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Soxb1-Driven Transcriptional Network Underlies Neural-Specific Interpretation of Morphogen Signals

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Soxb1-Driven Transcriptional Network Underlies Neural-Specific Interpretation of Morphogen Signals SoxB1-driven transcriptional network underlies neural-specific interpretation of morphogen signals Tony Oosterveena,1, Sanja Kurdijaa,1, Mats Ensteröa,b, Christopher W. Uhdea, Maria Bergslandb, Magnus Sandberga,b, Rickard Sandberga,b, Jonas Muhra,b, and Johan Ericsona,2 aDepartment of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and bLudwig Institute for Cancer Research, 171 77, Stockholm, Sweden Edited* by Thomas M. Jessell, Columbia University College of Physicians and Surgeons, New York, NY, and approved March 20, 2013 (received for review November 28, 2012) The reiterative deployment of a small cadre of morphogen signals and critically requires the direct synergistic input of SoxB1 pro- underlies patterning and growth of most tissues during embyo- teins at the transcriptional level (9). The SoxB1 group of TFs, Sox1 genesis, but how such inductive events result in tissue-specific to -3, are broadly expressed in the developing CNS and play an responses remains poorly understood. By characterizing cis-regula- important role in maintaining neural progenitor properties (10, tory modules (CRMs) associated with genes regulated by Sonic 11). Importantly, these TFs are expressed in a neural-specific hedgehog (Shh), retinoids, or bone morphogenetic proteins in the manner (Fig. 1B) (12), raising the possibility that SoxB1 proteins fi CNS, we provide evidence that the neural-specific interpretation of contribute to the tissue-speci c selection of target gene activation morphogen signaling reflects a direct integration of these pathways upon Shh pathway activation. with SoxB1 proteins at the CRM level. Moreover, expression of Graded Shh signaling underlies anteroposterior patterning of SoxB1 proteins in the limb bud confers on mesodermal cells the the developing limb bud, and Gli proteins are bound to the CRMs of class II genes Nkx2.2 and Nkx6.1, despite the fact that potential to activate neural-specific target genes upon Shh, retinoid, they are transcriptionally silent in this tissue (8, 9). We therefore or bone morphogenetic protein signaling, and the collocation of wished to determine whether expression of SoxB1 proteins in binding sites for SoxB1 and morphogen-mediatory transcription fac- mesodermal cells of the limb bud is sufficient to activate neural- fi BIOLOGY tors in CRMs faithfully predicts neural-speci c gene activity. Thus, an specific Shh-target genes in response to Shh signaling. To test unexpectedly simple transcriptional paradigm appears to conceptu- this theory, we electroporated expression constructs into the DEVELOPMENTAL fi ally explain the neural-speci c interpretation of pleiotropic signal- base of the presumptive limb bud at Hamburger–Hamilton ing during vertebrate development. Importantly, genes induced in (HH) stage 15 embryos (Fig. 1A) and collected embryos for anal- a SoxB1-dependent manner appear to constitute repressive gene ysis 6 or 40 h postelectroporation (hpe). The ambient level of regulatory networks that are directly interlinked at the CRM level Shh signaling was low at the base of the limb at 40 hpe, as indi- to constrain the regional expression of patterning genes. Accord- cated by low expression of the Shh receptor Patched 1 (Fig. S1A), ingly, not only does the topology of SoxB1-driven gene regulatory which is a primary response gene of Shh signaling (13). Activa- networks provide a tissue-specific mode of gene activation, but it tion of the Shh pathway by forced expression of a constitutively also determines the spatial expression pattern of target genes active form of Smoothened (SmoM2) induced Ptc1,aswellas within the developing neural tube. a Shh-responsive CRM associated with Ptc1 (Fig. 1 B and E) (14). However, neither SmoM2 nor Sox2 nor Sox3 expressed Sox3 | Gli | Sox2 | positional information individually in the limb bud were sufficient to activate expres- sion of class II genes (Fig. 1B, and Fig. S1 B–E). In contrast, misexpression of SmoM2 and SoxB1 in combination resulted in Results a striking induction of Nkx2.2, Nkx6.1, Nkx6.2,andOlig2 in the limb bud (Fig. 1B,andFig. S1 D and E). Similarly, CRMNkx2.2-, rowth and patterning of the developing embryo is ac- CRMNkx6.1-, and CRMNkx6.2-driven reporter constructs (Fig. 1C), complished by redeploying a remarkably small number of G E signaling pathways, but how these activities are interpreted in which are active in neural tissue but not mesodermal cells (Fig. 1 ) a tissue-specific manner is poorly understood (1, 2). During CNS (9), were activated in the limb bud by Shh signaling in a SoxB1- development, the morphogens Sonic hedgehog (Shh), retinoids, dependent manner (Fig. 1E). Direct binding of SoxB1 to CRMs was required for transcriptional activation, as indicated by the and bone morphogenetic proteins (BMPs) impart positional Nkx2.2 Nkx6.1 identity to progenitor cells at ventral, intermediate, and dorsal abolished activity of CRM and CRM following in- positions of the neural tube, respectively (3–5). These signals act activation of the Sox-binding sites (SBSs) in these elements by regulating the spatial expression patterns of homeodomain and (Fig. 1 C and F). Nkx2.2 is a known repressor of Olig2 in the basic-helix–loop–helix transcription factors (TFs) that specify cell neural tube (15), and we noted that expression of Olig2 was – lower than that of the other class II genes at 40 hpe, but identity along the dorsoventral (DV) axis of the neural tube (3 7), Olig2 and many of these TFs are activated by these pathways specifically CRM exhibited no activity, implying that Nkx2.2 represses Olig2 in the limb bud. Consistent with this finding, at 6 hpe we in neural progenitors but not other developing tissues. For ex- Olig2 ample, graded Shh signaling underlies patterning of the ventral observed that the class II genes, including Olig2 and CRM , neural tube by repressing or inducing expression of class I and were induced. Moreover, Nkx2.2 and Olig2 were expressed in class II TFs, respectively, but this set of genes is not induced during Shh-mediated patterning of the developing limb bud (8). We have recently identified cis-regulatory modules (CRMs) for Author contributions: T.O., S.K., M.E., M.B., M.S., R.S., J.M., and J.E. designed research; the Shh-regulated class I and II TFs Nkx2.2, Nkx2.9, Olig2, Nkx6.1, T.O., S.K., M.E., C.W.U., M.B., and M.S. performed research; M.E. contributed new re- agents/analytic tools; T.O., S.K., M.E., C.W.U., M.B., R.S., J.M., and J.E. analyzed data; Nkx6.2, Dbx1, Dbx2, and Pax6 (9). These CRMs recapitulate the and T.O., C.W.U., R.S., and J.E. wrote the paper. expression patterns of their respective endogenous genes in the fl neural tube, and are directly regulated by bifunctional Gli tran- The authors declare no con ict of interest. scription factors of the Shh pathway (5) via conserved Gli-binding *This Direct Submission article had a prearranged editor. sites (GBS) (Fig. 1C and Fig. S1F) (9). Importantly, functional 1T.O. and S.K. contributed equally to this work. analyses of these elements indicate that activation of Shh-induced 2To whom correspondence should be addressed. E-mail: [email protected]. Nkx2.2 Olig2 Nkx6.1 Nkx6.2 CRMs (CRM ,CRM ,CRM ,andCRM )byac- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tivator forms of Gli proteins (GliA) is sequence context-dependent 1073/pnas.1220010110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1220010110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. SoxB1 proteins confer competence to activate neural-specific Shh target genes in the limb bud. (A) Schematic illustrating experimental setup for electroporation of the proximal limb bud. (B) Expression of Sox3, Ptc1, and class II genes in the neural tube and limb bud following constitutive activation of Shh signaling by forced expression of SmoM2 alone (Left) or in combination with Sox3 (Right) in the proximal limb bud. Electroporated region of limb bud indicated by Ptc1 expression. Dashed line delineates proximal border of limb bud. NT, neural tube; LB, limb bud. (C) Schematics illustrate the position of SBSs and the GBS in each class II CRM. (D) Schematic of transverse section from chicken embryo. Blue boxes indicate regions of neural or limb bud tissue shown in E. (E) Activities of CRMs associated with Ptc1 and class II genes (white) in the ventral neural tube (Left, magnification 80×) and limb bud following electro- poration with SmoM2 alone (Center) or in combination with Sox3 (Right). GFP electroporation control (blue). CRM activity in limb is shown after 40 hpe (magnification 70×), except for CRMOlig2 that is 6 hpe (magnification 100×). (F, Upper) Schematics indicate the relative positions of SBSs (red) and GBSs (blue) in CRMs. X indicates mutationally inactive sites. Activity of wild-type or mutated CRMs (white) in the limb bud; GFP electroporation control (blue) (magni- fication 50×). (G) Expression of the neural-specific class II genes in the proximal limb bud 6 hpe in response to forced expression of Sox3 and SmoM2 (magnification 100×). (H and I) Expression of Sox3, dHand,andSox1 in the limb bud following forced expression of Sox3 (magnification 30×). Dotted red oval indicates electroporated area, as determined by Sox3 expression. a mutually exclusive fashion (Fig. S1D), indicating that cross- GBS and the nearest conserved SBS in CRMs associated with repressive interactions between neural patterning transcription Shh-induced class II genes was 36 bp (9), implying that collocation factors are recapitulated in this assay (see also Fig. S1E) (5). of conserved SBSs and GBSs in noncoding genomic sequences The rapid induction of class II genes in the limb bud (within 6 could be sufficient to identify genes regulated by Shh signaling hpe), together with the requirement for direct binding by SoxB1 in neural tissue.
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