How vertebrate limbs are formed is considered one of the better understood processes in development. It is generally accepted that the early patterning of a vertebrate limb requires a feedback loop between sonic hedgehog (Shh) and fibroblast growth factor (Fgf) signals. But the limb bud is prepatterned before this feedback loop is established, and the molecules responsible for this have until now remained unknown. New work by te Welscher and colleagues shows that the earlier patterning results from a mutual antagonism between two transcription factors — dHand and Gli3 — and that this interaction participates in positioning the Shh/Fgf signalling feedback loop.

During limb development, Shh signalling from the posterior mesenchyme regulates Fgf expression in the apical ectodermal ridge, whereas the Fgfs maintain Shh expression in the zone of polarizing activity. But Gremlin, a molecule that relays the Shh signal, and the Fgfs have asymmetric expression even before Shh comes on, suggesting that the limb bud is prepatterned. To investigate this asymmetry, te Welscher and colleagues looked at a transcription factor, dHand, because it is required to establish Shh signalling and because its initially uniform expression becomes restricted to the posterior of the limb bud just before Shh activation — but how? The authors reasoned that answering this question would provide information about the earliest events in limb bud patterning.

Shh is repressed in the anterior of the limb bud by another transcription factor, Gli3. The authors found that, unlike in wild-type mice, dHand is expressed in the anterior mesenchyme of Gli3-deficient limbs, which suggests that dHand is repressed by Gli3. In the posterior limb bud mesenchyme, Gli3 is in turn repressed by dHand.

How do these early events relate to the Shh/Fgf-mediated patterning? As both Gremlin and the Fgfs are expressed before Shh, the authors looked at their expression in Gli3-deficient mice. It turns out that Gli3 is required to repress Gremlin and Fgf4 in the anterior limb bud. The authors argue that the anterior expansion of Gremlin and Fgf4 in the absence of Gli3 could result from upregulating dHand in the anterior. Their hypothesis was confirmed by the anterior upregulation of Gremlin expression when dHand was ectopically expressed.

Interestingly, the authors show that Alx4, another transcription factor that is also expressed in anterior mesenchyme and participates in restricting Shh signalling, is not required for regulating dHand (or Gli3) expression. In fact, because Alx4 expression is reduced in Gli3-deficient mice it must lie genetically downstream of Gli3.

This study provides important insight into the very early patterning events in the vertebrate limb, but before we have the complete picture of limb development more factors will need to be incorporated into the already complex network of interactions.