Bao J et al. (2000), Regulation of neurogenesis by interactions betw...

XB-ART-11770

Development 2000 Jan 01;1272:425-35. doi: 10.1242/dev.127.2.425.

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Regulation of neurogenesis by interactions between HEN1 and neuronal LMO proteins.

Bao J , Talmage DA , Role LW , Gautier J .

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Basic-helix-loop-helix transcription factors regulate neurogenesis and neuronal differentiation by as yet unknown mechanisms. We show that an embryonic neuronal-specific basic-helix-loop-helix protein, HEN1 (also known as NSCL1 or NHLH), interacts with 'LIM only' proteins. Examination of the expression patterns of XHEN1 and XLMO-3, the Xenopus homologues of these human genes, reveals extensive overlap during early neurogenesis: at the onset of gastrulation on the dorsal side of the blastopore lip and, subsequently, in the prospective neural plate. Binding of XLMO-3 increases the transcriptional activity of XHEN1 in vivo. Co-expression of these two genes in Xenopus embryos induces a cascade of expression of neuronal-specific basic-helix-loop-helix proteins that leads to neuronal differentiation. We propose that XHEN1, in concert with XLMO-3, is a critical regulator of neurogenesis.

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Species referenced: Xenopus
Genes referenced: fos kit lmo1 lmo3 mespa myc neurod1 neurod6 neurog2 nhlh1 rpa1 tubb2b
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	Fig. 1. Specific interaction between HEN1 and LMO-1. (A) Testing the specific interaction of HEN1 with LMO-1 in yeast. The full-length LMO-1, or only one of its LIM domains (L1 or L2) fused in-frame with the transcription activator B42 in pJG4-5 vector, was transformed into the EGY48 yeast strain containing one of the following baits fused to LexA DNA-binding domain: HEN1 (full-length HEN1), HEN1-N (N-terminal of HEN1 without bHLH domain), DA (daughterless), NMYC, C-fos and CYC (cyclin C). Transformants were tested for specific interaction on glucose or galactose X-gal plates (galactose plate is shown here). The blue color indicates a positive protein-protein interaction. (B) Detection of complexes between HEN1 and LMO proteins by immunoprecipitation. Complexes were immunoprecipitated with anti myc-tag antibody from 293T mammalian cells cotransfected with HA-tagged HEN1 and one of the myc-tagged LMO proteins. The complexes were separated on the SDS-PAGE, and HEN1 protein was detected with anti-HA antibody. (C) Testing the binding of XLMO-3 to Xenopus neuronal-specific bHLH factors. The full-length XLMO-3 fused in-frame with the B42 in pJG4-5 was transformed into EGY48 yeast containing one of the following baits fused to LexA DNA-binding domain: XHEN1, X-NGNR-1, XNeuroD and XASH-3. Transformants were tested for specific interaction on galactose X-gal plates. The blue color indicates a positive protein-protein interaction. (D) Quantification of the strength of the interaction of XLMO-3 with Xenopus bHLH factors by measuring b- galactosidase activity. Yeast with XLMO-3 bait were transformed with Cyclin C (1), XHEN1 (2), X-NGNR-1 (3), XNeuroD (4) and XASH-3 (5). Five independent transformants from each group were picked and grown over 48 hours, and b- galactosidase activity was measured.
	Fig. 2. Sequence of XHEN1 and XLMO-3. (A) Alignment of the entire predicted amino acid sequences of Xenopus XHEN1 and human HEN1 and HEN2. Grey boxes indicate conserved amino acids. (B) Alignment of the bHLH regions of six Xenopus neuronal-specific bHLH proteins. (C) Alignment of the entire predicted amino acid sequences of Xenopus XLMO-3 and human LMO-1, LMO-2, LMO-3. Grey boxes indicate identical amino acids.
	Fig. 3. Spatial and temporal expression of XHEN-1 and XLMO-3 during early neurogenesis. (A) Detection of XHEN1, XLMO-3 and X-NGNR-1 during early development by RPA. The probes as indicated on the left side were made by the Dig-RNA labeling kit. XLMO-3 and XHEN1 are expressed earlier than X-NGNR-1. (B-J) Stage 10. 5, 11. 5 and 12 Xenopus embryos were hybridized with probes for XHEN1, XLMO-3 and X-NGNR-1. Uncleared embryos are shown in a dorsal view, with anterior to the left. XLMO-3 and XHEN1 are expressed in the same location.
	Fig. 4. Comparison of the expression patterns of XHEN1, XLMO-3, XNGNR- 1 and N-tubulin. (A-J) Stage 14, 25 and 35 Xenopus embryos were hybridized with probes for XLMO-3, XHEN1, X-NGNR-1 and N-tubulin. Embryos are shown in a dorsal (stage 14), dorsal-lateral (stage 25) or lateral (stage 35) view, with anterior to the left. At stage 14, XLMO-3 and XHEN1 are expressed only in two domains which will become interneurons (I) and motoneurons (M) of spinal cord, while X-NGNR-1 and N-tubulin are expressed also in the domains which will form sensory neurons (S) besides of these two medial domains. At stage 25, the expression patterns for all four probes are very similar. At stage 35, XHEN1 is only expressed in the brain region. (K,L) Transverse sections of Xenopus embryos hybridized with probes for XHEN1 and XLMO-3 at stage 20. In the neural tube, both of XHEN1 and XLMO-3 are located in the ventral part of spinal cord which is formed by interneurons and motoneurons.
	Fig. 5. Cooperative role of XHEN1 and XLMO-3 in vivo. (A) Transcriptional activity of XHEN1. The following plasmids were co-transfected with 10 mg pNEX-167, a reporter plasmid made of 5¢ regulatory region (167 bp) of NEX-1 gene fused to the firefly luciferase gene. C, control, reporter alone; H, 20 mg XHEN1; L, 20 mg XLMO-3; H/L, 20 mg each XHEN1 and XLMO-3; DH, 20 mg truncated XHEN1; DH/L, 20 mg each truncated XHEN1 and XLMO-3; N, 20 mg NEX. (B) Induction of N-CAM in animal caps by over-expression of XHEN1 and XLMO-3. EF-1a is a ubiquitously expressed mRNA and serves as a control for RT-PCR and loading. The bands from lanes of XHEN1 and XHEN1/XLMO-3 were quantified by NIH image 1.6. The optical density for the N-CAM and EF-1a band is 26.74 and 95.12 pixels/mm2 in the XHEN1 lane, 62.73 and 91.61 pixels/mm2 in XHEN1/XLMO-3 lane. After calibrating with the optical density of EF-1a band (the loading control), about 2.4 times more N-CAM was found to be induced by co-injection of XHEN1 and XLMO-3 than injection of XHEN1 alone. The mesodermal marker, muscle actin (M-Actin), is only induced in the positive control which are uninjected animal caps treated with activin (+). Untreated animal caps from uninjected eggs were used as the negative control (-). (C) Whole mount immunostaining with XAN-3 antibody of embryos injected with XHEN1 or/and XLMO-3. The main antigen for XAN-3 is N-CAM. The embryos were collected at stage 10 following the injection. There was no staining in the embryos injected only with XLMO-3 (left). Weak staining was found at stage 10 in the embryos injected only with XHEN1 (center), whereas strong staining was observed in the embryos co-injected with both XHEN1 and XLMO-3 (right).
	Fig. 6. Induction of X-NGNR-1 and XNeuroD Expression by XHEN1 and/or XLMO-3. (A-F) Embryos injected on one side with XLMO-3 (A,D), XHEN1 (B,E), or both (C,F) were hybridized at stage 9 with X-NGNR-1 (A-C) or XNeuroD (D-F) probe. Injection of XHEN1 mRNA induced endogenous X-NGNR-1 expression as well as endogenous XNeuroD expression, while co-injection of XHEN1 and XLMO-3 mRNAs induced more endogenous X-NGNR- 1 and XNeuroD. (G-K) Embryos injected on one side with XNGNR- 1 (G-I) or X-NeuroD (J,K) mRNAs were processed by in situ at stage 9 with probes for XNeuroD (G), XLMO-3 (XL; H,J) and XHEN1 (XI; I,K). X-NGNR-1 induced endogenous XNeuroD expression, but not endogenous XHEN1 and XLMO-3 expression.
	Fig. 7. Ectopic neurogenesis induced by XHEN1 and/or XLMO-3 mRNAs. (A) Ectopic neurogenesis was visualized at stage 14 and stage 25 embryos by whole mount in situ hybridization with an N-tubulin probe. Embryos were injected on one side (left side at the bottom) with XLMO-3, XHEN1, XHEN1/XLMO-3 or X-NGNR-1. All embryos are shown with anterior to the left. Only the injected half of stage 25 embryos is shown. (B) Downregulation of neurogenesis by overexpression of XHEN1-WPRW. Ectopic neurogenesis was visualized at stage 14 by N-tubulin staining. One cell of the 2-cell stage embryos were injected with 500 pg of mRNA encoding for XHEN1-WRPW. The injected half (bottom half) showed little N-tubulin expression (right panel) when compared to the uninjected control (left panel). (C) Temporal Induction of N-CAM and N-tubulin in animal caps by over-expression of XHEN1 and XLMO-3. EF-1a is a ubiquitously expressed mRNA and serves as a control for RT-PCR and loading.
	lmo3 (LIM domain only 3 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior left.