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First published online 1 June 2005
doi: 10.1242/dev.01878

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Gsh2 is required for the repression of Ngn1 and specification of dorsal interneuron fate in the spinal cord

Sonja Kriks¹, Guillermo M. Lanuza^1,*, Rumiko Mizuguchi^1,*, Masato Nakafuku² and Martyn Goulding^1,

¹ Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
² Department of Cell and Developmental Biology, Children's Medical Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA

View larger version (108K): [in a new window]   Fig. 1. Gsh2 is expressed in the progenitors of the dI3, dI4 and dI5 neurons in the embryonic spinal cord. (A-D) A subset of Pax7+ cells express Gsh2. (E,F) At E10.5 and E11.5, Gsh1/Gsh2 expression encompasses dI3, dI4 and dI5 progenitor domains. (G,H) Mash1 is expressed in the same dorsal progenitor domain as Gsh1/Gsh2. (I,J) The bHLH protein Ngn1 is expressed in adjacent domains dorsal and ventral to Gsh2+ and Mash1+ progenitors. (K) Gsh2 is homogeneously expressed in all Pax3+ dI3 progenitors. (L) Ngn1 marks 50% of Pax3+ cells in the dI2 ventricular domain. (M) Ngn1 and Gsh2 are expressed in adjacent domains in the dorsal neural tube; this image is an overlay of staining for Gsh1/Gsh2 and Ngn1 of the adjacent sections shown in K and L. (N) Ventricular dividing cells, marked by BrdU, express Gsh2 in dI3 progenitors. (O) Ngn1 is expressed by some of the cells marked after a 1.5-hour pulse of BrdU. (P-S) The Gsh1/Gsh2-positive domain gives rise to dI3, dI4 and dI5 neurons. (P,Q) dI4 neurons expressing both Lhx1/Lhx5 and Lbx1 arise ventrolateral to the most dorsal Gsh2+ domain, whereas the most ventral Gsh2+ domain gives rise to Lbx1+ dI5 neurons that do not express Lhx1/Lhx5. (R,S) Isl1+ and Brn3a+ dI3 neurons arise from the most dorsal Gsh2+ progenitor domain. (T) Schematic summary of the expression of several transcription factors in alar plate progenitors.

View larger version (111K): [in a new window]   Fig. 2. Gsh2 mutant embryos do not generate dI3 neurons. (A-F) Changes in dorsal neuron differentiation in E10.5 Gsh2 knockout (KO) embryos. (A) Lbx1+ dI4-dI6 neurons are still generated in the Gsh2-/- mutant spinal cord. (B) Foxd3+ dI2 neurons are increased in Gsh2-/- mutant embryos, whereas Isl1+ dI3 neurons are almost completely absent (C). The arrow in C indicates the few remaining dI3 cells. (D) Ventral Brn3a expression (arrow) is unchanged in the mutant, indicating a normal development of dI5 neurons. (E) The dorsal population of Tlx3+ neurons (arrow) is missing in the Gsh2 mutant cord, confirming the loss of dI3 neurons, while Tlx3 expression in dI5 neurons is not affected. (F) The loss of Isl1+ dI3 neurons is partially offset by an increase in Foxd3+ dI2 neurons. (G-L) Changes in transcription factor expression at E11.5 in Gsh2 mutant embryos. In Gsh2-/- mutants, Lhx1/5+ (G) and Foxd3+ neurons (H) are generated from the dI3 progenitor domain (arrows in G and H) at the expense of Isl1+ (I) and Otp+ (K) dI3 neurons (arrows). (J) There is a slight dorsal expansion of dI4 neurons expressing Pax2 (arrow). (L) Cell counts of Foxd3- and Isl1-positive cells in Gsh2 KO mice (-/-) show a loss of >95% of dI3 neurons and a concomitant increase in dI2 neurons, when compared with age matched wild-type embryos (+/+). Drg, dorsal root ganglion.

View larger version (70K): [in a new window]   Fig. 3. Gsh2 is required to establish the boundary between the dI2 and dI3 progenitor domains. (A-D) Gsh1 expression in dI4 and dI5 progenitors (brackets) is largely unchanged in Gsh2-/- embryos, although the Gsh1 domain shows a slight dorsal expansion when compared with wild-type (WT) embryos (arrow in D). (E,F) Ngn1 expression expands ventrally into the presumptive dI3 progenitor domain, as does Ngn2 expression (K,L), although to a lesser extent than Ngn1. (G,H) Mash1 expression is reduced in presumptive dI3 progenitors but is maintained in the adjacent dI4 and dI5 progenitor domains (brackets). (I,J) The expression of Dbx2 in dI6 progenitors is unchanged demonstrating that the boundary between the dI5 and dI6 progenitors is not altered in the Gsh2 mutant cord. The ventral boundary of Ngn1 at the dI5/dI6 border is also unchanged (E,F, see arrows). (M) Schematic summary of the changes in dorsal progenitor specification in the alar plate, showing a concomitant reduction of Mash1 expression and expansion of Ngn1 expression in the dI3 progenitor domain.

View larger version (132K): [in a new window]   Fig. 4. Gsh1 mutant embryos do not present any obvious defects in early dorsal interneuron development. (A-D) Expression of postmitotic marker proteins for the six different interneuron populations in the dorsal half of the spinal cord is unchanged in E11.5 Gsh1 mutant embryos. (E,F) Gsh2 and Mash1 also show normal expression patterns, confirming the normal development of dI3-dI5 progenitor populations.

View larger version (104K): [in a new window]   Fig. 5. Expression of Msx1, Olig3, Dbx2 and Ngn1 in the Gsh1/Gsh2 mutant spinal cord. (A-D) Msx1 and Olig3 expression in Class A progenitors is unchanged in E10.5 Gsh1/Gsh2 double mutants. The dashed line indicates the boundary between dI3 and dI4 progenitors. (E,F) Expression of Dbx2 in dI6 progenitors is unchanged in the double mutant spinal cord, indicating the Gsh proteins do not repress Dbx2. (G,H) Ngn1 expression expands into the dI3-dI5 progenitor domain in the Gsh1/Gsh2 mutant cord.

View larger version (101K): [in a new window]   Fig. 6. Reduced generation of dI3 neurons in Mash1-/- embryos. (A,C) Immunohistochemical detection of Isl1 protein shows that dI3 neurons are significantly reduced in Mash1 mutants at E10.5 and E11.5. (B,D) Gsh1/Gsh2 expression is slightly reduced in E10.5 Mash1 knockouts (B, arrowhead) but is unchanged in E11.5 Mash1-/- embryos (D). (G,H) Concomitant with the decrease in Isl1+ cells, there is an expansion of Foxd3+ (arrowhead) and Lhx1/Lhx5+ (asterisk) dI2 neurons into the prospective dI3 population. (E,F) At E10.5, Ngn1 expression expands ventrally into the prospective dI3 progenitors in the Mash1 mutant (arrow). (I-P) Electroporation (EP) of Mash1 in the chick neural tube; the electroporated side is shown on the right, control side on the left. (I,J) Isl1 is strongly induced after Mash1 overexpression, suggesting an induction of dI3 neurons (arrowhead). (K,L) Moreover, Mash1 misexpression leads to a reduction of Lhx1/Lhx5, indicating that Mash1 initiates the dI3 differentiation program at the expense of dI2 and dI4 neurons. (M,N) Ectopic Gsh2+ cells are found 24 hours after Mash1 overexpression (arrowheads in N), suggesting a positive-feedback mechanism onto Gsh2 to maintain dI3 progenitor identity. (O,P) Mash1 overexpression represses ventral and dorsal Ngn1 expression (arrow) 20 hours after electroporation.

View larger version (107K): [in a new window]   Fig. 7. Gsh2 can induce Isl1+ dI3 neurons. Framed areas in A and D are shown enlarged in C and F, respectively. (A-C) Misexpression of Gsh2 leads to an induction of ectopic Isl1+ dI3 neurons that is less dramatic than the induction seen following Mash1 overexpression. Arrowheads in B indicate ectopic Isl1+ cells. Additionally, this induction occurs in a non-cell autonomous manner (C, arrowheads). (D-F) Isl1 induction by Gsh2 is independent of Mash, as only very few ectopic Mash1+ cells are seen following Gsh2 overexpression. (G-I) Both Ngn1 and Ngn2 are strongly repressed (arrowheads) by Gsh2 misexpression, and this repression is apparent in both their dorsal and ventral expression domains.

View larger version (107K): [in a new window]   Fig. 8. Misexpression of Ngn1 in the chick neural tube leads to a repression of dI3 progenitor identity. Left and right sides in A-H show the control and electroporated halves of the neural tube, respectively. (A,B) Ngn1 overexpression leads to a reduction in Isl1+ dI3 neurons (arrowhead in B) due to decreases in Gsh2 (C,D, arrowhead in D) and Mash1 (E,F, arrowhead in F) protein expression. (G,H) Pax2+ dI4 and dI6 interneurons are unaffected by Ngn1 overexpression. (I-L) Expression patterns of Gsh2 and Mash1 in Ngn1/Ngn2 double knockouts. (I,J) Gsh2 expression expands beyond the dI5/dI6 boundary in Ngn1-/-/Ngn2-/- embryos (arrow), whereas the dorsal boundary is unchanged. (K,L) Mash1 is not markedly expanded dorsally in Ngn1/Ngn2 deficient mice, although a few ectopic Mash1+ cells are found dorsal to the dI2/dI3 boundary (arrowheads). Mash1 extends ventrally in the Ngn1/Ngn2 double-mutant spinal cord, when compared with wild type (arrow). (M,N) Tlx3 is hardly changed in Ngn1/Ngn2 double knockouts, although a few Tlx3+ cells are found dorsal to the dI2/dI3 boundary (arrowhead).

View larger version (16K): [in a new window]   Fig. 9. Schematic of the proposed genetic interactions between Gsh2, Ngn1 and Mash1. Arrows symbolize an induction; bars signify a repression. Solid lines indicate probable direct genetic effects, whereas dashed lines indicate likely indirect interactions.

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