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First published online 27 July 2004
doi: 10.1242/dev.01252

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Chick Dach1 interacts with the Smad complex and Sin3a to control AER formation and limb development along the proximodistal axis

¹ Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101, Japan
² Department of Developmental Neurobiology, Institute of Development, Aging and Cancer, Graduate School of Medicine, Tohoku University, 4-1, Seiryo, Aoba, Sendai, 980-8575, Japan

View larger version (22K): [in a new window]   Fig. 1. (A) Cos-7 cells were transfected with increasing amounts of an expression vector containing the Gal4 DNA-binding domain (DBD) fused to full-length Dach1 (Gal4-WT), along with the 4R-UAS-Luc reporter plasmid, which contains four Gal4-DBD-binding motifs. Transcription was repressed by Dal4-WT in a dose-dependent manner (12.5- to 23-fold repression). (B) Using the same reporter, a low amount of Gal4-Dach1 was expressed in Cos-7 cells, resulting in weak repression. However, co-expression of mouse Sin3a strongly enhanced the repressor activity. (C) Co-expression of Gal4 DBD-Smad1 and BMP4 activated the 4R-UAS-Luc reporter. Expression of Dach1 weakened this BMP4-mediated activation. Addition of mouse Sin3a further repressed this activation. When Dach1 expression was removed, mouse Sin3a-mediated repression was no longer observed. (D) An immunoprecipitation assay was carried out using HA-tagged Dach1 and Flag-tagged mouse Sin3a expressed in Cos-7 cells. An irrelevant IgG did not precipitate either Dach1 or Sin3a (IP IgG). When the anti-HA antibody was used, both Dach1 and Sin3a were detected (IP Dach1=HA) by the anti-Flag antibody (IB Flag=mSin3a) and the anti-HA antibody (IB HA), respectively. Input, 5% of the extract used in this immunoprecipitation assay. (E) Flag-tagged Smad1 and HA-tagged Dach1 were co-expressed in Cos-7 cells, and cell lysates were immunoprecipitated with the anti-HA antibody or an irrelevant IgG (as a negative control). In the precipitant of the irrelevant IgG, neither Smad1 nor Dach1 was detected (IP IgG). By contrast, both Smad1 and Dach1 were present in the precipitant when the anti-HA antibody was used for IP (IP Dach1=HA). Input, 5% of the extract used in this immunoprecipitation assay. (F) Addition of Dach1 repressed the BMP-4/Smad1/Smad4-mediated transcription of the Xvent2-Luc reporter. Co-expression of VP=Dach1 activated this reporter even in the presence of Dach1. (G) By contrast, co-expression of DD2 did not activate this promoter, but instead cancelled the Dach1-mediated repression.

View larger version (89K): [in a new window]   Fig. 2. VP=Dach1 was misexpressed in the ectoderm by pouring the plasmid solution over the ectoderm. Diagrams at the top show the experimental design of electroporation. (A) A dorsal view of an electroporated limb bud on the right-hand side (Exp.) compared with a normal limb bud on the left. Expression of Fgf8 is obscure. (B) In a lateral view, Fgf8 expression is faint and blurred without making a clear boundary. (C) At stage 24, a severe depression was evident in the dorsal end of the limb bud. (D) Strong EGFP signals, which were derived from co-electroporated pCAGGS-EGFP, were observed in the ectoderm. (E) In such limb buds, Fgf8 expression was lost in the central part and deformed in the anterior (red arrowheads), whereas Fgf8 was normally expressed in the posterior (blue arrowhead). (F) Expression of Bmp7 was also lost in the central part (red arrowhead) and in the anterior mesenchyme, but was normal in the posterior area (blue arrowhead). (G) Mesenchymal expression of Fgf10 was repressed with inhibition of the limb outgrowth (red arrowheads). (H) Shh was expressed normally posteriorly (blue arrowhead). A depression similar to C was evident in the distal end (red arrowhead). (I) At stage 37, distal truncation was obtained. In the wing, where misexpression of VP=Dach1 was weak, truncation of digit II was evident. In the leg, where misexpression was extensive, distal autopod structures were completely missing (red arrowhead). (J) Alcian Blue staining of these limbs revealed a short digit II in the wing (red arrow) and the complete loss of digits in the leg (red arrowhead). Approximately 60% of the misexpressed limbs showed these phenotypes (n=242).

View larger version (83K): [in a new window]   Fig. 3. Misexpression of DD2 in the limb mesenchyme by injecting plasmid solutions in the lateral plate mesoderm. Diagrams show the experimental design of electroporation. (A) We misexpressed the EGFP gene to monitor the expression of transgenes in the mesenchyme. (B) DD2 misexpression at stages 13-15 induced repression of Fgf10 expression at stage 20 (red arrows). (C) Wnt5a was also repressed (red arrow), with deformity of the AER. (D-F) At stage 19, expression of Fgf8, En-1 and Msx2 became unclear and distorted. (G) At stage 24, a broad and irregular AER was formed as judged by the Fgf8-positive domain, in which an Fgf8-negative line was observed. (H-K) At stage 20, Wnt7a and Lmx1b were expressed dorsally, and Bmp7 and Msx2 ventrally, without disturbance of the DV axis formation. Nonetheless, thickening of the ectoderm was observed near the margin of the expression of these markers (red arrowheads). Approximately 35% of the DD2-misexpressed limb buds displayed this phenotype in the AER (n=156).

View larger version (85K): [in a new window]   Fig. 4. (A) At stage 20, invagination of the ectoderm was obtained (red arrowhead) in the DD2-misexpressed limb bud. (B) Expression of Fgf8 was observed in the invaginated ectoderm (red arrowhead). (C) Cyclin D1 was expressed normally in the underlying mesenchyme. A red arrowhead indicates the invaginated epithelium. (D) In the normal limb bud, ßIII-tubulin proteins were detected by the TuJ1 antibody in both the ventral ectoderm and the motoneurons entering the limb. (E) In the DD2-misexpressed limb bud, ßIII-tubulin proteins were detected in the invaginated epithelium, suggesting that the invaginated ectoderm was derived from the ventral half. (F,G) The boxed shown in E was magnified. Clear TuJ1 staining was observed in the invaginated part (white arrowheads). In its distal-most end, thickening of the ectoderm was evident (blue arrowheads). EGFP proteins were visualized in green. (H) Fgf8 was expressed in this thick ectoderm (blue arrowhead). (I) In a ventral view, expression of Fgf8 was broad and flat. (J,K) At stage 24, two protrusions were formed with strong expression of Shh (J) and Gremlin (K) in the dorsal and the ventral protrusions, respectively. (L,M) When developed further, polydactylous limbs arose. Digits of such limbs were arranged in two rows, with digits a, b and c in one line, and digits a' and b' in another. A distal view was shown in M. (N,O) Skeletal patterns obtained by Alcian Blue staining. Lateral and posterior views are shown in N and O, respectively. (P) Schematic representation of the digit pattern in a dorsal view. Approximately 22% of the DD2-misexpressed limb buds displayed this phenotype (n=27).

View larger version (79K): [in a new window]   Fig. 5. (A-C) At stage 17, in the DD2-misexpressed limb, Meis2 was induced (B) in an area where co-expressed EGFP proteins (shown in green) were detected (A). Normal expression was shown in C. (D-F) At stage 19, similar but more extensive induction of Meis2 was observed. (G,H) Implantation of BMP4-soaked beads repressed Meis2 expression (red arrowheads) in both a proximal area (G) and a distal end (H), with a deformity of the AER (H). (I) Misexpression of DD2 cancelled the BMP4-mediated repression of Meis2, with expansion of Meis2 expression to the distal end. (J) A luciferase reporter plasmid containing 700 bp 5' region of human MEIS1 was transfected into HepG2 cells, along with BMP4, Smad1, Dach1, VP=Dach1 and mouse Sin3a expression plasmids. Expression of BMP4, Smad1 and Dach1 repressed this reporter. This repression was enhanced by the addition of mouse Sin3a. By contrast, when VP=Dach1 was expressed, activation of this reporter was obtained.