We have recently demonstrated that the retinoblastoma family of negative cell cycle regulators can form complexes with a class of developmental factors which contain paired‐like (PL) homeodomains (Wiggan et al. [1998] Oncogene 16:227–236). Our screens led to the isolation of a novel PL‐homeodomain protein which had been isolated independently by another group and called Alx‐4 (Qu et al. [1997] Development 124:3999–4008). Mice homozygous for a targeted null mutation of Alx‐4 have several abnormalities, including preaxial polydactyly, suggesting that Alx‐4 plays a role in pattern formation in limb buds. In data that we present here, we show that Alx‐4 is expressed in mesenchymal condensations of a diverse group of tissues whose development is dependent on epithelial–mesenchymal interactions, many of which are additionally dependent on expression of the HMG‐box–containing protein, LEF‐1. Alx‐4–expressing tissues include osteoblast precursors of most bones, the dermal papilla of hair and whisker follicles, the dental papilla of teeth, and a subset of mesenchymal cells in pubescent mammary glands. We show further that Alx‐4 strongly activates transcription from a promoter containing the homeodomain binding site, P2. Optimal activation requires specific sequences in the N‐terminal portion of Alx‐4, as well as a proline‐rich region downstream of the PL‐homeodomain, but not the paired‐tail at the C terminus. Taken together, our results demonstrate that Alx‐4 is a potent transcriptional activator that is expressed at sites of epithelial–mesenchymal interactions during murine embryonic development. Dev. Dyn. 1998;213:159–169. © 1998 Wiley‐Liss, Inc.
During murine embryogenesis, expression of the paired-like homeodomain protein Alx4 is restricted to tissues whose development depends on the expression of lymphoid enhancer factor-1 (LEF-1). Given the defects seen in hair follicle development in both LEF-1 and Alx4 knockout and mutant animals and the overlapping expression patterns, we predicted that LEF-1 and Alx4 might form physical complexes. We demonstrate here the interaction between LEF-1 and Alx4. This interaction is mediated through a specific proline-rich domain in the N-terminal region of Alx4 and requires the DNAbinding domain (HMG-box) of LEF-1. We also demonstrate that LEF-1 and Alx4 can bind simultaneously to adjacent sites on the neural cell adhesion molecule (N-CAM) promoter and that this binding alters N-CAM promoter activity. Furthermore, when expressed in primary mammary stromal cells, Alx4 decreases the expression of endogenous N-CAM protein. These results reveal a potential mechanism that gives rise to mesenchymal-specific activities of LEF-1.
We have recently demonstrated that the retinoblastoma family of negative cell cycle regulators can form complexes with a class of developmental factors which contain paired-like (PL) homeodomains (Wiggan et al. [1998] Oncogene 16:227-236). Our screens led to the isolation of a novel PL-homeodomain protein which had been isolated independently by another group and called Alx-4 (Qu et al. [1997] Development 124:3999-4008). Mice homozygous for a targeted null mutation of Alx-4 have several abnormalities, including preaxial polydactyly, suggesting that Alx-4 plays a role in pattern formation in limb buds. In data that we present here, we show that Alx-4 is expressed in mesenchymal condensations of a diverse group of tissues whose development is dependent on epithelial-mesenchymal interactions, many of which are additionally dependent on expression of the HMG-box-containing protein, LEF-1. Alx-4-expressing tissues include osteoblast precursors of most bones, the dermal papilla of hair and whisker follicles, the dental papilla of teeth, and a subset of mesenchymal cells in pubescent mammary glands. We show further that Alx-4 strongly activates transcription from a promoter containing the homeodomain binding site, P2. Optimal activation requires specific sequences in the N-terminal portion of Alx-4 as well as a proline-rich region downstream of the PL-homeodomain, but not the paired-tail at the C terminus. Taken together, our results demonstrate that Alx-4 is a potent transcriptional activator that is expressed at sites of epithelial-mesenchymal interactions during murine embryonic development.
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