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First published online 25 June 2008
doi: 10.1242/dev.021535

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Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline

¹ Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
² Kyoto University Graduate School of Biostudies, Kyoto 606-8502, Japan.
³ Japan Science and Technology Agency, CREST, Kyoto 606-8507, Japan.
⁴ RIKEN Brain Science Institute, Saitama 351-0198, Japan.

View larger version (51K): [in this window] [in a new window]   Fig. 1. Formation of Cajal-Retzius cells from Msx1+ domain of the dorsal telencephalic midline. (A,A') Orientation of sections. (B-D'') Coronal sections of E10.5 embryos. Msx1 was expressed in the prospective choroid plexus region (B). Some Msx1+ cells expressed Ngn2 (C-C'', arrowheads) but not DCX (D-D''). (E-M'') Transgenic mice carrying the Msx1 promoter-driven EGFP reporter were examined for lineage tracing of Msx1+ cells. At E10.0, EGFP was specifically expressed by Msx1+ cells (E-E''). At E10.5, EGFP was expressed in the prospective choroid plexus region (G) like the endogenous Msx1 (F). Subsets of EGFP+ cells expressed Ngn2 (H-H'',K-K'', arrowheads), Tuj1 (I-I'',L-L'', arrowheads) and p73 (J-J'',M-M'', arrowheads) at both E10.5 and E11.5, suggesting that some Msx1+ cells differentiated into Cajal-Retzius cells. di, diencephalon; tel, telencephalon. Scale bars: 50 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 2. Formation of Cajal-Retzius cells and choroid plexus epithelial cells in the dorsal telencephalic midline. pEF-EGFP was introduced into the dorsal telencephalic midline at E9.5 by in utero microelectroporation, and the fate of EGFP+ cells was examined at the indicated time points. For the orientation of the planes, see Fig. 1A,A'. (A-F) At E10.5, all EGFP+ cells resided in the Msx1+ region (A-C). At E11.5, many EGFP+ cells migrated tangentially into the cortical neuroepitheium (D,E), and some of them had already reached the marginal zone of the piriform cortex (D,F). (G-Q) At E12.5, all cells that migrated laterally seemed to have reached the piriform cortex (G, arrow). Only two regions, the choroid plexus epithelium (the origin, asterisk) and the piriform cortex (the destination, arrow) were labeled with EGFP (G). The cells that remained at the origin expressed transthyretin (Ttr) (H,I,O,P) but not reelin (Q). The cells that migrated into the piriform cortex expressed Lhx5 (J,K, adjacent sections), Tuj1 (L,L') and reelin (M,M', arrowheads) but not calretinin (N,N'). di, diencephalon; tel, telencephalon. Scale bars: 150 µm in A,D; 500 µm in G; 50 µm in L-Q.

View larger version (98K): [in this window] [in a new window]   Fig. 3. Expression of Hes1 and Hes5 in the dorsal telencephalic midline. (A) Scheme of a dorsal view of E12.5 forebrain. Coronal sections are made along the broken line. (B) Schemes of the coronal sections of the dorsomedial telencephalon at E10.5-E12.5. (C-F) At E10.5, the dorsal telencephalic midline expressed Bmp4 and Lmx1a (C,D). Hes1 was likewise expressed in this region as well as in the neighboring diencephalic and telencephalic neuroepithelium (E). Hes5 was expressed at a lower level in this region than in the telencephalic neuroepithelium (F). (G-N) At E11.5, the Bmp4 and Lmx1a expression domain was elongated (G,H). Msx1 and Ttr were expressed in differentiating choroid plexus epithelium (I,J), whereas Wnt2b was expressed in the prospective cortical hem (K). Hes1 expression was gradually downregulated in Ttr+ cells (L). Although Hes5 expression was upregulated in the telencephalon and the diencephalon, it was also downregulated in Ttr+ cells (M). Wnt2b+ and Msx1+ domains were clearly separated at this stage (N). (O-T) At E12.5, the choroid plexus epithelial cells became thin and cuboidal (R, Ttr+), whereas the cortical hem was still pseudostratified. Bmp4 and Lmx1a were expressed in both regions (O,P). Msx1 expression occurred mainly in the choroid plexus epithelium (Q), whereas Wnt2b expression occurred in the cortical hem (R). Hes1 and Hes5 expression was almost completely repressed in the choroid plexus epithelium (S,T). Scale bars: 100 µm in C-F; 200 µm in G-T.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Generation of Hes1 cKO mice. (A) Strategy for generation of Hes1 cKO mice. (B) Genomic DNA from drug-resistant cells was digested with HindIII and analyzed by Southern blot using a 0.6-kb HindIII-BamHI fragment as a 5'-probe, which detected wild-type and floxed fragments (6.0 kb), floxed-neo (7.8 kb) and deleted fragments (3.8 kb). (C-E) β-Galactosidase activity in the forebrain of Emx1-Cre;R26R mice at E10.5-E12.5. (F-O) Hes1 and Hes5 were expressed in the dorsal telencephalon of the control (Bmp4+, F,L) at E10.5 and E11.5 (G,H,M). In Hes1 cKO mice, Hes1 expression was downregulated around E10.5 (J, asterisk) and was lost by E11.5 (O, asterisk). Insets of M and O show immunohistochemistry for Hes1. Hes1 protein expression was lost in Hes1 cKO mice by E11.5. Hes5 expression was upregulated in Hes1 cKO mice (K). di, diencephalon; tel, telencephalon; hem, cortical hem. Scale bars: 100 µm in C,D,F-O; 200 µm in E.

View larger version (81K): [in this window] [in a new window]   Fig. 5. Defect of the choroid plexus and increase of Cajal-Retzius cell formation in Hes1;Hes3;Hes5 cKO mice. (A-F) HE staining of the dorsal telencephalic midline. In Hes1;Hes3;Hes5 cKO mice, the dorsal midline cells were not flattened but remained pseudostratified at E11.5 and E12.5 (D,D',E,E', asterisks). Even at E15.5, no choroid plexus was formed in Hes1;Hes3;Hes5 cKO mice (F, asterisk). (G-J) Ttr was expressed in the control at E11.5 and E12.5 (G,I) but not in Hes1;Hes3;Hes5 cKO mice (H,J). (K-N,R-U) Cajal-Retzius cells (reelin+,Lhx5+) of the mutant piriform cortex were increased in number (L,N,S,U arrows), compared with the control (K,M,R,T) at both E11.5 and E12.5. (O-Q) The number of reelin+ cells was quantified by counting DAPI+ cells on every five sections from four independent embryos for each genotype. *P<0.01, t-test. Scale bars: 100 µm in A,D; 200 µm in B,E,G-N; 250 µm in C,F; 50 µm in R-U.

View larger version (129K): [in this window] [in a new window]   Fig. 6. Bmp signaling and homeodomain gene expression in Hes1;Hes3;Hes5 cKO mice. (A-G') At E11.5, in Hes1;Hes3;Hes5 cKO mice, the expression domains of Bmp4, Lmx1a, Msx1, Msx2 and Wnt3a were reduced in size, compared with the control, although Foxg1 and Lhx2 expression (cortex) was not significantly affected. (H-M') At E12.5, in the control, the telencephalic midline region was clearly separated into the choroid plexus epithelium and the cortical hem, and Bmp4 and Lmx1a were expressed in both regions (H,I). In Hes1;Hes3;Hes5 cKO mice, the prospective choroid plexus region remained pseudostratified, and Bmp4 and Lmx1a expression domain was smaller in size (H',I'). Msx1 and Msx2 expression domains were also reduced in size (J',K', asterisks), whereas expression of Wnt3a (cortical hem) and Lhx5 (eminentia thalami) was not significantly affected (L',M'). Scale bars: 100 µm in A-G'; 200 µm in H-M'.

View larger version (67K): [in this window] [in a new window]   Fig. 7. Upregulation of proneural genes in the dorsal telencephalic midline of Hes1;Hes3;Hes5 cKO mice. (A-D) Neurogenesis (Tuj1+) was enhanced in the dorsal telencephalic midline (brackets) of Hes1;Hes3;Hes5 cKO mice at E10.5 and E11.5 (B,D, asterisks), compared with the control (A,C). (E,F) Double immunostaining for Ngn2 and Hes1 in wild-type embryos. Many cells co-expressed Ngn2 and Hes1 at E10.5 (arrowheads), but the expression was mostly segregated at E11.5 (arrows). (G-S) In the dorsal telencephalic midline region (Lmx1a+) of control mice, Ngn1 and Ngn2 expression occurred at low levels in subsets of cells at E10.5 (G-I) and was down-regulated at E11.5 (J,K,Q,Q'). By contrast, Ngn1 and Ngn2 expression was highly upregulated in Hes1;Hes3;Hes5 cKO mice at both E10.5 and E11.5 (L-P, asterisks; R,R',S). *P<0.01; **P<0.001, t-test. Scale bars: 50 µm in A-D,E1,F1,G-I,L-N; 100 µm in J,K,O,P; 20 µm in Q-R'.

View larger version (34K): [in this window] [in a new window]   Fig. 8. Misexpression of Ngn2 inhibits choroid plexus formation and enhances Cajal-Retzius cell formation in the dorsal telencephalic midline. (A-G) pEF-EGFP alone (A-C) or the Ngn2 expression vector together with pEF-EGFP (D-F) was introduced into the dorsal telencephalic midline at E9.5 by in utero microelectroporation, and the region was analyzed at E12.5. Misexpression of Ngn2 inhibited formation of the choroid plexus (E', asterisk) and increased the number of Cajal-Retzius cells in the piriform cortex (F,G). (H-K) Ngn2 was overexpressed in the prospective choroid plexus and cortical hem regions by electroporation at E11.5, and the coronal sections were examined at E12.5. Forced expression of Ngn2 at E11.5 increased Cajal-Retzius cell (reelin+) formation in the cortical hem (H,I, arrows) but did not affect the choroid plexus development (J,K). *P<0.05, t-test. Scale bars: 200 µm in A,B,D,E,H-K; 50 µm in C,F.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Summary of developmental defects of the dorsal telencephalic midline region of Hes1;Hes3;Hes5 cKO mice. (A) In Hes1;Hes3;Hes5 cKO mice, the choroid plexus epithelium (cpe) is lacking, and Cajal-Retzius (CR) cell formation in the piriform cortex (pir) is enhanced. (B) Ngn2 promotes Cajal-Retzius cell formation, whereas Hes genes regulate specification of the choroid plexus epithelium by antagonizing Ngn2. In Hes1;Hes3;Hes5 cKO mice, Ngn2 expression is upregulated and Cajal-Retzius cell formation is enhanced, whereas choroid plexus epithelial cells are lacking. ctx, neocortex; hem, cortical hem; emt, eminentia thalami.

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