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First published online 11 September 2008
doi: 10.1242/dev.027078

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Temporal progression of hypothalamic patterning by a dual action of BMP

MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.

View larger version (75K): [in this window] [in a new window]   Fig. 1. Temporal expression of Shh, BMP signal pathway components and Pax7 in the chick hypothalamus. (A-H) At stage 9-10, Shh, Ptc1 and Gli1 are detected in prechordal mesoderm (white arrow, A) and/or hypothalamic floor plate (blue arrow, A). Bmp7 is detected in prechordal mesoderm (arrow, E), but Bmp7, Msx1/2 and pSmad1/5/8 are not detected in the hypothalamic floor plate. Pax7 is detected in roof plate cells (H), but not floor-plate cells, marked by expression of Nkx2.1 (red). (I-P) At stage 15-17, Shh is expressed in Nkx2.1+ ventrolateral cells (arrow, I; Nkx2.1 expression shown in red in J); Ptc1 and Gli1 show some overlap with Shh, but extend more dorsally. Expression of Bmp7, Msx1/2 and pSmad1/5/8 is detected in hypothalamic floor plate cells. (Q-W) At stage 22-25, ventrolateral cells that span the neuroepithelium express Shh. Expression is largely detected in cell bodies and end-feet. Shh mRNA appears to be largely confined to VZ cells. Ptc1 and Gli1 are detected dorsal to Shh-expressing cells. Bmp7, Msx1/2 and pSmad1/5/8 are detected in ventrolateral cells, which now express Pax7. (X) Robust expression of Pax7 is detected in ventrolateral cells at stage 30. (A') Pax7+ cells form in Nkx2.1+ cells located furthest from the VZ. Many Pax7+ cells co-express Nkx2.1, but a minority of Pax7-single positive cells can be detected. (B'-G') Pax7 marks basal progenitors of the ventrolateral hypothalamus. Gsh+ cells are located at the VZ/SVZ boundary. The majority of Gsh+ cells lie just medial to Pax7+ cells, but some cells co-express Gsh and Pax7 (arrows, E'). Pax7+ cells co-express Ki67 (F', arrows), and are distinct from postmitotic Isl1+ cells (G').

View larger version (81K): [in this window] [in a new window]   Fig. 2. Bmp7 upregulates Gli3 and specifies Pax7+ basal progenitors. (A) Schematic showing pHyp dissection (green box) from stage 5-7 embryos and culture in collagen. (B-J) Sections through explants, analysed by double-colour immunofluorescent labelling for expression of Nkx2.1/Pax7 (B-F) or Gsh/Pax7 (G-J). Pax7+ cells, either adjacent to, or co-expressed with, Nkx2.1 and Gsh (arrows), are detected in the presence of prechordal mesoderm (PM; B,G), Bmp7 (D,I) and cyclopamine (F). (H,I) Quantitative analysis of pHyp explants cultured with (I) or without (H) Bmp7. Quantitative analysis reveals that expression of Pax7 does not occur at the expense of Gsh+ cells (mean number of Gsh+ cells: in control=150±5.8, in explants with Bmp7=156±8.2; mean number of Pax7+ cells: in control=0.4±0.2, in explants with Bmp7=171±9.1). (F) Pax7+ cells, many of which co-express Nkx2.1 are found in pHyp explants cultured in the presence of cyclopamine. (K) Quantitative analysis on pHyp explants cultured alone, with Bmp7 or with cyclopamine. Bars show mean number of Pax7+ cells/section. Cyclopamine can significantly increase the number of Pax7+ cells (control=4.9±1.3 cells; cyclopamine=47.7±10.2 cells; P<0.0024; unpaired t-test), but approximately threefold more Pax7+ cells are detected with Bmp7. (L-S) Gli3 expression in vivo (L-O) and in pHyp explants (P-S). (L) At stage 8, Gli3 is not detected in the ventral/ventrolateral hypothalamus (arrow). (M-O) At stage 25, Gli3 expression is detected in ventral and ventrolateral regions of the hypothalamus, expression confined to VZ/SVZ cells (arrows in M,N). (P,Q) Gli3 expression is not detected in a stage 6 pHyp explant cultured alone (P), but is upregulated in the presence of Bmp7 (Q). (R,S) Stage 15 ventral hypothalamic explants, dissected from region shown (box in inset, R). Gli3 expression is detected when ventral hypothalamic regions are explanted at stage 15 and cultured to a stage 25 equivalent (R). Expression is abolished when explants are cultured with chordin (S).

View larger version (119K): [in this window] [in a new window]   Fig. 3. Antagonistic actions of GliA and GliR regulate Pax7 expression. (A-I;K-N) Transverse section through hypothalamus of chicks electroporated at stage 10-12 and developed until stage 25-30. White dots indicate lumen; electroporated sides are to the left of the lumen in images A-N. (A-D) Electroporation of Gli1 (GliA) abolishes Pax7 expression (A,B). A control GFP construct has no apparent effect on Pax7 (C,D). (E-H) Electroporation of a Gli3 RNAi contruct leads to the downregulation of Gli3 (E,F; adjacent sections). (G,H) Electroporated cells show a marked downregulation of Pax7; neighbouring non-electroporated cells develop with normal Pax7+ identity. (I) Co-electroporation of Gli3 RNAi and a Gli3R shows rescue of Pax7+ cells; i.e. similar numbers of Pax7+ cells are detected on non-electroporated and electroporated sides. (J) Percentage of Pax7+ cells on electroporated versus non-electroporated sides of the hypothalamus. (Gli1 overexpression=20±8 (s.e.m.); GFP=97±3; Gli3 RNAi=27±6; Gli3 RNAi + Gli3=93±4). Residual Pax7 expression detected after Gli1 overexpression or Gli3 knockdown appears to be in non-electroporated cells (e.g. G). (K, L) Gli3 RNAi has no effect on Lim1+ expression. (M, N) A control luciferase RNAi construct does not affect Pax7 expression.

View larger version (71K): [in this window] [in a new window]   Fig. 4. Dual BMP action specifies Pax7+ ventral progenitors in the hypothalamus. (A-J) Transverse sections through stage 25 hypothalamus, after electroporation at stage 10. (A, B) Co-electroporation of Gli3R and an RFP expression plasmid. Ectopic Pax7+ cells are observed (white arrows), but are restricted to the dorsal region of the hypothalamus, close to endogenous sites of Pax7 expression at the roof plate. (C, D) Co-electroporation of Smad5 and RFP. No ectopic Pax7+ cells are detected. (E-H) Co-electroporation of GFP, Gli3R and Smad5. Ectopic Pax7+ cells are detected throughout the dorsoventral axis. G and H show high-power views of boxed regions (E',F'). White arrows show cells co-expressing Pax7 and GFP. (I, J) Co-electroporation of Smad5 and GFP. No ectopic expression of Gli3 is detected. (K) Model for the specification of Pax7+ ventral progenitors in the developing hypothalamus. Asterisk (*) indicates that Gli3R de-represses Pax7. (L-O) Stage 6 pHyp explants cultured with Bmp7 or a combination of cyclopamine (Cyc) and dorsomorphin (DM). No expression of Pax7 or pSmad1/5/8 is detected with cyclopamine and dorsomorphin. Inset shows that expression of Gli3 is detected under both conditions.

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