XB-ART-10360Development. October 1, 2000; 127 (19): 4203-16.
Hes6 acts in a positive feedback loop with the neurogenins to promote neuronal differentiation.
During the development of the vertebrate nervous system, neurogenesis is promoted by proneural bHLH proteins such as the neurogenins, which act as potent transcriptional activators of neuronal differentiation genes. The pattern by which these proteins promote neuronal differentiation is thought to be governed by inhibitors, including a class of transcriptional repressors called the WRPW-bHLH proteins, which are similar to Drosophila proteins encoded by hairy and genes in the enhancer of split complex (E-(SPL)-C). Here, we describe the isolation and characterization of Hes6, which encodes a novel WRPW-bHLH protein expressed during neurogenesis in mouse and Xenopus embryos. We show that Hes6 expression follows that of neurogenins but precedes that of the neuronal differentiation genes. We provide several lines of evidence to show that Hes6 expression occurs in developing neurons and is induced by the proneural bHLH proteins but not by the Notch pathway. When ectopically expressed in Xenopus embryos, Hes6 promotes neurogenesis. The properties of Hes6 distinguish it from other members of the WRPW-bHLH family in vertebrates, and suggest that it acts in a positive-feedback loop with the proneural bHLH proteins to promote neuronal differentiation.
PubMed ID: 10976052
Article link: Development.
Genes referenced: drg1 eef1a2 elavl3 esr1 hes1 hes4 hes5.2 hes6.1 hes6.2 hist2h2ab lgals4 myt1 neurod1 neurog1 nhlh1 notch1 runx1 snai2 sox10 stmn2 tbx2 tcf3 tubb2b zic2
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|Fig. 1. Xenopus and mouse Hes6 define a new family of WRPW-bHLH proteins. (A) Mouse (m) and Xenopus (x) cDNAs encode related WRPW-bHLH proteins with 72% sequence identity in the bHLH domains and 52% overall. (B) Alignment of the basic helix-loop-helix regions of the WRPW-bHLH proteins shown in C. Residues identical to those in mouse Hes6 are boxed. Note the overall higher level of sequence similarity between mouse and Xenopus Hes6 relative to the other WRPW-bHLH proteins, and the shorter loop domain. (C) Sequence similarity tree showing the relationship of Xenopus and mouse Hes6 to other WRPW-bHLH proteins identified in Xenopus (xEsr1, 4, 5, 6e and 7, Xhairy1 and Xhairy2a), rat (r) (Hes1 and Hes5), chick (cHairy1) and Drosophila (Dm) hairy, and Esplm8). Note that mouse and Xenopus Hes6 define a new subfamily. (D) Lanes 1-9, Hes6, Xhairy1 and Xhairy2A were expressed in bacteria as GST fusion proteins and purified by affinity chromatography on glutathioneagarose beads. 150 ng of each protein was mixed with a 32P labeled oligonucleotide containing an N-boxbinding site (Van Doren et al., 1994). For competition, each reaction included a 20-fold excess of unlabeled oligonucleotide, either wild type (wt) or mutant (mu) (see Materials and Methods). Up to 2 mg of Hes6 tested in this assay failed to give detectable binding. Lanes 10- 12; in vitro translated Hes6 (lane 11) and E12 (lane12) proteins were mixed with an E-box probe. Closed arrow denotes specific binding by the E12 protein. Open arrows indicate nonspecific shift, which is also observed in mock translated extract (lane 10).|
|Fig. 2. Expression of Mouse and Xenopus Hes6 during embryogenesis. (A-F) Hes6 RNA expression in Mouse embryos. (A) Expression is first seen at E8.75 in the midbrain (A, arrowhead) and primordia of sensory cranial ganglia (arrows). (B) Expression in DRG is noted at E9.25 (arrow). (C) By E9.5, Hes6 is expressed in the hindbrain and neural tube (arrowheads), and expression in DRG is clearly established (arrow). (D) By E10.5, extensive expression of Hes6 occurs in the DRG indicated by arrow and somite by arrowhead. (E) Expression of Hes6 in the intermediate zone in the neural tube (arrowhead; arrow points to DRG) and (F) in subregions of hindbrain (arrow points to Hes6- expressing cells and arrowhead indicates ventricular side) is shown in E10.5 embryo sectioned following whole-mount in situ hybridization. (G-J) In situ hybridization of adjacent sections at the hindlimb level of a E10.5 mouse embryo. Expression of NeuroD (H, arrowhead) and Hes6 (I, arrowhead) overlaps with that of Ngn1 (G) but is highest in the region adjacent to marginal zone where terminally differentiated neurons are marked by SCG10 (J, arrowhead). (K-M) Mouse embryos at 28-somite stage were probed with Hes6 (K), NeuroD (L), and Scg10 (M). The 12th DRG for each of the embryo is indicated by the arrows. Note that expression of Hes6 and NeuroD precedes that of Scg10 by three to four somites. Note also expression of Hes6 in the somites at this stage (K; arrowhead). (N-P) Hes6 RNA expression was localized in Xenopus embryos by whole-mount in situ hybridization. (N) Expression of Hes6 in the nervous system is first detected at open neural plate stages in scattered cells within the three domains where primary neurons form, including a medial domain (m) corresponding to motoneurons, and a lateral domain (l) corresponding to sensory neurons. (O) By late neurulae stages, higher levels of Hes6 expression are detected during secondary neurogenesis in the brain (arrow) and eye (arrowhead). Note also the expression of Hes6 in the tailbud domain (TBD). (P) Tissue section of late neurulae embryo at the level of the hindbrain (otic vesicle: OV) shows extensive Hes6 expression within the intermediate zone of the neural tube along the entire dorsal ventral axis but excluded from the floor plate (fp) and roof plate.|
|Fig. 3. Regulation of Hes6 expression in Xenopus and mouse embryos (A-D) Xenopus embryos were injected with RNA encoding ICD (A,B), Xngn1 (C) or Xash3 (D), at the two-cell stage along with lacZ RNA as a tracer. At neural plate stages the embryos were fixed, stained for X-gal, which produces a lightblue reaction product, and for the expression of Hes6 (A,C,D) or Esr7 (B) by whole-mount in situ hybridization, which produces a dark blue-purple staining pattern. Shown are dorsal views with the injected side oriented up, and anterior to the left. Note that both Xngn1 (39/41embryos) and Xash3 (50/51 embryos) induce the expression of Hes6, and that ICD induces the expression of Esr7 but not that of Hes6. (E-J) Requirement of Ngn1 for Hes6 expression in proximal cranial ganglia. (E) Ngn1+/-;Hes6tlacz/tlacZ embryo at E10 shows staining for X-gal in both proximal (arrowheads) and distal ganglia (arrows). (F) Ngn1-/-;Hes6tlacz/tlacZ embryo shows staining only in the distal ganglia (arrows). Hes6tlacz/tlacZ and Hes6tlacZ/+ embryos show identical X-gal staining patterns (data not shown). (G-J) Adjacent transverse sections of Ngn1+/-;Hes6tlacZ/+ embryo (G and I) and Ngn1-/-;Hes6tlacZ/+ embryo (H and J) at E9.5 are shown. Trigeminal neural crest cells are visualized by Sox10 in both embryos (G, H; arrows), but b-galactosidase is detected only in the Ngn1+/-;Hes6tlacZ/+ embryo (I,J; arrows).|
|Fig. 5. Hes6 promotes neuronal differentiation. (A-H) Two-cell-stage embryos were injected with Hes6 RNA along with lacZ RNA as a tracer. Embryos were fixed and stained with X-gal at neural plate stages and then processed for the staining for probes that correspond to various genes expressed during primary neurogenesis as indicated. Hes6 induces an increase in the number of cells expressing Xmyt1 (11/15 embryos), Drebrin (8/9), Nscl1 (10/10), Xmmot-1 (12/13), Elrc (11/11), N-tubulin 90/108), Xngn1 (15/17) and Xaml (10/10). (I,J) For comparison, embryos were injected with RNA encoding a dominant negative form of Su(H), XSu(H)DBM, and stained with probes of N-tubulin and Xngn1 (Wettstein et al., 1997). Note that the levels of Xngn1 increase on the injected side, but the striped pattern of expression is retained, and that N-tubulin-expressing cells subsequently form at a higher density in each stripe.|
|Fig. 6. Hes6 induces the expression of the hairy genes. (A-D) Hes6 was ectopically expressed along with lacZ in Xenopus embryos, which were processed at the neural plate stage as described in the legend to Fig. 3. Note that the expression of both Xhairy1 (B, 53/54 embryos) and Xhairy2a (C, 39/42 embryos) is upregulated in response to Hes6, while Zic2 expression (A, 34/37 embryos) and Slug expression (D, 23/30 embryos) do not change appreciably. (E) RNase protection analysis of Xhairy1 RNA levels in animal caps injected with RNA encoding Xhairy2a (0.5 ng, H2A), Xhairy2a-Gal4 (0.5 ng, H2AG4), Hes6 (0.5 ng, H6), Xhairy2a and Hes6 (0.5/0.5 ng, H6/H2A), a DNA-binding mutant of Hes6 (2 ng, H6DBM), or both Xhairy2A and a DNA-binding mutant of Hes6 (2/0.5 ng, H6DBM/H2A). Xhairy1 RNA levels are expressed in arbitrary units after normalizing to the levels of an endogenous control Ef1a RNA (Materials and Methods). Note that both Hes6 and Hes6DBM induce Xhairy1 expression above that in control animal caps (CONT), while Xhairy2A represses. Hes6 and the DNA-binding mutant reverses the inhibitory effect of Xhairy2a.|
|Fig. 7. DNA-binding mutant of Hes6 promotes neuronal differentiation. (A-D) RNAs as indicated were ectopically expressed along with lacZ in Xenopus embryos, which were processed at the neural plate stage as described in the legend to Fig. 3 for N-tubulin expression. Note that Hes6 (23/26 embryos) and the DNA-binding mutant of Hes6 (23/36 embryos) promote neuronal differentiation, while Esr7 inhibits (13/15 embryos). A DNA-binding mutant of Esr7 does not produce significant changes (14/15 embryos unchanged).|
|Fig. 8. Physical interactions between Hes6 and the hairy proteins. (A) Equivalent amounts of GST fusion protein bound to beads were incubated with 35S-labeled Hes6 and Xhairy2A and washed; the bound proteins were detected by gel electrophoresis and autoradiography (see Materials and Methods). 20% of the 35S labelled Hes6 and Xhairy2A added to each binding reaction is shown on the left, and the bound proteins are shown on the right. Note that both Hes6 and Xhairy2A do not show appreciable binding to GST alone, but bind to varying extents to each other. Notably Hes6 binds better to the hairy proteins than to itself, while Xhairy2A binds to all proteins. (B) Embryos were injected with RNA encoding a Myctagged form of Hes6 alone, or along with RNA encoding His-tagged forms of Xhairy2A, Xhairy1, Esr7 or Hes6. Note that Hes6 binds to both His-tagged Xhairy2a (H2A), and Xhairy1 (H1), but not to Esr7, or to itself.|