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Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling

¹ Developmental Genetics Program and the Department of Cell Biology, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA
² Harvard University, Department of Molecular and Cellular Biology, Cambridge, Curie Building, 45 Moulton Street, Cambridge, MA 02138, USA

View larger version (90K): [in a new window]   Fig. 2. Effects of ectopic Shh signaling on regional patterning in the telencephalon. Embryos were injected with ActSmo-expressing virus at E9.5 and analyzed at E14.5. Histochemical staining for PLAP reveals the location of virally infected cells co-expressing ActSmo (A,D,G,I,K,N). Adjacent sections were assayed for regionally expressed homeodomain genes (A-J) or for the expression of downstream targets of Shh signaling pathway (K-O). (A-C) Confocal immunohistochemistry for PLAP (A) and Pax6 (C) proteins within the lateral neocortex shows a repression of Pax6 expression in clusters of cells infected by ActSmo (asterisks). (G-J) Coronal sections of ActSmo-infected brains stained for ventral genes expression. Ectopic Nkx2.1 expression is restricted to the LGE and lateral neocortex (H, arrows), whereas Gsh2 expression can be induced throughout the neocortex (J, arrows). Dotted bracket illustrates weaker levels of induction within the medial neocortex. Sagittal sections (D-F) reveal a similar restriction along the rostrocaudal extent of the telencephalon. Asterisks in D-J represent the sites of endogenous expression. (K-O) Ectopic expression of ActSmo induces the upregulation of Gli1 and Ptch expression all along the dorsoventral (L,M, arrows and bracket) or anteroposterior (O) axes, showing that the telencephalon has the competency to respond to Shh signaling at this age. The mismatch between areas of viral infection and Ptch or Gli1 induction reflects the fact that these are visualized in adjacent sections. This is unavoidable because of the incompatibility of PLAP histochemistry and in situ hybridization. (P,Q) Schematic representations of a sagittal (P) and coronal (Q) section showing Nkx2.1 and Gsh2 endogenous domains of expression as well as the regions where their expression can be induced by Shh signaling.

View larger version (43K): [in a new window]   Fig. 1. Differential effects of Shh- or ActSmo-expressing retroviruses on brain morphology. Animals were infected in utero with control virus (A,B) and viruses expressing ActSmo (C,D) or Shh (E,F). Injections were performed at E8.5 (A,C,E) or E9.5 (B,D,F) and analyzed 4 days later (E12.5 or E13.5, respectively). Embryos injected with Shh virus showed a dramatic increase in the size of their heads, with substantially enlarged telencephalic vesicles (E,F). Despite the fact that the ActSmo virus was injected at a 10-fold greater titer than Shh virus, heads from these embryos appeared to have largely normal morphology (C,D).

View larger version (50K): [in a new window]   Fig. 3. Shh-/- and Gli3-/- mutants show opposite telencephalic phenotypes. Pan-ventral gene expression was analyzed in E10.5 Shh-/- (A-F) and E12.5 Gli3-/- (G-L) mutants. Dlx2 and Gsh2 expression is maintained but displaced ventrally in Shh-/- embryos (B,D,F). By contrast, Dlx2 and Gsh2 expression expands dorsally in Gli3-/- mutants (H,J,L). (K,L): Gsh2 and Pax6 expression domains abut at the corticostriatal boundary in wild-type animals (K, arrow). This boundary is affected in Gli3-/- embryos (L), with Gsh2 expression expanding dorsally. Dlx2 expression was assayed either by X-gal staining on whole-mount embryos (A,B) or by in situ hybridization on coronal sections (C,D,G,H). Gsh2 expression was assayed by immunofluorescence against the Gsh2 protein (E,F,I-L).

View larger version (74K): [in a new window]   Fig. 4. Dorsoventral patterning of the telencephalon is largely restored in Shh-/-;Gli3+/- mutants. Lateral (A-C) and dorsal (A'-C') views of the heads from wild-type, Shh-/- and Shh-/-;Gli3+/- E12.5 embryos, showing the extent of the phenotypic rescue. The telencephalon of Shh-/-;Gli3+/- embryos is formed of two paired vesicles (arrows), in sharp contrast to the phenotype of Shh-/- embryos. Note that the eye phenotype is also partially rescued as indicated by the presence of two distinct eyes, albeit fused at the midline and the proboscis observed in Shh-/- animals is reduced in size (asterisks in B,C; arrows in E,G,K,M; arrowheads in B-C'). (D-O) Coronal sections of wild-type and Shh-/-;Gli3+/- E12.5 embryos assayed for various region-specific homeobox gene expression. Dlx2 (D,E), Mash1 (J,K) and Gsh2 (L,M) expression in the mutants closely resembles that in wild-type embryos, showing that ventrolateral patterning is established normally in this context. Double immunohistochemistry for Gsh2 and Pax6 further demonstrate that LGE- and cortex-like structures are properly established in Shh-/-;Gli3+/- animals (N,O). Arrowheads delineate the boundary between the LGE and the cortex. In the ventral midline, a small region of Nkx2.1 expression is observed in Shh-/-;Gli3+/- embryos (G,I, asterisks). Overlay of Nkx2.1 (red) and Dlx2 (green) RNA in situ hybridization from adjacent sections using Adobe Photoshop 4, shows the nested pattern of Nkx2.1 within a broader Dlx2 domain in both wild-type and mutant embryos (H,I). (P-S) Coronal sections of wild-type and Shh-/-;Gli3+/- E12.5 embryos assayed for Gli1 (P,Q) and Ptch (R,S) expression, showing that the Shh pathway is not active. Brackets in P,R indicate the extent of Gli1 (P) or Ptch (R) expression.

View larger version (52K): [in a new window]   Fig. 5. Dorsoventral patterning is established properly in Shh-/-;Gli3-/- and Smo-/-;Gli3-/- double mutants. (A-L) Coronal sections of wild-type and Shh-/-;Gli3-/- E10.5 embryos. The telencephalic morphology appears relatively normal in the mutants, except for the loss of dorsal midline structures (arrows). (A-L) Analysis of various homeodomain genes expression. Dlx2 expression assayed by RNA in situ hybridization (A,B) and Gsh2 expression assayed by immunofluorescence (C,D) show that normal ventrolateral patterning is established in the double homozygous mutants. Arrowheads delineate the boundary between lateral and dorsal regions in both wild-type and mutant embryos. Nkx2.1 expression also appears normal (E,F). Overlay of Nkx2.1 (red) and Dlx2 (green) RNA in situ hybridization on adjacent sections using Adobe Photoshop 4 (G,H) shows a similar nested pattern of expression for these two genes in wild-type and Shh-/-;Gli3-/- double homozygous embryos. This reveals the existence of ventral and lateral structures similar to the MGE and LGE in the wild-type embryos (asterisk and arrowhead, respectively). Gli1 expression is absent in Shh-/-;Gli3-/- double mutants (I,J), showing that the Shh pathway is not active. Brackets in I indicate Gli1 expression. (K,L) Ptch is expressed at low levels in Shh-/-;Gli3-/- mutants, with higher expression in ventral areas (bracket), similar to wild-type embryos. (M,N) Analysis of ventral genes expression in Smo-/-;Gli3-/- E10.5 embryos. As a result of exencephaly, the dorsal telencephalic structures are disrupted. Despite this, the ventral structures can still be analyzed. Nkx2.1 and Dlx2 are expressed in ventral areas of the telencephalon. Despite the exencephalic morphology of the mutants ventral pattern appears unperturbed, as evidenced by the presence of Nkx2.1 expression nested within the broader Dlx2 domain. This suggests that MGE and LGE cell fates are specified properly in Smo-/-;Gli3-/- double mutants.

View larger version (15K): [in a new window]   Fig. 6. Model of genetic interactions between Shh and Gli3 in patterning the mouse telencephalon. (A) Schematic representation of a coronal section through an E10.5 mouse telencephalon, highlighting different domains along the dorsoventral axis. The prospective regions of the cortex (blue), the LGE (green) and the MGE (red) are shown. On the right, homeodomain genes are indicated that are expressed in these regions and have been shown to play an essential role in patterning the telencephalon (reviewed by Wilson and Rubenstein, 2000). Note that expression of Gsh2 is only restricted to the lateral telencephalon for a short window of embryonic development (E10.0-E10.5) (Corbin et al., 2000). Ultimately Gsh2 is expressed in both the LGE and MGE. (B) The results of the present study demonstrate that Shh is inducing ventral (Nkx2.1) and lateral (Gsh2, Dlx2) telencephalic patterning indirectly, through the inhibition of Gli3 repressor activity. Note that the regulation of Nkx2.1 expression appears to be different from that of pan-ventral genes, as it is not affected in Gli3-/- single mutants (bold blunt arrow). Gsh2 expression and Pax6 expression at the corticostriatal boundary are repressed by each other. However, the repression of Gsh2 expression by Pax6 appears to be regulated by Gli3 (asterisk), as co-expression of these two genes is observed in the cortex of Gli3-/- mutants. Our data reveal the existence of an unknown hedgehog-independent pathway (X).