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. 2011;6(11):e27880.
doi: 10.1371/journal.pone.0027880. Epub 2011 Nov 16.

Hes6 is required for the neurogenic activity of neurogenin and NeuroD

Kasumi Murai  1 , Anna Philpott, Philip H Jones
Affiliations

Hes6 is required for the neurogenic activity of neurogenin and NeuroD

Kasumi Murai et al. PLoS One. 2011.

Abstract

In the embryonic neural plate, a subset of precursor cells with neurogenic potential differentiates into neurons. This process of primary neurogenesis requires both the specification of cells for neural differentiation, regulated by Notch signaling, and the activity of neurogenic transcription factors such as neurogenin and NeuroD which drive the program of neural gene expression. Here we study the role of Hes6, a member of the hairy enhancer of split family of transcription factors, in primary neurogenesis in Xenopus embryos. Hes6 is an atypical Hes gene in that it is not regulated by Notch signaling and promotes neural differentiation in mouse cell culture models. We show that depletion of Xenopus Hes6 (Xhes6) by morpholino antisense oligonucleotides results in a failure of neural differentiation, a phenotype rescued by both wild type Xhes6 and a Xhes6 mutant unable to bind DNA. However, an Xhes6 mutant that lacks the ability to bind Groucho/TLE transcriptional co-regulators is only partly able to rescue the phenotype. Further analysis reveals that Xhes6 is essential for the induction of neurons by both neurogenin and NeuroD, acting via at least two distinct mechanisms, the inhibition of antineurogenic Xhairy proteins and by interaction with Groucho/TLE family proteins. We conclude Xhes6 is essential for neurogenesis in vivo, acting via multiple mechanisms to relieve inhibition of proneural transcription factor activity within the neural plate.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Conserved domains in Xhes6 and mutants used in this study.
Xhes6 contains a conserved basic domain, required for DNA binding, a helix-loop-helix domain, implicated in dimerization with Xhes6 and other bHLH proteins, an “orange” domain, comprising the third and fourth helices of the protein, required for protein-protein interaction, and a C terminal WRPW motif, required for binding to Groucho/TLE family transcriptional coregulatory proteins. In this study a mutant of the basic domain which is unable to bind DNA (Xhes6 DBM) and a mutant lacking the WRPW motif (Xhes6 ΔWRPW) were used.
Figure 2
Figure 2. Xhes6 expression is required for neural differentiation.
Whole mount in situ hybridization of neurula stage embryos for neural markers neural β-tubulin (N-tubulin, A-H) or NeuroD (I-P). A, B, I, J: One cell of two cell stage embryos was injected with control (CTL, A, I) or Xhes6 (MO1, B, J) morpholinos along with β-gal tracer mRNA (red staining). Injection of MO1 decreases both N-tubulin and NeuroD expression on the injected side (yellow box). C-H, K-P: Rescue of MO1 phenotype. 500 pg of mRNA encoding wild type Xhes6 or DNA binding domain (DBM) or Groucho binding domain, (ΔWRPW) mutants was injected with or without MO1. Overexpression of Xhes6 (C, K) or DBM (E,M) alone enhances neural differentiation, both Xhes6 and DBM rescue neural maker expression in Xhes6 morphants (D, F, L, N). The ΔWRPW mutant has minimal effect when injected alone (G, O) and is less efficient in rescuing the MO1 phenotype. Q,R quantitation of phenotypes observed, scored as in Fig. S2, Q, N-tubulin mRNA expression, corresponding to panels A-H, R, NeuroD mRNA expression, corresponding to panels I-P. Full data on the frequency of phenotypes and the number of embryos analyzed is given in Table 1.
Figure 3
Figure 3. Xhes6 is required for the function of Xngn2 and NeuroD.
Xngn2 or NeuroD mRNA was injected into one cell of two cell stage embryos with or without control (STD CTL) or Xhes6 (MO1) morpholinos. At neurula stage, embryos were analysed for expression of transcripts encoding N-tubulin (A−F) or NeuroD (G−J) by in situ hybridization. Injection of mRNAs encoding Xngn2 and NeuroD increases the number of primary neurons (C, E) but this effect is blocked by co-injection of the Xhes6 MO (D, F). Xhes6 is also required for Xngn2 to induce its target gene, NeuroD (J). K-L quantitation of phenotypes seen, scored as shown in Fig. S1; K, L effects of MO1 on Xngn2 induced expression of N-tubulin (K) and NeuroD (L) mRNA, M, effect of MO1 on N-tubulin mRNA expression induced by NeuroD. Full data on the frequency of phenotypes and the number of embryos analyzed is given in Table 2.
Figure 4
Figure 4. Effects of Hes6 and Xhairy1 proteins on Xngn2/E12 DNA-binding activity.
A DNA fragment containing E-box element from the mouse NeuroD promoter was labeled with 32P and incubated with the in vitro translated proteins shown. The reaction mixture was then analyzed on a native polyacrylamide gel. (A) Effect of Xhes6 on DNA-binding activity of Xngn2/E12. Arrow indicates complex of Xngn2/E12 and DNA. Free probes are shown by arrowhead. (B) Effect of Xhairy1 on DNA-binding activity of Xngn2/E12. Faint bands with slow mobility (thin arrow) are non-specific protein and DNA complexes. Thick arrow indicates Xngn2/XE12 probe complex. Neither Xhes6 and Xhairy1 affect heterodimer formation or the DNA binding activity of Xngn2/XE12 proteins.
Figure 5
Figure 5. Xhairy1 inhibits the proneurogenic activity of Xngn2 and NeuroD.
Embryos either uninjected (A) or with mRNAs encoding Xhairy1 (B), Xngn2 (C), Xhairy1 plus Xngn2 (D), NeuroD (E) or NeuroD plus Xhairy1 (F) injected into one cell at the two cell stage were analyzed for N-tubulin expression by in situ hybridization at neurula stage. G,H quantitation of phenotypes seen, scored as shown in Figure S2; Full data on the frequency of phenotypes and the number of embryos analyzed is given in Tables 3, 4, and 5.
Figure 6
Figure 6. Physical Interaction of Xhes6 and Xhairy1.
HA tagged Xhairy1 and myc tagged Xhes6 proteins (wild type, DNA binding mutant (DBM) and WRPW deletion mutant (ΔWRPW)) were translated in vitro and mixed as indicated. Following incubation with the antibody shown immunocomplexes analyzed by sodium dodecyl sulfate gel electrophoresis after which tagged proteins were detected by Western blotting. Wild type and both mutant forms of Xhes6 protein bind Xhairy1.
Figure 7
Figure 7. Inhibition of Xhairy1 by Xhes6 does not require the Xhes6 Groucho binding domain.
Expression of N-tubulin transcript in uninjected embryos (A) or embryos in which mRNA encoding Xhairy1 (B), Xhes6 (C), Xhairy1 plus Xhes6 (D), Xhes6ΔWRPW mutant (E), Xhairy1 plus Xhes6ΔWRPW (F) or Xhairy1ΔWRPW mutant (G) was injected into one cell at the two cell stage. H: quantitation of changes in N-tubulin mRNA expression, scored as shown in Figure S2. Full data on the frequency of phenotypes and the number of embryos analyzed is given in Table 6.
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