The Hedgehog (Hh) signaling pathway has critical functions during embryogenesis of both invertebrate and vertebrate species [1]; defects in this pathway in humans can cause developmental disorders as well as neoplasia [2]. Although the Gli1, Gli2, and Gli3 zinc finger proteins are known to be effectors of Hh signaling in vertebrates, the mechanisms regulating activity of these transcription factors remain poorly understood [3] [4]. In Drosophila, activity of the Gli homolog Cubitus interruptus (Ci) is likely to be modulated by its interaction with a cytoplasmic complex containing several other proteins [5] [6], including Costal2, Fused (Fu), and Suppressor of fused (Su(fu)), the last of which has been shown to interact directly with Ci [7]. We have cloned mouse Suppressor of fused (mSu(fu)) and detected its 4.5 kb transcript throughout embryogenesis and in several adult tissues. In cultured cells, mSu(fu) overexpression inhibited transcriptional activation mediated by Sonic hedgehog (Shh), Gli1 and Gli2. Co-immunoprecipitation of epitope-tagged proteins indicated that mSu(fu) interacts with Gli1, Gli2, and Gli3, and that the inhibitory effects of mSu(fu) on Gli1's transcriptional activity were mediated through interactions with both amino- and carboxy-terminal regions of Gli1. Gli1 was localized primarily to the nucleus of both HeLa cells and the Shh-responsive cell line MNS-70; co-expression with mSu(fu) resulted in a striking increase in cytoplasmic Gli1 immunostaining. Our findings indicate that mSu(fu) can function as a negative regulator of Shh signaling and suggest that this effect is mediated by interaction with Gli transcription factors.

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein predominantly expressed in tendons and ligaments. We found that scleraxis (Scx), a member of the Twist-family of basic helix-loop-helix transcription factors, is a transcriptional activator of Tnmd expression in tenocytes. During embryonic development, Scx expression preceded that of Tnmd. Tnmd expression was nearly absent in tendons and ligaments of Scx-deficient mice generated by transcription activator-like effector nucleases-mediated gene disruption. Tnmd mRNA levels were dramatically decreased during serial passages of rat tenocytes. Scx silencing by small interfering RNA significantly suppressed endogenous Tnmd mRNA levels in tenocytes. Mouse Tnmd contains five E-box sites in the ~1-kb 5′-flanking region. A 174-base pair genomic fragment containing a TATA box drives transcription in tenocytes. Enhancer activity was increased in the upstream region (−1030 to −295) of Tnmd in tenocytes, but not in NIH3T3 and C3H10T1/2 cells. Preferential binding of both Scx and Twist1 as a heterodimer with E12 or E47 to CAGATG or CATCTG and transactivation of the 5′-flanking region were confirmed by electrophoresis mobility shift and dual luciferase assays, respectively. Scx directly transactivates Tnmd via these E-boxes to positively regulate tenocyte differentiation and maturation.

NE enhancer activation and notochord development in transgenic mouse embryos. Furthermore, manipulation of Tead activity in zebrafish embryos led to altered expression of foxa2 in the embryonic shield. These results suggest that Tead activates the Foxa2 enhancer core element in the mouse node in cooperation with a second factor that binds to the 5Ј Ј element, and that a similar mechanism also operates in the zebrafish shield. Research articleDevelopment 4720 led to the identification of an evolutionarily conserved sequence motif, CS3, which is essential for enhancer activity (Nishizaki et al., 2001). Here, we identified the Tead family transcription factors as proteins that bind to CS3. Tead family transcription factors all contain a DNA-binding domain called a TEA domain, and consist of four members (Tead1-Tead4) in both mouse and human (Jacquemin et al., 1998;Kaneko and DePamphilis, 1998). The founding member of this family, Tead1 [also known as transcriptional enhancer factor 1 (TEF-1)], was originally identified as an activator of simian virus 40 (SV40) enhancer (Davidson et al., 1988;Xiao et al., 1991). A Drosophila Tead protein, Scalloped (Sd), interacts with a co-activator protein, Vestigial (Vg), and regulates wing development (Halder et al., 1998;Simmonds et al., 1998). Vertebrate Tead proteins also require co-factors to act as activators, and the candidates are the four Vg homologs (Maeda et al., 2002;Vaudin et al., 1999) and Yes-associated protein 65 (YAP65) (Maeda et al., 2002;Vassilev et al., 2001;Vaudin et al., 1999). Several other mechanisms are also suggested for regulation of Tead activity, including interaction with other transcription factors and modification by protein kinases (Gupta et al., 2001; Gupta et al., 1997;Jiang et al., 2001;Thompson et al., 2003). Tead genes are expressed widely, from preimplantation embryos to various adult tissues, with distinct patterns (Jacquemin et al., 1998;Kaneko et al., 1997). Tead proteins are suggested to be involved in activation of the cardiac and skeletal muscle genes, CTP:phosphocholine cytidylyltransferase (Pcyt -Mouse Genome Informatics) and Pax3 in neural crest cells (Jiang et al., 2000; Milewski et al., 2004;Stewart et al., 1994;Sugimoto et al., 2001), and Tead1 mutant embryos die between E11 and 12 due to resulting heart defects (Chen et al., 1994). However, the roles played by Tead genes during early embryogenesis have not yet been revealed.In this study, we first showed that the core element (CE) of the Foxa2 enhancer drives gene expression in the node. Two transcription factors activate the CE in a cooperative fashion, and Tead proteins are one of these factors. The Tead-binding site in the CE was essential for node/notochord enhancer (NE) activity, and inhibition of Tead function in mouse embryos disturbed notochord development. In zebrafish embryos, manipulation of Tead activity changed the expression of foxa2. These results suggest that the key mechanism of Foxa2 expression in the node/notochord is activation of the enhancer core element ...

Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling in vertebrates. The question remains unanswered, however, as to how these Gli proteins participate in the Shh signaling pathway. In this study, regulatory activities associated with the Gli2 protein were investigated in relation to the Shh signaling. Although Gli2 acts as a weak transcriptional activator, it is in fact a composite of positive and negative regulatory domains. In cultured cells, truncation of the activation domain in the C-terminal half results in a protein with repressor activity, while removal of the repression domain at the N terminus converts Gli2 into a strong activator. In transgenic mouse embryos, N-terminally truncated Gli2, unlike the full length protein, activates a Shh target gene, HNF3beta, in the dorsal neural tube, thus mimicking the effect of Shh signal. This suggests that unmasking of the strong activation potential of Gli2 through modulation of the N-terminal repression domain is one of the key mechanisms of the Shh signaling. A similar regulatory mechanism involving the N-terminal region was also found for Gli3, but not for Gli1. When the Shh signal derived from the notochord is received by the neural plate, the widely expressed Gli2 and Gli3 proteins are presumably converted to their active forms in the ventral cells, leading to activation of transcription of their target genes, including Gli1.

Chondromodulin I (ChM-I) was supposed from its limited expression in cartilage and its functions in cultured chondrocytes as a major regulator in cartilage development. Here, we generated mice deficient in ChM-I by targeted disruption of the ChM-I gene. No overt abnormality was detected in endochondral bone formation during embryogenesis and cartilage development during growth stages of ChM-I−/− mice. However, a significant increase in bone mineral density with lowered bone resorption with respect to formation was unexpectedly found in adult ChM-I−/− mice. Thus, the present study established that ChM-I is a bone remodeling factor