Lee J et al. (1997), Gli1 is a target of Sonic hedgehog that induces...

XB-ART-16319

Development 1997 Jul 01;12413:2537-52. doi: 10.1242/dev.124.13.2537.

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Gli1 is a target of Sonic hedgehog that induces ventral neural tube development.

Lee J , Platt KA , Censullo P , Ruiz i Altaba A .

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The vertebrate zinc finger genes of the Gli family are homologs of the Drosophila gene cubitus interruptus. In frog embryos, Gli1 is expressed transiently in the prospective floor plate during gastrulation and in cells lateral to the midline during late gastrula and neurula stages. In contrast, Gli2 and Gli3 are absent from the neural plate midline with Gli2 expressed widely and Gli3 in a graded fashion with highest levels in lateral regions. In mouse embryos, the three Gli genes show a similar pattern of expression in the neural tube but are coexpressed throughout the early neural plate. Because Gli1 is the only Gli gene expressed in prospective floor plate cells of frog embryos, we have investigated a possible involvement of this gene in ventral neural tube development. Here we show that Shh signaling activates Gli1 transcription and that widespread expression of endogenous frog or human glioma Gli1, but not Gli3, in developing frog embryos results in the ectopic differentiation of floor plate cells and ventral neurons within the neural tube. Floor-plate-inducing ability is retained when cytoplasmic Gli1 proteins are forced into the nucleus or are fused to the VP16 transactivating domain. Thus, our results identify Gli1 as a midline target of Shh and suggest that it mediates the induction of floor plate cells and ventral neurons by Shh acting as a transcriptional regulator.

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Species referenced: Xenopus laevis
Genes referenced: foxa2 foxa4 gli1 gli2 gli3 myc nkx2-1 shh spon1 srsf2 tbx2 tra
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	Fig. 1. Comparison of deduced amino acid sequences of Gli1 proteins. (A) Comparison of the amino acid sequences of the zinc finger DNAbinding domains of Gli family members. The five zinc fingers are denoted by roman numerals and the consensus sequence is shown on top. Dots refer to conserved residues. Spaces introduced to maximize alignment are denoted by dashes. The cysteines and histidines of the finger structure predicted to establish contacts with the zinc ion are shown in bold. Note the high degree of conservation in the last three zinc fingers, which are the only ones to contact DNA as determined in a protein-DNA co-crystal structure (Pavletich and Pabo, 1993). The frog fGli1, fGli2 and fGli3 sequences derive from this work. The human hGli1 and hGli3 sequences derive from Kinzler et al. (1988) and Ruppert et al. (1990). The human tax-helper-protein (THP) derives from Tanimura et al.(1993). The mouse mGli1, mGli2 and mGli3 sequences derive from Hui et al. (1994). The fly cubitus interruptus (ci) sequence derives from Orenic et al. (1990). The nematode tra-1 sequence derives from Zarkower and Hodgkin (1992). (B) The deduced amino acid sequences of the full-length frog and human Gli1 proteins are shown aligned to highlight conserved regions. The five zinc finger DNA-binding domain is shown boxed and other conserved regions underlined. The human Gli1 (hGli1) sequence is from Kinzler et al. (1988). The frog Gli1 (fGli1) sequence has been submitted to GenBank under accession number U57454.
	Fig. 2. Gli gene expression in frog embryos; expression of Sonic hedgehog (Shh), Gli1, Pintallavis (Plvs), Gli2, Gli3, neural-tubulin (N-tub) and HNF-3b at different stages as indicated on the side. (AE) At early gastrula stages (~stage 10.5) Shh RNA (A) is not yet detected and that of Pintallavis is clearly seen in the organizer region (C). Expression of Gli1 (B) and Gli3 (E) but not Gli2 (D) is detected in the animal cap, mostly on the dorsal side. All panels show a vegetal view with dorsal side up. (F-J) At midgastrula stages (stage ~11.5), expression of Shh (F) and Pintallavis (H) is detected in the nascent notochord (nc) as seen in cleared embryos. Noncleared embryos show the expression of Gli1 in midline (md) neural plate cells (G), the expression of Gli2 in non-midline neural plate (np) cells (I) and that of Gli3 (J) in the neural plate folds (nf) and posterior mesoderm (pm). All panels show dorsal views from different angles and the direction of the A-P axis is indicated (a, anterior; p, posterior). (K-O) At neurula stages (stage ~14), expression of Shh and Pintallavis is detected in midline (md) neural plate cells (K, M) whereas that of Gli1 is found in cells lateral to the midline (lmd; L). Gli2 (N) is found in neural plate cells with the exception of the midline (md) and anterior area overlying the prechordal plate whereas Gli3 (O) is detected in a graded fashion with highest levels laterally and absent from the midline. All panels show dorsal views with anterior end up. (P-T) Cross sections show expression of Shh (P) in deep (d) and not superficial (s) midline neural plate cells overlying the notochord (n) at stage ~14. Gli1 expression is also detected at stage ~11.5 in deep midline cells (Q) but, by stage 14, expression is predominantly found in cells lateral to the midline (an anterior section is shown here, R; compare to panel V). Expression in the somites (so) is found also adjacent to the notochord (n). Gli2 (S) is detected throughout the neural plate and somites but not in midline neural plate cells or notochord and Gli3 (T) is detected in a graded fashion with highest levels laterally both within the neural plate and somites. (U-X) Gli1 expression at stage 12.5 is normally found in cells lateral to the midline (lmd; V) but it is not detected in the neural ectoderm (ne) of complete exogastrulae (exog; U) at this time or at earlier stages (not shown). This lateral to the midline expression coincides with the position of the first Ntubulin expressing cells (W), most likely primary motor neurons, which precede the appearance of the well-characterized three stripes of primary neurons in the midneurula embryo located in a medial (m), intermediate (I) and lateral (l) position (stage ~14, X). Expression is also detected in trigeminal ganglion neurons (tg). All panels show dorsal views with anterior end up. bp: blastopore. (YZa) Expression of Gli1 (Y), Gli2 (Z) and Gli3 (Za) in the anterior area of a tadpole (stages ~32-36) embryo. Note the similarity in the expression of Gli1 with that of Shh (Fig. 6C). Gli1 is expressed in the lateral and dorsal diencephalon, medial hindbrain, dorsal rhombomeres 1 and 3, otic vesicle and eye. It is also expressed in the frontonasal (fn) process and branchial arches (not shown) but not in the infundibulum (i). Gli2 (Z) and Gli3 (Za) are mostly coexpressed in the dorsal neural tube including the telencephalon (tel). High Gli3 expression is detected at the midbrain/hindbrain boundary (mb/hb) and diencephalon/midbrain boundary. Coexpression of the three Gli genes is detected in the eye, otic vesicle, frontonasal process and branchial arches. (Zb-Zg) Cross sections of the hindbrain-cervical spinal cord of tadpoles (stages ~34-36). (Zb,Zc) The expression of HNF-3b protein and Shh mRNA is detected in floor plate (fp) cells. Note the basal position of nuclei expressing HNF-3b and the ventricular position of the Shh mRNAcontaining cytoplasm. Gli1 (Zd) is expressed in the ventricular zone with the exception of the floor plate. Gli2 (Ze) and Gli3 (Zf) are coexpressed in the dorsal ventricular zone with low levels or no expression in the roof plate (rp). Differentiated neurons (dn) expressing N-tubulin are found distal form the ventricle (Zg). Scale bar is 0.5 mm in A-E,H
	Fig. 3. Neural expression of Gli genes in mouse embryos. Expression of transcripts in the posterior neural plate at E8.5-8.75 (E8.5-P; A-E), in the anterior neural plate at E8.5 (E8.5-A; F-H) and in the spinal cord at E9.5 (E9.5-SC; I-M) and E11.5 (E11.5-SC; N, O). (A,F,I,N) Expression of Gli1 mRNA. Gli1 is expressed throughout the early neural plate (A), is absent from the floor plate in slightly older stages (F) and is then found restricted to the ventral ventricular zone including cells adjacent to the floor plate (I,O). Strong Gli1 labeling is detected in the condensing sclerotome surrounding the neural tube (O). (B,G,J) Expression of Gli2 mRNA. Gli2 is expressed throughout the early neural plate (B) and is absent from the floor plate in slightly older stages (G). In the closed neural tube, Gli2 is expressed in the dorsal aspect including the ventricular zone (J). (C,H,K) Expression of Gli3 mRNA. Gli3 is also expressed throughout the early neural plate (C) and is absent from the floor plate in slightly older stages (H). In the closed neural tube, Gli3 is expressed in a manner similar to that of Gli2 (J) with highest expression in the dorsal ventricular zone (K). (D,L) Expression of HNF-3b mRNA. HNF-3b is first expressed in the neural ectoderm in midline cells of the neural plate shortly after floor plate (fp) induction by the underlying notochord, which also expresses this gene. In the early neural plate (D) midline cells express HNF-3b and in the closed neural tube it is found in the floor plate and in a small group of immediately adjacent cells (compare L and M). (E,M,O) Expression of Shh mRNA. The early neural plate does not express Shh but high expression is detected in the underlying notochord (nc, E). In the closed neural tube (M), Shh expression is maintained in the notochord (nc) and new expression is detected in the floor plate. At later stages, weak expression of Shh is detected in the motor neuron pools (mn) in limb regions lateral to the floor plate (fp), which maintains high expression (O). Scale bars are 30 mm in (A-H) and 50 mm in (I-O).
	Fig. 4. Diagram showing the ability (+) or inability (-) of different Gli family members and chimeric proteins to induce ectopic HNF-3b expression in injected embryos. The maps are shown to scale centered around the conserved 5 zinc finger DNA-binding domain (black box in fGli1). See text for details.
	Fig. 5. Subcellular localization of Gli proteins. (A,C,E,G) Localization of Gli proteins in gastrula (stage ~12) injected frog embryos. Insets at lower right show a higher magnification of labeled cells. Labeling was visualized with the peroxidase reaction. All panels show a dorsal view. (B,D,F,H,I-L) Localization in transfected mammalian COS cells. Nuclei in B, D, H and K were counterstained with DAPI, shown in blue. Labeling was visualized by epifluorescence. A,B) Myc-tagged frog Gli1 protein localizes both to the cytoplasm (left inset) and nuclei (right inset). (C,D) A myc-tagged frog Gli1 protein bearing a nuclear localization signal (NLS) in its N terminus localizes primarily to the nucleus although some cytoplasmic labeling is evident. (E,F) Human glioma Gli1 localizes to both the nuclei and cytoplasm with cell-to-cell variability in the amount of cytoplasmic labeling (F). (G,H) Myc-tagged human Gli3 protein localizes to the cytoplasm primarily although a small number of cells showed weak nuclear labeling. (I) Frog HNF-3b protein localizes to the nuclei. (J) Human Gli1 protein (in green) is sometimes detected in nuclear subdomains distinct from those containing the splicing protein SC35 (in red). (K) Cotransfected human Gli1 and Gli3 proteins show a colocalization in the cytoplasm. Gli1 (green) is primarily nuclear whereas Gli3 (red) is mostly cytoplasmic. Part of the cytoplasm is colored yellow due to the normal presence of some Gli1 protein in this compartment (see J). A dark-blue stain depicts weak DAPI labeling to show the position of unlabelled nuclei. (L) Myc-tagged hGli3 protein is detected in a fibrelike pattern in the cytoplasm in apparent association with the cytoskeleton. Scale bar is 160 mm in A,C,E,G; 10 mm in B,D,F,H; 6 mm in I,K,L; 4 mm in J.
	Fig. 6. Ectopic differentiation of floor plate cells driven by injected Gli1. Expression of HNF-3b (A,B,D,E), Shh (C,F,G) and F-spondin (J,K) in normal controls (c; A,C,J) and hGli1-injected (inj) tadpole (stage ~36) embryos (B,D-G, K) viewed in whole mount. Arrows point to sites of ectopic expression. All panels show side views for whole mounts with anterior end to the left and dorsal side up for cross sections. (A) Normal distribution of HNF- 3b protein in the floor plate (fp) as well as in cells, possibly neurons, in the ventral midbrain and near the zona limitans intrathalamica (zli) in the diencephalon (di). HNF-3b protein can also be seen in the forming gut tube (g) and in the endoderm of the gill area (ge). Expression is completely absent from the telencephalon (tel). (B,D,E) Expression of HNF- 3b protein after injection of hGli1 (B,E) or myc-fGli1 (D). Note the ectopic expression in the neural tube in the dorsal diencephalon and midbrain as well as in more medial areas. Cells expressing ectopic HNF-3b are detected throughout the brain (B,D), including the diencephalon and telencephalon (B,D), hindbrain (hb) and spinal cord (sc; D,E). (C) Normal distribution of Shh mRNA in tadpole embryos. Within the neural tube, Shh mRNA is found in a similar pattern to that of HNF-3b including expression in the floor plate (fp), the zone limitans intrathalamica (zli) and in a stripe in the lateral diencephalon (ldi). There is no expression in dorsal areas or in the telencephalon. Shh mRNA is also found in the notochord (nc), frontonasal process (fn), branchial arches (ba) and in a small region of the ventral anterior endoderm (ven). See also Ruiz i Altaba et al.(1995a). (F,G) Ectopic expression of Shh mRNA in hGli1-injected tadpoles. Ectopic expression is detected in the brain in the telencephalon (G), dorsal diencephalon (F,G), midbrain and hindbrain (F,G). (H,I) Ectopic expression of HNF-3b in the epidermis (epi) of hGli1-injected embryos. The expression in the floor plate (fp) is seen out of focus (H). (I) A cross section showing HNF-3b + cells in the epidermis. (J) Normal expression of F-spondin mRNA in the floor plate (fp). Expression is also detected in a small group of cells near the zona limitans intrathalamica and in the forebrain commissures (c). tel, telencephalon. (K,L) Ectopic expression of F-spondin in hGli-injected embryos seen in whole-mount (K) or cross section (L). Ectopic expression is detected throughout the dorsal neural tube. Endogenous expression in the floor plate (fp) in K is out of focus. (M-P) Expression of ectopic HNF-3b in cross sections of embryos injected with hGli1 plus RLDx (M,N) or alone (O,P). Since embryos were injected into one cell at the 2-cell stage, the ectopic expression of HNF-3b is usually unilateral. The injected side is marked (inj). (M,N) Localization of ectopic HNF-3b expression in the telencephalon (M) and spinal cord (N) with expressing cells colabeled with coinjected RLDx (in pink; M, N) and not colabeled (right most dorsal cell in N). Of all counted cells (n=27), 48% of HNF-3b+ cells were labeled with RLDx and 52% were not. (O,P) Ectopic expression of HNF-3b throughout the D-V extent of the neural tube (midbrain) in the injected half (arrows in O). In addition to cells showing the nuclear size and intensity of labeling of those in the floor plate, there are others with large nuclei normally located in a position suggestive of their neuronal nature (asterisks). These cells are also found at ectopic locations in the injected side (asterisk in P). Along the A-P axis, sites of ectopic expression of HNF-3b, Shh and F-spondin in the telencephalon constituted 13%, 25% and 18%, in the dorsal diencephalon 13%, 25% and 17%, in the ventral diencephalon 6%, 3% and not determined, in the dorsal midbrain 10%, 17% and 23%, in the dorsal hindbrain 21%, 17% and 32% and in the spinal cord 37%, 13% and 10% of all sites, respectively (with n=106, 40 and 147, respectively). Along the D-V axis, the roof plate displayed a disproportionate incidence of ectopic HNF-3b sites (31% of sites, n=246) given its small size (<1/10th the size of the basal and alar plates), as compared to the basal (25% of sites) or alar (19% of sites) plates. The ventricular zone showed a small number of sites (15%). Scale bar is 1 mm in (A-H,J,K) and 25 mm for (I,L-O).
	Fig. 7. Ventral neuronal differentiation induced by widespread expression of Gli1. Expression of serotonin (5HT; A-E) and Nkx2.1 (F-H) in control and Gli-1injected embryos. Arrows depict ectopic expression. Panels show lateral (A-D; F-H) views for whole mounts with anterior end to the left; in E dorsal side is up. (A) Normal expression of 5HT+ neurons in the ventral anterior hindbrain of stage ~36 tadpoles. Immunoreactivity is detected in the soma and axons growing both cranially and caudally. Control embryos: 19 neurons unilaterally ±0.6 s.e.m.; n=26. (B) Normal and ectopic differentiation of 5HT+ neurons (arrow) in the hindbrain of an injected tadpole. Note the dorsal position of the ectopic cell bodies. Injected embryos: 8% ectopic sites, n=302 with 19 neurons unilaterally ±2.4 s.e.m.; n=13. The relatively low percentage of embryos displaying ectopic 5HT+ neurons is expected and significant. The incidence of ectopic Shh or HNF-3b in the hindbrain was 17% and 21%, respectively. Since the expected incidence of ectopic Shh and HNF-3b expression in the anterior hindbrain, where 5HT+ neurons differentiate, is less than half of that detected in the entire hindbrain or less than 9% and 11%, these values are very close to the observed 8% incidence of ectopic 5HT+ neuronal differentiation. (C,D) Details of the ectopic differentiation of 5HT+ neurons in the hindbrain of injected embryos. Note the aberrant pathway of labeled axons (arrows). (E) Cross section through the anterior hindbrain of an injected embryo showing the unilateral ectopic differentiation of 5HT+ neurons in the alar plate (arrow) and the bilateral presence of endogenous 5HT+ neurons adjacent to the floor plate (fp). (F-H) Expression of Nkx2.1 protein in the forebrain of normal control (F) and embryos injected with NLSmyc- fGli1 (G) or hGli1 (H). Note the normal expression in ventral diencephalic (vdi) and ventral telencephalic (vt) cells. The boundary of the telencephalon is denoted by a broken line in F-H. The notochord (n) sheath shows cross reaction with the antisera. Scale bar is 0.5 mm in A,B, 200 mm in C,D,H, 20 mm in E and 270 mm in F,G.
	Fig. 8. Ectopic midline gene expression induced by Shh and Gli1 at neural plate stages. (A-C) Ectopic expression of Shh (A) and Pintallavis (B) after injection of hGli1 (A) or VP16- fGli1 (B). Ectopic gene expression was detected in small patches or single cells, mostly outside of the neural plate. The normal expression of Shh and Plvs in midline cells (md) is shown out of focus (A,B). (C) Ectopic Gli1 (D) expression driven by injected Shh. Arrows point to sites of ectopic expression. Panels show dorsolateral views with anterior en up. a; anterior; p: posterior. Scale bar is 200 mm.
	Fig. 9. Schematic diagram of Gli1 function in ventral neural tube patterning by Shh. The diagram depicts Shh signaling from the notochord to the medial neural plate, the future ventral neural tube, with Gli1 functioning in the nuclei of responding cells downstream of the cytoplasmic signal transduction machinery. Gli1 is proposed to induce both floor plate and immediately adjacent ventral neuronal cell types. In a pathway for floor plate differentiation, Gli1 is proposed to act upstream of Pintallavis/HNF-3b which in turn regulate downstream floor plate genes such as F-spondin and Sonic hedgehog. The lack of Gli1 expression in floor plate cells and differentiated ventral neurons at late gastrula stages suggests Gli1 transcription is repressed in these differentiated cells. It is unclear at present how the concentration-dependent effects of Shh on ventral cell type differentiation are interpreted by Gli1 although differential levels of Gli1 activity remain a possibility. See text for details.