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Misexpression of dHAND induces ectopic digits in the developing limb bud in the absence of direct DNA binding

¹ Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9148, USA
² Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9148, USA
³ Department of Cell Biology and Genetics, Erasmus University Rotterdam, Dr Molewaterplein 50, 3015GE, Rotterdam, The Netherlands

View larger version (20K): [in a new window]   Fig. 1. DNA binding and transcriptional activity of dHAND mutant proteins. (A) Wild-type and mutant dHAND proteins were translated in vitro with or without E12, as indicated, and tested for DNA binding in gel mobility shift assays to a radiolabeled oligonucleotide probe containing the eHAND/Th1 E-box sequence. HAND/E12 complex mobility is indicated by an asterisk and a nonspecific complex is marked by ns. (B) 10T1/2 cells were transiently transfected with the L8E6-luciferase reporter and pcDNA3.1 expression vector encoding wild-type and mutant dHAND proteins as indicated (see Fig. 2), and luciferase activity was determined in cell extracts. Values are expressed as fold activation of the reporter gene relative to the level of expression with the reporter alone. (C) 10T1/2 cells were transiently transfected with the L8G4-luciferase reporter and the pGE1b-GAL4 expression vector encoding regions of the dHAND-coding region fused to the GAL4 DNA-binding domain and luciferase activity was determined in cell extracts. Values in B and C represent the mean±s.e.m. determined from at least three independent experiments.

View larger version (15K): [in a new window]   Fig. 2. Summary of the activities of dHAND mutant proteins. Mutant dHAND proteins and their activities are shown. All mutants contained an N-terminal Myc epitope tag. M, Myc epitope tag; B, basic region; HLH, helix-loop-helix.

View larger version (60K): [in a new window]   Fig. 3. Preaxial polydactyly induced by a dHAND mutant protein lacking the N-terminal transcription activation domain. F0 transgenic mouse embryos were sacrificed at E16.5 and limbs were stained for bone and cartilage. (A) Limb from a wild-type nontransgenic embryo. Embryos harboring prx-dHAND (B) and prx-1-90 (C) transgenes showed preaxial polydactyly. All panels show forelimbs. Ectopic digits are indicated with an asterisk.

View larger version (81K): [in a new window]   Fig. 4. Preaxial polydactyly and ectopic Shh expression induced by dHAND-VP16. F0 transgenic mouse embryos were sacrificed at E16.5 and limbs were stained for bone and cartilage. (A) Limb from an embryo harboring the prx-dHAND-VP16 transgene. This construct induced severe preaxial polydactyly. Note the presense of four ectopic digits that have similar morphology. Ectopic digits are indicated with an asterisk. (B,C) Detection of Shh transcripts by whole-mount in situ hybridization to wild-type and prx-dHAND-VP16 transgenic embryos, respectively, at E11.5. In wild-type embryos, Shh transcripts are localized to the ZPA at the posterior of the limb bud. In prx-dHAND-VP16 embryos, Shh transcripts are seen throughout the peripheral region of the limb bud. Blue arrowheads indicate Shh expression domain. fl, forelimb bud; hl, hindlimb bud.

View larger version (110K): [in a new window]   Fig. 5. Limb abnormalities induced by a dHAND mutant protein lacking the C-terminal region. F0 transgenic mouse embryos were sacrificed at E16.5 and limbs were stained for bone and cartilage. (A) A forelimb from an embryo harboring prx-155-217. This construct induced preaxial polydactyly, as well as truncation of the zeugopod (indicated by black arrowhead). (B) Detection of dHAND transcripts by in situ hybridization to a longitudinal section through the developing ulna of an E13.5 embryo. dHAND expression is detected in the chondrocytes and perichondrium (white arrowhead). Note highest levels of expression in the hyperplastic zone of the chondrocytes (white arrow). Ectopic digits are indicated with an asterisk.

View larger version (63K): [in a new window]   Fig. 6. Limb abnormalities induced by dHAND mutant proteins that fail to bind DNA. (A) Amino acid sequence of the basic regions of wild-type and mutant dHAND proteins. X designates residues that were deleted in the basic mutant. (B,C) F0 transgenic mouse embryos harboring prx-RRR109EDE (B) and prx-basic (C) transgenes, respectively, were sacrificed at E16.5 and limbs were stained for bone and cartilage. Both constructs induced preaxial polydactyly, as well as truncation of the zeugopod. Both panels show forelimbs. Ectopic digits are indicated with an asterisk. Arrowheads indicate zeugopod abnormalities.

View larger version (102K): [in a new window]   Fig. 7. Analysis of the role of the HLH region on limb patterning. F0 transgenic mouse embryos were sacrificed at E16.5 and limbs were stained for bone and cartilage. Transgenes are indicated in each panel. The prx-HLH (A), prx-F119P (B) and prx-bHLH (C) transgenes had no effect on limb patterning. The prx-eHAND transgene (D) resulted in preaxial polydactyly indistinguishable from that resulting from prx-dHAND. The prx-paraxis transgene resulted in shortened zeugopodial bones (E) and only a single extra digit in one out of 11 transgenic embryos (F). (G) Embryos harboring prx-Id transgenes displayed malformed zeugopodial structures and normally patterned digits that are labeled numerically. All panels show forelimbs. Ectopic digits are indicated with an asterisk.

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