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doi: 10.1242/10.1242/dev.00590

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Distinct regulators of Shh transcription in the floor plate and notochord indicate separate origins for these tissues in the mouse node

Yongsu Jeong and Douglas J. Epstein

Department of Genetics, University of Pennsylvania School of Medicine, Clinical Research Building, Room 470, 415 Curie Blvd, Philadelphia, PA 19104, USA



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  		Fig. 1. Conservation of Shh regulatory sequences across phyla. (A) A schematic of the Shh locus showing the location of coding exons (black boxes), noncoding sequences (solid line) and intronic enhancers (gray oval, Sbe1; yellow oval, Sfpe2). The sequence of the 746 bp region of intron 2 overlapping Sfpe2 activity in the mouse was compared with human, chicken and zebrafish, identifying three regions of high sequence homology corresponding to homology region-a (HR-a, blue), HR-b (green) and HR-c (red). (B) Reporter constructs (RC) designed to assay Sfpe2 activity. DNA segments from the 746 bp region were cloned downstream of a reporter cassette containing a minimal Shh promoter (Ius), lacZ gene (light-blue box) and Sbe1 sequences (gray oval). To the right of the constructs are the results of the transgenic expression analysis indicating the total number of transgenic embryos generated for each construct (#Tg) and the number of embryos that stained in the ventral midline of the spinal cord (SC) and midbrain (MB). The consistency of staining intensity and minimal time to initiate staining were used to subjectively classify the spinal cord expression as strong (+++), moderate (++), weak and patchy (+/–), or absent (–). The lacZ expression generated in the midbrain is regulated by Sbe1 and serves as a positive control for transgene expression. Rc1 and Rc2 were reported previously (Epstein et al., 1999Go). (C) X-gal staining of transgenic embryos carrying reporter constructs at 9.5 dpc. The cranial staining depicted by the embryo carrying Rc1 is also representative of the staining pattern seen in embryos expressing Rc3-6.

 


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  		Fig. 2. Functional conservation of the 88 bp homology region-c (HR-c) element. X-gal staining of transgenic embryos at 9.5 dpc carrying three copies of HR-c from mouse (A,D), chicken (B,E) and zebrafish (C,F). Transverse sections through the spinal cord at the level of the forelimb of representative embryos revealed strong floor plate and notochord expression from mouse (D) and chicken (E) but not zebrafish (F) DNA. The ectopic staining from Rc11 was seen in all six transgenic embryos. The broken line in D,E mark the boundary between the floor plate and notochord.

 


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  		Fig. 3. Homology region-c (HR-c) is responsive to Shh signaling. Neural explants isolated from wild-type (A,C) or Shh-/- (B,D) embryos carrying Rc9 were cultured in the presence (+) or absence (–) of N-Shh for 30 hours. A few wild-type cells showed X-gal staining when cultured in the absence of N-Shh (possibly reflecting their prior exposure to Shh in vivo), however this number was significantly increased when cultured in the presence of N-Shh (compare A with C, n=16). No cells showed X-gal staining in explants derived from Shh-/-, RC9+ embryos grown in the absence of N-Shh (B), in marked contrast to the significant number of positive stained cells from the corresponding explant grown in the presence of N-Shh (D, n=3).

 


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  		Fig. 4. Requirement of conserved DNA binding sites in homology region-c (HR-c) on floor plate and notochord expression. Alignment of HR-c sequences from mouse, human, chicken and zebrafish is shown at the top. Conserved sequences are shaded in gray. DNA recognition sequences matching the consensus for homeobox (blue), Foxh1 (green), T-box (pink) and Foxa (yellow) transcription factors are shown. (A-E) X-gal staining of transgenic embryos at 9.5 dpc carrying reporter constructs with (A) wild-type Rc9, or (B-E) mutant DNA binding sites. Adjacent to each embryo is a transverse section through lumbar regions of the spinal cord. Underneath each figure is a schematic of the reporter construct. The table to the right indicates the total number of transgenic embryos generated for each construct and their patterns of expression. The arrow in B points to the floor plate, which is devoid of X-gal staining. NC, notochord.

 


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  		Fig. 6. The homeodomain binding-sites in homology region-c (HR-c) are required at the onset of floor plate expression. Whole-mount X-gal staining of embryos between 6 and 13 somites from reporter lines of mice expressing wild-type Rc9 (A,C,E,G), or mutated homeobox Rc12 (B,D,F,H) transgenes. Ventral midline expression from Sfpe2 is delayed as early as the 9-somite stage in embryos expressing Rc12 (compare C with D). Ectopic lacZ expression was detected in the somites of one of the lines carrying the homeobox mutation. G and H show ventral views of the floor plate after the notochord has been removed as shown in the diagram. Transverse sections of spinal cords from Rc9 (I) and Rc12 (J and K) expressing embryos stained for lacZ mRNA by whole-mount in situ hybridization. The floor plate expression of lacZ in Rc12 transgenics was patchy and thus appeared on sections as either absent (J) or weak (K). Brackets in C,D indicate extent of cranial reporter activity. Broken lines in E,F indicate the thoracic regions of the spinal cord that are magnified in G,H.

 


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  		Fig. 5. Loss of a T-box binding site in homology region-c (HR-c) results in ectopic expression in the diencephalon. (A,B) Whole-mount salmon-gal staining of transgenic embryos at 9.5 dpc expressing either (A) wild-type (Rc9) or (B) mutated T-box (Rc14) constructs. The arrows in A and B point to the rostral limit of salmon-gal staining detected at the level of prosomeres 3 and 5, respectively. (C-D) Transverse sections through the rostral diencephalon of transgenic embryos expressing either (C) wild-type Rc9, or (D) mutated T-box (Rc14), double-stained for lacZ reporter activity (salmon-gal) and Shh mRNA (BM-purple). The blue arrowhead points to the stripe of lateral expression of endogenous Shh mRNA adjacent to the ventral midline in this region of the diencephalon (di). The salmon-colored arrowhead in D points to the ectopic reporter activity in the ventral midline of the diencephalon. The asterisk marks the floor plate of the hindbrain (hb).

 


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  		Fig. 7. Lineage tracing of Shh-expressing cells in the mouse node. (A,B) Posterior-ventral views of 5-6 somite stage mouse embryos showing (A) Sfpe2 reporter activity and (B) Shh expression in the (i) node and (ii) ventral midline. Asterisk in A marks ectopic X-gal staining in the somites. Sections generated through the embryos in A and B at the level of the (i) node and (ii) second most newly formed somite show that X-gal staining and Shh expression are restricted to the ventral (mesodermal) layer of the node and notochordal plate, respectively. (C-E) Shh and ß-gal immunostaining colocalize to the ventral layer of the node in a 5-6 somite stage embryo carrying the Sfpe2 reporter. (F,G) Ventral views of 5-6 somite stage mouse embryos showing (F) Ptc-lacZ and (G) Foxa2 expression in the (i) node and (ii) ventral midline. Sections taken through the embryos in F and G are at the level of the (i) node and (ii) second most newly formed somite. Both Ptc-lacZ staining and Foxa2 expression localize to the dorsal and ventral layers of the node and to their derivatives, the floor plate and notochord, respectively. Red arrowheads point to the expression of Ptc-lacZ and Foxa2 in the presumptive floor plate. DL, dorsal layer; VL, ventral layer.

 


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  		Fig. 8. Homeogenetic induction of Shh expression in the floor plate. (A) Schematic of the mouse node depicting the segregation of gene expression patterns in the dorsal (blue) and ventral (red) layers (DL and VL, respectively). Whereas Shh and Sfpe2 reporter activity are restricted to the ventral layer, the Shh target genes Ptc and Foxa2 are expressed in both the DL and VL. (B) Schematic of the notochord and floor plate showing the transcriptional cascade resulting in Shh expression. In the notochord, Foxa2 activates Shh, which then signals to the overlying neural plate. For simplicity, only transcriptional components of the Shh pathway resulting in its expression in the floor plate is shown. In response to Shh signaling, Gli2 activates Foxa2 expression in the neural plate. Foxa2, in cooperation with a homeodomain (HD) protein and at least one other transcriptional activator (X) bind Shh regulatory elements (Sbe1, gray oval and Sfpe2, yellow oval) to stimulate Shh transcription in the floor plate.

 

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