in the chick neural tube. Altered functional domains are depicted in red. (B) Percentage of embryos showing up- or down-regulation of cHes5-1 expression upon transfection with the different variants of cHES6-1 (black bars), cHES6-2 (grey) and mHES6 (white).
Figure 8. Model of cHES6-1 and cHES6-2 function during neurogenesis.During neuronal differentiation in the developing spinal cord, cHES6-1 and cHES6-2 act sequentially to relieve the cell from Notch signalling: cHES6-2 acts first to repress the transcription of cHes5 genes and cHES6-1 subsequently sequesters and inactivates cHES5 and cHES6-2 proteins.
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