During development, Hedgehog (Hh) signaling controls both cell fate and proliferation, but how cells decide whether to divide or differentiate in response to signaling is not clear. Stückemann et al. report that, in the zebrafish gastrula, Hh signaling promoted proliferation of endoderm and inhibited proliferation of nonendodermal cells (mesoderm and ectoderm). Because the chemokine stromal cell–derived factor 1 (SDF-1) has been implicated in Hh-induced proliferation in other cell types, and because the SDF-1–activated G protein–coupled receptor CXCR4a is present in the endoderm, the authors investigated the role of CXCR4a in the context of early endoderm development. Hh signaling is induced when Hh binds to its transmembrane receptor Patched (Ptc), thus relieving Ptc-mediated repression of the transmembrane protein Smoothened (Smo), which initiates intracellular signal transduction. Knocking down Ptc, therefore, activates Hh signaling. In endodermal cells, morpholino-mediated knockdown of CXCR4a reduced proliferation and blocked the increase in proliferation elicited by knocking down Ptc. Whereas activation of Hh signaling by Ptc knockdown reduced proliferation of nonendodermal cells, expression of cxcr4a in conjunction with Ptc knockdown, but not without Ptc knockdown, increased proliferation in these cells, suggesting that CXCR4a switched the response to Hh signaling from inhibition to stimulation of proliferation in these cells. As assayed by embryonic phenotypes and Hh target gene expression, experiments using various combinations of Hh pathway mutations, morpholino-mediated knockdowns, and cxcr4a overexpression indicated that CXCR4a promoted expression of gli1, which encodes a Gli transcription factor that lacks the repressor domain and is not cleaved, and thus acts only as a transcriptional activator and never as a repressor. In contrast, protein kinase A (PKA) inhibits Hh signaling by phosphorylating the Gli2 and Gli3 transcription factors, which promotes their proteolytic processing into repressor forms. PKA is active when bound to cyclic adenosine monophosphate (cAMP), and the enzyme that produces cAMP, adenylyl cyclase (AC), is inhibited by Gαi-coupled GPCRs. Experiments employing expression of a dominant-negative form of PKA and a PKA biosensor indicated that inactivation of PKA downstream of both Smo and CXCR4a was required for the CXCR4a-mediated proliferative response to Hh signaling. Additionally, CXCR4a repressed PKA activity upstream of AC. The CXCR4a ligand CXCL12b is produced by mesodermal cells, opening the possibility that the adjacent mesoderm may promote endodermal cell proliferation in response to Hh by activating CXCR4.
T. Stückemann, T. Wegleiter, E. Stefan, O. Nägele, K. Tarbashevich, G. Böck, E. Raz, P. Aanstad, Zebrafish Cxcr4a determines the proliferative response to Hedgehog signalling. Development 139, 2711–2720 (2012). [Abstract] [Full Text]