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First published online 11 January 2006
doi: 10.1242/dev.02239

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A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers

¹ Department of Genetics, University of Pennsylvania School of Medicine, Clinical Research Building, Room 470, 415 Curie Boulevard, Philadelphia, PA 19104, USA.
² Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892-3717, USA.

View larger version (56K): [in a new window]   Fig. 1. Functional analysis of locally acting Shh enhancers in the context of Bac 429M20eGFP. Schematic of Bac clones 429M20eGFP and 389P3eGFP, which contain an eGFP insertion into Shh exon 1 and extend 180 kb upstream and 160 kb downstream of Shh, respectively. Reporter constructs carrying enhancer deletions generated in Bac 429M20eGFP are listed. (A-F) Whole-mount views of embryos carrying (A) Bac 389P3eGFP, (B) 429M20eGFP and (C-F) 429M20eGFP deletion constructs. (G-I) In embryos carrying 389P3eGFP or 429M20eGFP, strong eGFP expression was observed in the floor plate (G,I), in a pattern comparable with endogenous Shh protein (H). Embryos carrying a deletion of SFPE1 (C,K) or SFPE2 (D,L) show similar patterns of eGFP fluorescence to each other and to embryos carrying the wild type Bac (B). (J) The ventral midbrain and caudal diencephalon also show strong eGFP expression in embryos carrying 429M20eGFP and 389P3eGFP. Embryos carrying deletions of both SFPE1 and SFPE2 showed a dramatic reduction in eGFP expression in the floor plate (E,M). Embryos carrying a deletion of SBE1 showed a complete loss of eGFP staining in the ventral midbrain and caudal diencephalon (compare F,N,O with B,I,J). The absence of hindgut staining in embryos depicted in E and F is a consequence of the variable nature of the hindgut enhancer and does not reflect a dependency on sequences mediating Shh floor-plate enhancer activity. eGFP staining on sections was performed by immunohistochemistry using an anti eGFP antibody. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (A-F). di, diencephalon; fp, floor plate; mb, midbrain; nc, notochord.

View larger version (38K): [in a new window]   Fig. 2. A screen for long-range acting Shh regulatory sequences. Physical map displaying the distribution of genes and Bac clones spanning 1 Mb upstream of Shh. Reporter cassettes (lacZ, blue rectangle) were introduced into overlapping Bacs by random transposon insertion. (A-F) Whole-mount views of embryos stained for (A) Shh mRNA and (B-F) lacZ expression (X-gal) in representative Bac transgenic embryos harvested at 10.5 dpc. X-gal staining in the notochord was observed in embryos carrying any of three overlapping Bacs (208K5ßlacZ, 447L17ßlacZ, 265M1ßlacZ), suggesting the existence of at least two long-range acting notochord enhancers (B-D). X-gal staining in the ventral forebrain was observed in embryos carrying 447L17ßlacZ (B) and 265M1ßlacZ (D). X-gal staining in the posterior limb bud was observed in embryos carrying 265M10ßlacZ (F). None of the five embryos carrying 214O17ßlacZ exhibited reporter activity (E). di, diencephalon; fl, forelimb; hl, hindlimb; nc, notochord; tel, telencephalon. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (B-F).

View larger version (92K): [in a new window]   Fig. 3. Isolation of regulatory sequences controlling Shh reporter activity in the forebrain. Schematic of Bac clones 447L17 and 265M1. Whole-genome alignments were performed using the ECR Browser with human sequence (hg17-May'04, chr7:155395662-155783102) as the base. ECRs (>75% similarity over a minimum of 350 bp) shared between human and mouse (pink) or between human, mouse and chicken (green) are represented by vertical bars. ECRs in the overlap between 447L17 and 265M1 are numbered ECR1 to ECR7. (A) Shh mRNA expression in the head of a 10.5 dpc embryo with accompanying transverse sections through the forebrain at the level of the (G) hypothalamus, (M) optic vesicles and (S) telencephalon. (B-X) X-gal staining in the forebrain of transgenic embryos carrying (B,H,N,T) 447L17ßlacZ; (C,I,O,U) 265M1ßlacZ; (D,J,P,V) ECR1/SBE2; (E,K,Q,W) ECR3/SBE3; and (F,L,R,X) SBE4 reporter constructs at 10.5 dpc. Shh forebrain reporter activity is divided into ventral diencephalic (SBE2, SBE4) and ventral telencephalic (SBE3, SBE4) regulatory elements. Unlike Shh and 447L17ßlacZ, 265M1ßlacZ transgene was not expressed in ventricular zone (vz) of the medial ganglionic eminence (mge) (compare U with S,T). SBE3 activity is restricted to the subventricular zone (svz) of the mge (W). The asterisk in E indicates ectopic X-gal staining in the hindbrain. ht, hypothalamus; ov, optic vesicle; poa, preoptic area; vz, ventricular zone; svz, subventricular zone. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (D-F).

View larger version (58K): [in a new window]   Fig. 4. Requirement of Shh forebrain enhancers in the context of Bac 447L17ßlacZ. Schematic representation of the SBE2, SBE3 and SBE4 deletion constructs. X-gal staining in the forebrain of embryos carrying (A-D) 447L17ßlacZSBE2, (E-H) 447L17ßlacZSBE2, SBE3, (I-L) 447L17ßlacZSBE4, (M-P) 447L17ßlacZSBE3, SBE4, (Q-T) 447L17ßlacZSBE2, SBE4 and (U-X) 447L17ßlacZSBE2, SBE3, SBE4. Deletion of SBE2 resulted in the loss of reporter activity at most levels of the diencephalon, including the preoptic area (POA; arrowhead in D). Compare A-D with the pattern of X-gal staining in embryos carrying the wild-type 447L17ßlacZ transgene (Fig. 3B,H,N,T). Embryos carrying 447L17ßlacZSBE2, SBE3 (E-H) showed patterns of X-gal staining that were similar to those carrying 447L17ßlacZSBE2 (A-D). Embryos carrying 447L17ßlacZSBE4 (I-L) showed an absence of staining in the ventricular zone (vz) of the mge (L). In embryos carrying 447L17ßlacZSBE3, SBE4 (M-P), X-gal staining was not detected in the vz or subventricular zone (svz) of the medial ganglionic eminence (mge) (P). Deletion of SBE2 and SBE4 resulted in a loss of expression in the vz of the mge and the entire diencephalon (Q-T). Embryos carrying 447L17ßlacZSBE2, SBE3, SBE4 (U-X) showed no expression in the diencephalon or telecephalon. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (A,E,I,M,Q,U).

View larger version (40K): [in a new window]   Fig. 5. Conservation of SBE2 sequence and function. (A) Vista plots comparing the alignment of human SBE2 sequences with mouse, chicken, frog and tetraodon. (B) Comparison of the distance between SBE2 and the Shh transcription start site in human, mouse, chicken, frog and tetraodon. (C-F) SBE2 reporter activity derived from (C) human, (D) chicken, (E) frog and (F) pufferfish sequences. X-gal staining in the otic vesicle of the embryo shown in F is ectopic. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (C-F).

View larger version (39K): [in a new window]   Fig. 6. Conservation of SBE4 sequence and function. (A) Vista plots comparing the alignment of human SBE4 sequences with mouse, chicken, frog and tetraodon. (B) Comparison of the distance between SBE4 and the Shh transcription start site in human, mouse, chicken, frog and tetraodon. (C-E) SBE4 reporter activity derived from (C) chicken, (D) frog and (E) pufferfish sequences. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct (C-E).

View larger version (61K): [in a new window]   Fig. 7. An Nkx2 binding site is required for SBE3 enhancer function. (A) Vista plot of the alignment between human and mouse SBE3 sequences. The target recognition sequence for the Nkx2 homeodomain protein (red) is present in both human and mouse SBE3 sequences. (B) Binding of Nkx2.1 protein to a site in the SBE3 sequence. Cell lysates transfected with pCMV (lane 1) or pCMV-Nkx2.1 (lanes 2-6) were analyzed for binding to a 39 bp probe overlapping the Nkx2 recognition sequence in SBE3. Wild-type cold competitor (lanes 3, 4) interfered with binding of Nkx2.1 protein, while mutant competitor with nucleotide substitutions in the core binding site (AAGTAGGGAGCA) did not alter the shifted complex (lanes 5, 6). (C-F) X-gal staining of transgenic embryos carrying a wild-type (C,E) or mutant SBE3 reporter construct in which the Nkx2 core recognition sequence (AAGTAG) was deleted (D,F). Embryos carrying the wild-type SBE3 reporter construct show consistent X-gal staining in the svz of the mge (C,E). By contrast, embryos carrying an SBE3 reporter construct lacking the Nkx2 site (Nkx2) showed no staining in the ventral forebrain (D,F). Ectopic X-gal staining in the hindbrain (asterisk) of embryos carrying either wild-type or mutant SBE3 transgenes was detected in equal frequency and thus served as an internal staining control. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct.

View larger version (25K): [in a new window]   Fig. 8. Shh expression in the CNS is controlled by multiple regulatory modules. Schematic view of Shh expression in the CNS, color-coded to depict the distinct regulatory elements governing Shh transcription along the AP axis of the mouse neural tube. Hatched patterns in the floor plate of the spinal cord and hindbrain, in the p3 domain of the diencephalon and in the svz of the telencephalon represent the sites of Shh expression regulated by more than one enhancer. Solid patterns in the ventral midbrain, diencephalon and telencephalon represent sites of Shh expression controlled by single regulatory elements. The location of the six CNS enhancers with respect to the Shh transcription start site is also indicated. The arrows indicate the position of the translocation breakpoints (T1, T2, T3, T4) identified in individuals with HPE (Roessler et al., 1997).

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