Distinct Roles of Hand2 in Initiating Polarity and Posterior Shh Expression during the Onset of Mouse Limb Bud Development

Abstract

The polarization of nascent embryonic fields and the endowment of cells with organizer properties are key to initiation of vertebrate organogenesis. One such event is antero-posterior (AP) polarization of early limb buds and activation of morphogenetic Sonic Hedgehog (SHH) signaling in the posterior mesenchyme, which in turn promotes outgrowth and specifies the pentadactylous autopod. Inactivation of the Hand2 transcriptional regulator from the onset of mouse forelimb bud development disrupts establishment of posterior identity and Shh expression, which results in a skeletal phenotype identical to Shh deficient limb buds. In wild-type limb buds, Hand2 is part of the protein complexes containing Hoxd13, another essential regulator of Shh activation in limb buds. Chromatin immunoprecipitation shows that Hand2-containing chromatin complexes are bound to the far upstream cis-regulatory region (ZRS), which is specifically required for Shh expression in the limb bud. Cell-biochemical studies indicate that Hand2 and Hoxd13 can efficiently transactivate gene expression via the ZRS, while the Gli3 repressor isoform interferes with this positive transcriptional regulation. Indeed, analysis of mouse forelimb buds lacking both Hand2 and Gli3 reveals the complete absence of antero-posterior (AP) polarity along the entire proximo-distal axis and extreme digit polydactyly without AP identities. Our study uncovers essential components of the transcriptional machinery and key interactions that set-up limb bud asymmetry upstream of establishing the SHH signaling limb bud organizer.

Transcriptional components of anteroposterior positional information during zebrafish fin regeneration

“…Second, as mentioned above, a rich field of embryonic limb development has detailed the relationship between transcription factors and AP skeletal patterning (Qu et al, 1997;Charité et al, 2000;Fernandez-Teran et al, 2000;Zákány et al, 2004;Tzchori et al, 2009;Galli et al, 2010;Sheth et al, 2012). Third, our dataset revealed many examples of developmental transcription factors with expression differences along the AP axis.…”

“…hand2 regulates the localization of sonic hedgehog (shh) in the posterior region of developing embryonic forelimbs, wings or pectoral fins (Charité et al, 2000;Fernandez-Teran et al, 2000;Yelon et al, 2000;Galli et al, 2010). However, shha is expressed in epidermal cells at the distal tip of each uninjured and regenerating adult fin ray, regardless of whether that ray expresses hand2 (Quint et al, 2002;Lee et al, 2009).…”

Msx1 and Msx2 in limb mesenchyme modulate digit number and identity

“…In this respect, it should be noted that Gli3R, the truncated form of the Gli3 protein, accumulates in the anteriormost region of the limb, where Msx activity is high, and that Gli3 À/À mutant embryos also display ectopic Shh expression anteriorly in the limbs (Buscher et al, 1997). Furthermore, Gli3R has been shown to interfere with positive transcriptional regulation at the ZRS (Galli et al, 2010). Interactions between Gli3R and Msx might be required for Shh repression anteriorly.…”

Evolution of vertebrate appendicular structures: Insight from genetic and palaeontological data

“…Regulatory cross-talk among these genes is largely unknown for fish fin (left and middle panel), whereas genetic studies in mice have elucidated some aspects of cross-regulatory interactions (right panel). For tetrapod limb bud (right panel), regulatory links depicted in red and green are the ones confirmed functionally (Capellini et al, 2006;Vokes et al, 2008;Galli et al, 2010) whereas black links indicate putative cross-talk through cis-regulatory sequences, speculated here. Solid lines (right panel) indicate direct regulatory interactions where a given gene product (transcriptional factor) mediates the expression by directly binding to the concerned cis-acting site, whereas dashed lines indicate indirect interactions where a given gene product is not the transcriptional factor (Shh, Gre, Bmp, Fgf, Wnt7a), but interacts with cis-acting sites of concerned genes through their downstream effectors.…”