Zebrafish Cxcr4a determines the proliferative response to Hedgehog signalling

Abstract

The Hedgehog (Hh) pathway plays dual roles in proliferation and patterning during embryonic development, but the mechanism(s) that distinguish the mitogenic and patterning activities of Hh signalling are not fully understood. An additional level of complexity is provided by the observation that Hh signalling can both promote and inhibit cell proliferation. One model to account for this apparent paradox is that Hh signalling primarily regulates cell cycle kinetics, such that activation of Hh signalling promotes fast cycling and an earlier cell cycle exit. Here we report that activation of Hh signalling promotes endodermal cell proliferation but inhibits proliferation in neighbouring non-endodermal cells, suggesting that the cell cycle kinetics model is insufficient to account for the opposing proliferative responses to Hh signalling. We show that expression of the chemokine receptor Cxcr4a is a critical parameter that determines the proliferative response to Hh signalling, and that loss of Cxcr4a function attenuates the transcription of cell cycle regulator targets of Hh signalling without affecting general transcriptional targets. We show that Cxcr4a inhibits PKA activity independently of Hh signalling, and propose that Cxcr4a enhances Hh-dependent proliferation by promoting the activity of Gli1. Our results indicate that Cxcr4a is required for Hh-dependent cell proliferation but not for Hh-dependent patterning, and suggest that the parallel activation of Cxcr4a is required to modulate the Hh pathway to distinguish between patterning and proliferation.

Somite-Derived Retinoic Acid Regulates Zebrafish Hematopoietic Stem Cell Formation

“…The Ct value data were analyzed using the comparative Ct method (2 -ΔΔCt method) [38], using eukaryotic translation elongation factor 1 alpha 1a ( ef1a ) as an endogenous control. Previously published qPCR primer sequences are cxcr4a -F, ; cxcr4a -R [39]; ef1a -F, ; ef1a -R, [34]. Intron-spanning her9 ( her9 -F, ; her9 -R, ) qPCR primers were selected from the Universal Probe Library Assay Design Center for Zebrafish (Roche), Prior to real-time qPCR analysis, these primer sets were validated as follows: An amplification plot was produced from a standard cDNA two-fold dilution series.…”

In Vivo Regulation of the Zebrafish Endoderm Progenitor Niche by T-Box Transcription Factors

“…These, together with Ta and Tbx16 binding coordinates and associations with histone marks, are shown in Data S1. Among target genes with common Ta/Tbx16 peaks at functional chromatin are FGF target gene etv4 (Roehl and Nüsslein-Volhard, 2001), mesodermally expressed endodermal regulator cxcl12b (Mizoguchi et al., 2008, Nair and Schilling, 2008, Stückemann et al., 2012), mesodermal progenitor regulator eve1 (Seebald and Szeto, 2011), and migration-associated marker ph4a2 (Chang et al., 2011; Figures 1G and 1H), all of which play roles in key Ta and Tbx16 activities.…”

“…Intriguingly, our analyses also indicate that a subset of CRMs are bound by combinations of Eomesa, Smad2, Mixl1, Nanog, Mxtx2, and Pou5f3, including those proximal to other genes implicated in endoderm formation, such as dusp4 (Brown et al., 2008), cxcr4a (Stückemann et al., 2012), and spns2 (Osborne et al., 2008). It will be interesting to learn more about how these TFs collectively contribute to the function of the identified CRMs.…”

“…We discovered that Ta/Tbx16 genomic binding substantially overlaps and provide evidence that use of common cis -regulatory modules (CRMs) accounts for their functional redundancy (Garnett et al., 2009). Here, we describe a profound loss of endoderm in ta/tbx16 double mutants and present findings demonstrating that Ta/Tbx16 directly regulate the cell-intrinsic endodermal regulator Mixl1 (Kikuchi et al., 2000), as well as extrinsic regulators of endoderm proliferation, the Cxcr4a ligands Cxcl12a/b (Mizoguchi et al., 2008, Stückemann et al., 2012). …”

Decoding the phosphorylation code in Hedgehog signal transduction

“…Furthermore, downregulation of PKA does not explain how Hh stimulates Smo phosphorylation. Nevertheless, the involvement of PKA in Hh signaling does allow the pathway activity to be regulated by other GPCRs, as has been implicated by several recent studies 145,146 . The question of how Smo phosphorylation is regulated is intimately linked to the question of how Ptc inhibits Smo.…”