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First published online 23 June 2004
doi: 10.1242/dev.01230

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Transduction of graded Hedgehog signaling by a combination of Gli2 and Gli3 activator functions in the developing spinal cord

Department of Neuroscience and Cell Biology, UMDNJ/Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA

View larger version (69K): [in a new window]   Fig. 1. Defects in Shh signaling in Gli2-/-;Gli3xt/xt mutant embryos. (A,B) Widespread and overlapping Gli2 and Gli3 expression in the ventral spinal cord at E10.5. Gli2 expression extends further ventral than Gli3 at this stage (yellow bars mark similar DV position). (C-J) Shh protein expression in the notochord and floorplate (FP). (C,D) Wild-type embryos express Shh in the notochord and FP. (E,F) In Gli2-/- mutants, FP expression of Shh is selectively lost (arrowhead), while notochord expression is retained. (G,H) In Gli3xt/xt mutants, Shh expression in the notochord and FP is similar to that in wild type. (I,J) Gli2-/-;Gli3xt/xt mutants do not show Shh expression in FP (arrowhead) but notochord expression is similar to Gli2-/- mutants. (K-N) Ptch1 mRNA expression. Yellow lines indicate wild-type dorsal expression boundary. (K) In wild-type embryos, Ptch1 expression is restricted to the ventral VZ, with higher levels in cells dorsal to the FP (lower arrowhead). Ptch1 is absent in the dorsal VZ (upper arrowhead). (L) In Gli2-/- mutants, strong ventral Ptch1 expression is not detected (arrowhead), but remaining VZ expression is similar to wild type. (M) In Gli3xt/xt mutants, Ptch1 expression in the ventral VZ is similar to wild type, but ectopic expression is detected in the dorsal VZ (arrowhead). (N) In Gli2-/-;Gli3xt/xt mutants, weak uniform Ptch1 expression is seen (n=3) even in dorsal regions (upper arrowhead). All sections are from E10.5 embryos.

View larger version (71K): [in a new window]   Fig. 3. MNs, V2 and V1 cells are generated in the ventral spinal cord of Gli2-/-;Gli3xt/xt mutant embryos but are intermingled. (A-C) MNs (HB9+) and V1 (En1+) cells are generated in double mutants but extend into the ventral midline. (C) Quantification of MN number in thoracic and lumbar regions of wild-type and Gli2-/-;Gli3xt/xt mutants. (D,E) In wild-type embryos, V2a (Chx10) and V2b (Gata3) cells develop as intermingled sub-populations dorsal to V1 cells. In Gli2-/-;Gli3xt/xt mutants, both classes of V2 cells were generated in the ventral midline but retained their intermingled organization. (F) Quantification of V2a and V2b cells in Gli2-/-;Gli3xt/xt mutants, compared with wild type. (G,H) In wild-type embryos, V0 (Evx1+) and V1 (En1+) cells differentiate as discrete populations. In Gli2-/-;Gli3xt/xt mutants, V0 cells show a limited expansion into the V1 domain. V0 and V1 intermingling is less pronounced at lumbar levels (data not shown). (I) Quantification of V1 cells in Gli2-/-;Gli3xt/xt mutants. (J,K) MNs, V2 and V1 neurons intermingle in Gli2-/-;Gli3xt/xt mutants. (L-P) Altered number of Olig2+ pMN cells in Gli2-/-;Gli3xt/xt mutants. Sections through mid-thoracic (L,M) and lumbar (O,P) regions. (N) Quantification Olig2 pMN numbers in wild-type and Gli2-/-;Gli3xt/xt mutants. Histograms indicate average cell counts/section, error bars indicate s.e.m. (Q,R) Pax6 expression in pMN cells. Pax6 is normally expressed weakly in dorsally derived MNs (bracket, Q). In Gli2-/-;Gli3xt/xt mutants, strong Pax6 expression extends to the ventral midline (R).

View larger version (53K): [in a new window]   Fig. 2. Complete absence of V3 interneurons in Gli2-/-;Gli3xt/xt mutants. (A,B) In wild-type embryos, Nkx2.2 and Nkx2.9 mark V3 ventral interneuron progenitors that form adjacent to the FP. (C,D) In Gli2-/- mutants, Nkx2.2 expression is greatly reduced, and only a few Nkx2.9 cells could be detected at E10 (C) but not at E10.5 (D). (E,F) In Gli2-/-;Gli3xt/xt mutants, neither protein was detected at any stages or axial level examined.

View larger version (104K): [in a new window]   Fig. 4. p1 and p2 progenitors expand into the ventral spinal cord and intermingle in Gli2-/-;Gli3xt/xt mutants. (A,B) In wild-type embryos, Mash1 is co-expressed with Nkx6.1 in p2 progenitors. In Gli2-/-;Gli3xt/xt mutants, Mash1+/Nkx6.1+ p2 progenitors are scattered throughout the Nkx6.1 domain. (C,D) In wild-type embryos, p1, p0 and pd6 progenitors express Dbx2 and are distinct from Mash1+ p2 progenitors ventrally and dorsally. In Gli2-/-;Gli3xt/xt mutants, Dbx2+ and Mash1+ progenitors are intermingled in the ventral spinal cord, but co-expression of these two factors was not seen. (E,F) In wild-type embryos, Jag1 is expressed between Mash1 domains, but is excluded from the p0 domain. In Gli2-/-;Gli3xt/xt mutants, only the ventral Jag1 expression domain in p1 progenitors has expanded ventrally, while the more dorsal domain, as well as the intervening Jag1-negative domain, were similar to wild type. Co-expression of Jag1 and Mash1 was not detected.

View larger version (94K): [in a new window]   Fig. 5. pMN progenitors intermingle with p2 and p0 progenitor in the ventral VZ in Gli2-/-;Gli3xt/xt mutants. (A) In wild-type embryos, Olig2 and Mash1 are expressed in non-overlapping domains in the ventral spinal cord. (B) In Gli2-/- mutants, the Olig2 pMN domain expands into the ventral midline, but normal segregation between p2 Mash1+ and Olig2+ pMN cells is maintained. (C) In Gli2-/-;Gli3xt/xt mutants, Olig2- and Mash1-expressing cells are intermingled, but no cells co-expressing these factors were found. (D) In wild-type embryos, Nkx6.2 marks the p1 and p2 progenitor domains in the VZ, as well as some post-mitotic MNs that form outside the Olig2+ pMN domain (arrowhead). (E) In Gli2-/- mutants, the dorsal Nkx6.2 domain in p0 and p1 progenitors is not altered, but ventrally Nkx6.2+ MNs extend across the midline (arrowhead), matching the ventral shift in Olig2+ pMN cells. (F) In Gli2-/-;Gli3xt/xt mutants, VZ cells expressing Nkx6.2 expanded into the ventral midline, and these cells are intermingled with Olig2+ pMN cells. No cells were found that co-express these factors. Nkx6.2 expression was also detected in some post-mitotic MNs that were located outside of the Olig2+ expression VZ domain in Gli2-/-;Gli3xt/xt mutants (arrowhead). (G-I) Schematics summarizing progenitor patterning in wild type, Gli2-/- and Gli2-/-;Gli3xt/xt mutants.

View larger version (93K): [in a new window]   Fig. 6. Misexpression of dominant activator forms of Gli2 and Gli3 activates ventral and represses dorsal factors. (A-H') Transfections of Gli2 and Gli3 cDNAs encoding N-terminally-truncated forms of the proteins (see schematic) using in-ovo electroporation in chick embryos. (A,B) Neither Gli2N-term nor Gli3N-term induced ectopic Shh expression when transfected at HH stage 12-14. Expansion of epithelium results from Gli2N-term transfections (arrowhead; also see C',E'). (C,D) Gli2N-term induced weak expression of Foxa2/Hnf3ß throughout the spinal cord, while Gli3N-term did not. (E,F) Both Gli2N-term and Gli3N-term induced robust ectopic Nkx2.2 expression throughout the spinal cord. Insets show higher power view of area indicated by arrowhead, excluding the blue channel. (G,H) Gli2N-term, but not Gli3N-term, induced Nkx6.1 expression in the dorsal spinal cord. (I-L) Both Gli2N-term and Gli3N-term suppressed Pax6 and Pax7 expression in the dorsal spinal cord. (M,N) Gli2N-term induced patchy upregulation of Ptch1 and Ptch2 transcription. (O,P) Gli3N-term induced Ptch1 weakly but Ptch2 strongly.

View larger version (65K): [in a new window]   Fig. 7. Activation of the Shh pathway in dorsal progenitors in the chick neural tube elicits cell clustering. (A,B) Transfection of Shh (green) elicits widespread, relatively uniform activation of Nkx2.2 (red) and repression of Pax7 (red) cell autonomously and non-autonomously. (C) GFP-only transfections have no effect on endogenous Pax7 or Nkx2.2 (not shown) expression. (D) Schematic illustrating broad activation of the Shh pathway (indicated by Nkx2.2 expression) in transfected (via autocrine signaling) and untransfected (via paracrine signaling) cells. Transfected cell is indicated by a lightening bolt. (E,F) Cotransfection of Shh with ptcloop2 (green) induces Nkx2.2 expression (red) only in untransfected cells. In this case Nkx2.2+ cells in the dorsal (F) but not ventral (F') spinal cord segregate into clusters. (G) Expression of Pax7 (red) in co-transfected cells (yellow) indicates autocrine Shh signaling is blocked. (H) Schematic representation of signaling differences between untransfected and Shh + ptcloop2 co-transfected cells. Only paracrine Shh signaling occurs. (I-K) Cells transfected with Gli2N-term or Gli3N-term (green) activators form clusters in the dorsal spinal cord (J) that do not express Pax7 (red), while ventral cells do not cluster (not shown). (L) Schematic showing cell-autonomous, ligand-independent activation of the Shh pathway by transfections of dominant-activator Gli2 and Gli3 proteins. Untransfected cells retain their dorsal identity. (M,N) Schematic summarizing results of chick transfection studies and Shh/Gli pathway in ventral spinal cord progenitor cells. Shh pathway activation is indicated by blue `+' areas. Blue outlined areas in the dorsal region represent clusters of cells, while circles indicate individual cells. In M, arrow thickness indicates relative roles of Gli2 and Gli3 activators in inducing ventral target genes.