SoxB1-driven transcriptional network underlies neural-specific interpretation of morphogen signals

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)
April 15, 2013
110 (18) 7330-7335

Abstract

The reiterative deployment of a small cadre of morphogen signals underlies patterning and growth of most tissues during embyogenesis, but how such inductive events result in tissue-specific responses remains poorly understood. By characterizing cis-regulatory modules (CRMs) associated with genes regulated by Sonic hedgehog (Shh), retinoids, or bone morphogenetic proteins in the CNS, we provide evidence that the neural-specific interpretation of morphogen signaling reflects a direct integration of these pathways with SoxB1 proteins at the CRM level. Moreover, expression of SoxB1 proteins in the limb bud confers on mesodermal cells the potential to activate neural-specific target genes upon Shh, retinoid, or bone morphogenetic protein signaling, and the collocation of binding sites for SoxB1 and morphogen-mediatory transcription factors in CRMs faithfully predicts neural-specific gene activity. Thus, an unexpectedly simple transcriptional paradigm appears to conceptually explain the neural-specific interpretation of pleiotropic signaling during vertebrate development. Importantly, genes induced in a SoxB1-dependent manner appear to constitute repressive gene regulatory networks that are directly interlinked at the CRM level to constrain the regional expression of patterning genes. Accordingly, not only does the topology of SoxB1-driven gene regulatory networks provide a tissue-specific mode of gene activation, but it also determines the spatial expression pattern of target genes within the developing neural tube.

Results

Growth and patterning of the developing embryo is accomplished by redeploying a remarkably small number of signaling pathways, but how these activities are interpreted in a tissue-specific manner is poorly understood (1, 2). During CNS development, the morphogens Sonic hedgehog (Shh), retinoids, and bone morphogenetic proteins (BMPs) impart positional identity to progenitor cells at ventral, intermediate, and dorsal positions of the neural tube, respectively (35). These signals act by regulating the spatial expression patterns of homeodomain and basic-helix–loop–helix transcription factors (TFs) that specify cell identity along the dorsoventral (DV) axis of the neural tube (37), and many of these TFs are activated by these pathways specifically in neural progenitors but not other developing tissues. For example, graded Shh signaling underlies patterning of the ventral neural tube by repressing or inducing expression of class I and 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 the Shh-regulated class I and II TFs Nkx2.2, Nkx2.9, Olig2, Nkx6.1, Nkx6.2, Dbx1, Dbx2, and Pax6 (9). These CRMs recapitulate the expression patterns of their respective endogenous genes in the neural tube, and are directly regulated by bifunctional Gli transcription factors of the Shh pathway (5) via conserved Gli-binding sites (GBS) (Fig. 1C and Fig. S1F) (9). Importantly, functional analyses of these elements indicate that activation of Shh-induced CRMs (CRMNkx2.2, CRMOlig2, CRMNkx6.1, and CRMNkx6.2) by activator forms of Gli proteins (GliA) is sequence context-dependent and critically requires the direct synergistic input of SoxB1 proteins at the transcriptional level (9). The SoxB1 group of TFs, Sox1 to -3, are broadly expressed in the developing CNS and play an important role in maintaining neural progenitor properties (10, 11). Importantly, these TFs are expressed in a neural-specific manner (Fig. 1B) (12), raising the possibility that SoxB1 proteins contribute to the tissue-specific selection of target gene activation upon Shh pathway activation.
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 electroporation 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) (magnification 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, and Sox1 in the limb bud following forced expression of Sox3 (magnification 30×). Dotted red oval indicates electroporated area, as determined by Sox3 expression.
Graded Shh signaling underlies anteroposterior patterning of 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 they are transcriptionally silent in this tissue (8, 9). We therefore wished to determine whether expression of SoxB1 proteins in mesodermal cells of the limb bud is sufficient to activate neural-specific Shh-target genes in response to Shh signaling. To test this theory, we electroporated expression constructs into the base of the presumptive limb bud at Hamburger–Hamilton (HH) stage 15 embryos (Fig. 1A) and collected embryos for analysis 6 or 40 h postelectroporation (hpe). The ambient level of Shh signaling was low at the base of the limb at 40 hpe, as indicated by low expression of the Shh receptor Patched 1 (Fig. S1A), which is a primary response gene of Shh signaling (13). Activation of the Shh pathway by forced expression of a constitutively active form of Smoothened (SmoM2) induced Ptc1, as well as a Shh-responsive CRM associated with Ptc1 (Fig. 1 B and E) (14). However, neither SmoM2 nor Sox2 nor Sox3 expressed individually in the limb bud were sufficient to activate expression of class II genes (Fig. 1B, and Fig. S1 B–E). In contrast, misexpression of SmoM2 and SoxB1 in combination resulted in a striking induction of Nkx2.2, Nkx6.1, Nkx6.2, and Olig2 in the limb bud (Fig. 1B, and Fig. S1 D and E). Similarly, CRMNkx2.2-, CRMNkx6.1-, and CRMNkx6.2-driven reporter constructs (Fig. 1C), which are active in neural tissue but not mesodermal cells (Fig. 1E) (9), were activated in the limb bud by Shh signaling in a SoxB1-dependent manner (Fig. 1E). Direct binding of SoxB1 to CRMs was required for transcriptional activation, as indicated by the abolished activity of CRMNkx2.2 and CRMNkx6.1 following inactivation of the Sox-binding sites (SBSs) in these elements (Fig. 1 C and F). Nkx2.2 is a known repressor of Olig2 in the neural tube (15), and we noted that expression of Olig2 was lower than that of the other class II genes at 40 hpe, but CRMOlig2 exhibited no activity, implying that Nkx2.2 represses Olig2 in the limb bud. Consistent with this finding, at 6 hpe we observed that the class II genes, including Olig2 and CRMOlig2, were induced. Moreover, Nkx2.2 and Olig2 were expressed in a mutually exclusive fashion (Fig. S1D), indicating that cross-repressive interactions between neural patterning transcription factors are recapitulated in this assay (see also Fig. S1E) (5).
The rapid induction of class II genes in the limb bud (within 6 hpe), together with the requirement for direct binding by SoxB1 and the fact that Gli is bound to CRMNkx2.2 and CRMNkx6.1 elements in the limb bud (8, 9), strongly indicates that the tissue-specific expression of Shh-regulated genes is primarily determined by the combinatorial activity of Sox and Gli at the transcriptional level. It is notable that expression of the mesodermal marker dHand was maintained in Sox3-expressing cells 40 hpe (Fig. 1 H and I) and that a negligible induction of the neural marker Sox1 was detected at this stage. Importantly, these data argue that the induction of neural-specific class II genes in the limb cannot be explained by reprogramming of mesodermal cells into bona fide neural progenitors, although we do not exclude the possibility that long-term expression of SoxB1 proteins in the limb could eventually result in reprogramming of mesodermal cells.
Our data show that SoxB1 proteins account for the neural-specific activation of class II genes in response to Shh signaling, suggesting that other genes may be regulated according to a similar transcriptional logic. The farthest distance between a functional GBS and the nearest conserved SBS in CRMs associated with Shh-induced class II genes was 36 bp (9), implying that collocation of conserved SBSs and GBSs in noncoding genomic sequences could be sufficient to identify genes regulated by Shh signaling in neural tissue. Binding of the transcriptional coactivator p300 has been shown to accurately predict tissue specific enhancer activity during embryogenesis (16), and we therefore devised a computational method to identify TF-binding sites (TFBSs) that were enriched within 50 bp of conserved GBSs in p300-bound elements from limb bud (n = 25) and neural tissue (n = 72) (SI Experimental Procedures and Fig. S2A). This TFBSs analysis revealed a significant overrepresentation of SBSs around GBSs in elements bound by p300 in neural compared with limb tissue (Fig. 2A and Table S1).
Fig. 2.
The collocation of a SBS and GBS in CRMs defines a genome-wide transcriptional code for Shh-regulated genes active in the CNS. (A) In silico screen of p300-bound mouse elements from limb or CNS (as determined by ref. 16) for association strength between GBSs and TFBSs that are conserved between mouse, human, and opossum. Significant values for association of Sox-binding sites with GBSs are highlighted in red. Error bars indicate SD (n = 10). (B) Statistics for database and literature survey of the tissue- and region-specific expression patterns of transcription factors associated with collocated SBS and GBS sites. (C) Neural expression of genes identified following a genome-wide screen for a SBS and GBS located at a maximum distance of 36 bp from one another. GBS affinity scores are shown in Table S10. (Upper) SBS and GBS sequence logos. (D) Expression of SoxB1-Gli target genes in the unelectroporated limb bud (control) or following forced expression of SoxB1 and SmoM2. (E) Transcriptional assays in P19 cells on CRMs containing a SBS-GBS signature, indicating a synergistic relationship between SoxB1 and the Shh pathway for activation of these CRMs. RLU: relative luciferase units. Error bars indicate SD (n = 2).
To directly test the predictive value of a GBS-SBS signature, we performed a genome-wide search for consensus GBS and SBS-containing noncoding elements conserved between mouse, human, and opossum, and examined the expression patterns and regulation of nearby genes (Table S2). Eighty-three presumptive CRMs were identified when the maximum distance between the GBS and SBS was set at 36 bp (Table S3), and the number of positive regions decreased as a function of increased distance between conserved Gli and SoxB1 binding sites (Fig. S2 B and C). A survey of gene-expression databases and the literature indicated that genes encoding TFs that could be linked to mammalian GBS-SBS elements have a statistically significant higher probability (Fisher’s exact test, P < 0.01) of being expressed in the ventral neural tube (57%) compared with the posterior limb bud (7%), where Shh signaling levels are high (Fig. 2B and Table S4) (8, 17). A similar search for GBS-SBS signatures between chick and mouse identified 45 presumptive CRMs enriched for conserved GBSs and SBSs (Table S5). This relatively low number of regions probably reflects the incomplete sequence information of the chick genome at the time of analysis. Nevertheless, we used this dataset to evaluate whether GBS/SBS elements promote neural gene expression in a Shh-dependent manner. Of the closest neighboring genes, we selected those located within 150 kb of a Sox-Gli signature (except for one located ∼1 × 106 bp away). After excluding from our dataset the class I and II genes for which Shh-regulated CRMs have been identified previously (9), we obtained 15 functional riboprobes from the remaining 20 neighboring genes identified. Expression analysis of this set of genes revealed that 87% (13 of 15) of genes showed a clear ventral bias of expression in the neural tube (Fig. 2C). Strikingly, most of these genes could be ectopically activated in the limb bud in response to Sox3 and SmoM2 expression (Fig. 2D). Moreover, although Ppap2b was one of two genes that were not induced in the limb bud assay, isolation of the CRM associated with phosphatidic acid phosphatase type 2B (Ppap2b, CRMPpap2b) showed that this element could be synergistically activated by Sox3 and an activator form of Gli3 (Gli3-High, Gli3H) in P19 cells (Fig. 2E), and similar results were obtained for three CRMs associated with Ebf3 (CRMEbf3), C20orf19 (CRMC20orf19), and Zfp238 (CRMZfp238) (Fig. 2E). Taken together, these data demonstrate that enrichment and proximity of GBSs and SBSs in conserved noncoding sequences can faithfully predict genes regulated by Shh signaling in the developing CNS, providing evidence that a common transcriptional strategy underlies tissue-specific activation of Shh target genes in the developing CNS.
We next wished to determine whether SoxB1 proteins contribute to the neural-specific interpretation of other pleiotropic signals active in the developing CNS. The class II genes Dbx1 and Dbx2 are expressed in the intermediate neural tube, but whereas repressor forms of Gli proteins (GliR) are important to suppress expression of these genes in the dorsal neural tube (9, 18), Shh signaling is not required to activate them (3, 9). Instead, retinoic acid (RA) signaling by paraxial mesoderm has been implicated in the induction of these genes in the neural tube (3). RA binds nuclear receptors directly to activate transcription (19) and examination of CRMDbx1 and CRMDbx2 revealed that each contains two conserved nuclear receptor binding sites (NRBS) (Fig. 3A and Fig. S1F) resembling RARE (retinoic acid response element) half sites. Mutational inactivation of these sites abolished the activity of these CRMs in vivo (Fig. 3A), providing evidence that RA directly regulates transcription of Dbx1 and Dbx2. Like CRMs for Shh-induced class II genes (9), CRMDbx1 and CRMDbx2 contain four and three conserved SBSs, respectively, and ChIP experiments showed that SoxB1 proteins bound these elements in vivo (Fig. 3C). Inactivation of the three SBSs in CRMDbx2 led to a complete loss of CRM activity (Fig. 3B), whereas mutation in CRMDbx1 of SBS4, which lies nearby a typical RARE (DR2) (Fig. 3A), eliminated CRM-driven reporter activity in the neural tube (Fig. 3A). Analyses of CRMDbx1 in transcriptional assays in P19 cells indicated that exposure of cells to RA was not sufficient to induce CRMDbx1 in vitro, whereas forced expression of Sox3 alone resulted in a moderate up-regulation of transcriptional activity (Fig. 3B). Importantly, however, there was a synergistic and dose-dependent activation of CRMDbx1 when P19 cells overexpressing Sox3 were also exposed to increasing concentrations of RA (Fig. 3B). Furthermore, this synergistic activation was abolished in CRMDbx1 carrying either the inactivated RARE or the closely located SBS4 (Fig. 3B). Thus, SoxB1 proteins are required for and appear to act synergistically with RA to activate transcription of Dbx genes in the developing neural tube.
Fig. 3.
SoxB1 proteins are required for activation of retinoid and BMP target genes in the CNS. (A) (Upper) Schematics indicating positions of SBSs and NRBSs in CRMDbx1 and CRMDbx2. (Lower) Neural activity of CRMDbx1 (24 hpe, magnification 100×) and CRMDbx2 (40 hpe, magnification 90×). For each CRM (Left) two wild-type CRMs driving expression of distinct reporter genes (white and blue); (Center) NRBS-inactivated and wt control CRM (white and blue, respectively); (Right) SBS-inactivated and wild-type control CRM (white and blue, respectively). (B) Transcriptional assays in P19 cells on wild-type CRMDbx1 or elements carrying either an inactivated SBS4 or inactivated NRBS, as indicated, following addition of RA alone (Left) and in combination with Sox3 (Center, Right). (C) Sox3 ChIP on mouse tissue for CRMDbx1 and CRMDbx2. (D) Expression of Sox3 and class I genes in the neural tube and limb bud following forced expression of Sox3 in the proximal limb bud. Electroporated region of limb bud indicated bySox3 expression. Dashed line delineates proximal border of limb bud. NT, neural tube; LB, limb bud. (E) Activity in the limb of a wild-type (Top, Middle) or NRBS-inactivated (Bottom) CRMDbx1 (red) and GFP electroporation control (green), electroporated alone or in combination with Sox3 (magnification 70×). (F) In silico screen of p300-bound mouse elements from limb or CNS (as determined by ref. 16) for association strength between RARE and TFBSs that are conserved between mouse, human, and opossum. Significant values for association of Sox- and Fox-binding sites with RAREs are highlighted in red and blue, respectively. Error bars indicate SD (n = 10). (G) Enrichment of functional annotation as CNS or limb of genes associated with an SBS-RARE signature following a genome-wide screen for these sites that are conserved between mouse and chick and either ≤50 bp or >50 bp of one another. (H) Expression of Msx1 and Gsh1 following forced expression of a constitutively active BMP type 1 receptor (Alk-2CA) alone (Left) or in combination with Sox3 (Right) in the proximal limb bud. Only the electroporated regions of limb bud are shown (magnification 70×). (I, Upper) Schematics illustrate the position of the isolated CRM in the Msx1 locus and the location of its SBS and SmBSs. Expression of Msx1 (Far Left) and activity of its associated CRMMsx1 in the dorsal neural tube (magnification 90×). (Center Left) Two wild-type CRMs driving expression of distinct reporter genes (white and blue); (Center Right) SBS-inactivated and wild-type CRM (white and blue, respectively); (Far Right) SmBS-inactivated and wt CRM (white and blue, respectively).
Analysis of conserved RAREs in p300-bound elements from limb bud and neural tissue revealed an overrepresentation of SBSs located in proximity to RAREs in elements active in neural tissue (Fig. 3F and Table S6), suggesting that an SBS-RARE transcriptional code may be of predictive value in determining neural-specific gene expression. Indeed, a complementary genome-wide analysis identified 545 RAREs conserved between mouse and chick (Table S7), 52 of which are located within 50 bp of a conserved SBS, and these collocated sites lie nearby genes significantly enriched for functions in neural development but not limb development (Fig. 3G and SI Experimental Procedures), among them Pax6, which has been implicated in the regulation of DV patterning in the neural tube (5). In contrast, genes lying nearby RAREs located >50 bp away from an SBS showed essentially no difference in the level of enrichment for functional annotation of neural versus limb tissue (Fig. 3G). Retinoids have also been shown to critically regulate limb bud development and to be present at relative high concentrations in the proximal limb bud (20), and consistent with this, we found that expression of Sox3 alone in the base of the presumptive limb was sufficient to activate Dbx1, Dbx2, and Pax6 in this tissue (Fig. 3D). CRMDbx1 was also activated in the limb bud in response to Sox3 expression, and, importantly, this activation required the RARE in this element (Fig. 3E). Taken together, these analyses imply that SoxB1 proteins and retinoids may cooperatively regulate a broad array of genes.
BMP signaling is mediated by nuclear translocation of Smad TFs (Smad 1/5/8) (21), and studies of differentiating hematopoietic cells have indicated that the lineage-specific TFs GATA1 and C/EBPα function to recruit SMAD1 to cell type-specific enhancers active in erythroid and myeloid cells, respectively (22). BMP signaling induces members of the Msx, Pax, and Gsh families of TFs in the dorsal neural tube (4, 7), and we found that Msx1 and -2, Pax3 and -7, and Gsh1 and -2 were associated with presumptive CRMs containing conserved SBS and SMAD binding sites (SmBSs) (Table S8). Msx proteins act as repressors of Nkx6.1 and Dbx1 expression in the neural tube (9), and we isolated a noncoding sequence associated with Msx1 (CRMMsx1) containing two conserved SmBSs and one SBS (Fig. 3I and Fig. S1F) and assessed the transcriptional activity of this element in the neural tube. CRMMsx1 precisely recapitulated the endogenous expression pattern of Msx1 in the dorsal neural tube (Fig. S3A), and this activity was lost when either the two SmBSs or the unique consensus SBS in CRMMsx1 was inactivated (Fig. 3I). Msx genes are also induced in nonneural tissues in response to BMP signaling (23, 24), and in gain-of-function experiments, activation of BMP signaling by forced expression of a constitutively active form of BMP receptor type I (Alk-2CA) alone induced expression of the endogenous Msx1 gene, as well as both wild-type and SBS-mutated forms of CRMMsx1 in both neural tissue and limb bud (Fig. 3H and Fig. S3B) (23, 25). Taken together, these data argue that SoxB1 promotes Msx1 expression but is not absolutely required for Smad-mediated induction of Msx1 in the neural tube. Compared with Msx genes, Gsh genes are expressed in a more neural-specific fashion (26, 27), and we found that induction of Gsh1 in limb bud mesoderm by BMPs required the cooperative activity of SoxB1 proteins (Fig. 3H), implying that expression of at least a subset of genes induced by BMP signaling in neural tissue critically requires the cooperative activity of SoxB1 proteins.

Discussion

Collectively, our data provide evidence for a functional integration of SoxB1 proteins and nuclear mediators of Shh, retinoid, and BMP signaling at the CRM level, offering an unexpectedly simple transcriptional paradigm for the neural-specific interpretation of pleiotropic morphogen signals during embryonic development. Accordingly, SoxB1 is sufficient to confer on mesodermal cells the potential to up-regulate neural-specific target genes induced by morphogen signaling in the limb bud in a manner that recapitulates the DV patterning activity of Shh, RA, and BMPs in the neural tube (Fig. 4B). The neural-specific interpretation of Shh and retinoid signaling appears to be contingent on genome-wide signatures consisting of a collocated SBS-GBS or SBS-RARE in genomic sequences, respectively. In the case of Shh signaling, such motifs can faithfully predict expression of associated genes in the ventral neural tube. Similarly, genes associated with presumptive SBS-RARE–regulated CRMs have a higher probability of being active in neural tissue compared with limb bud.
Fig. 4.
Recapitulation of dorsoventral neural patterning in the limb bud and a model of the neural-specific responses to morphogen signaling. (A) Schematic of transverse section from chicken embryo. Blue boxes indicate regions of neural or limb bud tissue shown in B. (B) Forced expression of Sox3 confers on mesodermal cells the potential to recapitulate the DV patterning of the neural tube (see Left), as indicated by the induction of the Shh-regulated Nkx2.2, retinoid-regulated Dbx2, and BMP-regulated Gsh1/2 genes in the chick limb bud (Right). Shh*, Shh pathway activated by SmoM2; RA*, endogenous levels of RA signaling; Bmp*, Bmp pathway activated by Alk-2CA. (C) Neural-specific gene activation: Shh, RA, and BMPs emanate from local sources and act in a graded fashion to pattern cells along the DV axis of the neural tube. Many cell fate-determining TFs operating downstream of these morphogens are specifically induced in neural tissue via the cooperative activity of SoxB1 proteins and signal-transducing TFs at the CRM level, enabling the neural-specific selection of target gene activation in response to these pleiotropic signals. Regional restriction of gene expression: Genes induced in a SoxB1-dependent manner constitute functional, repressive GRN that act locally to constrain the regional expression domains of cell fate-determining TFs operating downstream of morphogen signals in neural patterning, and the repressive inputs of these Shh-, RA-, and BMP-driven networks are directly interconnected at the CRM level to control the patterning of target genes along the DV axis of the neural tube. Accordingly, not only does the topology of SoxB1-driven GRNs provide a tissue-specific mode of gene activation, but it also determines the spatial expression pattern of target genes within that tissue. G, Gli; NR, nuclear receptor; S, SoxB1; Sm, Smad.
It has long been established that cross-repressive interactions between Shh-regulated TFs are themselves an integral feature of gene regulatory networks (GRNs) that interpret positional information in neural patterning (6, 15, 2832), but it has remained elusive how these repressive inputs operate together with morphogen signals at the genomic level to regulate patterns of gene expression. Class I and II TFs determine the positional identity of progenitors downstream of the Shh gradient (5), and our recent analyses indicated that these genes are induced by a synergistic interaction between SoxB1 and Gli proteins at the CRM level; positional information is primarily imparted at the CRM level by cooperative transcriptional repression mediated by Gli-repressors and cross-repressive interactions between class I and II proteins themselves (9). In addition to the induction of these repressive factors, it is notable that many in silico-identified SBS-GBS–associated genes have been implicated previously in Shh signaling and ventral pattern formation, including Tcf4, Tle4, Nkx2.1, Six3, Nf1a, hedgehog interacting protein (Hhip), and beta-transducin repeat containing protein (Btrc) (Table S9). Furthermore, we observed a functional recapitulation of tissue patterning and cross-repressive interactions between class I and II proteins in the limb bud (Fig. 4B and Fig. S1E). SoxB1 and Gli proteins therefore appear to define the central node of a neural-specific GRN required to translate graded Shh signaling into regional gene-expression patterns in the ventral neural tube (Fig. 4C). Although SoxB1 and Gli proteins are sufficient to trigger activation of this network in the limb, this does not mean that they are the only activators involved, as many genes cooperatively activated by SoxB1 and Gli proteins in neural tissue (and in the limb) encode transcriptional activators or repressors that themselves are integral components of the network. Such proteins are likely to act in a more CRM context-dependent manner to influence the regional expression pattern of Shh-regulated genes within the neural tube, as exemplified by Tcf proteins, which act cooperatively with SoxB1 and Gli to activate transcription of Nkx2.2 in the ventral-most neural tube (9) and which are induced upon SoxB1 and SmoM2 expression in the limb bud. Such a node-driven process provides a mechanistic rationale for how established GRNs can be coopted and redeployed by cells simply by up-regulating core activator components of the GRN (33). Positional information provided by retinoids and BMP signaling in neural tissue is likely to be similarly interpreted by SoxB1-driven repressive GRNs (Fig. 4C). Importantly, these GRNs are directly interlinked at the CRM level, as illustrated by Nkx6.1, which is induced by SoxB1 and Gli proteins but positionally restricted to ventral progenitors by RA- and BMP-induced genes (9), and Dbx1, which is regulated by both RA and Gli-repressors (present study) (9). Accordingly, the SoxB1 transcriptional code provides not only a strategy for the tissue-specific selection of target genes, but also for the induction of GRNs that drive morphogen interpretation and determine the positional identity of cells (Fig. 4C).

Experimental Procedures

For additional information regarding cell transfection assays, ChIP, bioinformatics, DNA-constructs, RNA-probes, antibodies, and additional reagents, please refer to SI Experimental Procedures.

In Ovo Electroporation.

pCAGGS expression vectors and reporter constructs (BGZA or BG-eGFP) containing wild-type or mutated CRMs were electroporated either alone or in combination according to the text, into HH stage 10–12 (neural tube) (6) or HH stage 15 (limb bud) (34) chicken embryos. After 6, 24, or 40 h of incubation, embryos were isolated and processed for immunohistochemistry and in situ hybridization. Reporter constructs containing mutated CRMs were electroporated at concentrations that would result in clear detectable activities for their wild-type versions. For each experimental condition, at least three different embryos from two independent experiments were analyzed.

Immunofluorescence and in Situ Hybridization.

Immunofluorescence and in situ hybridization were performed essentially as previously described (6, 35).

P19 Cell Transfection Assays.

P19 cells were transfected by using Lipofectamine and Plus Reagent (Invitrogen).

ChIP.

ChIP experiments were performed as previously described (9).

Acknowledgments

We thank J. Briscoe, M. Goulding, B. Novitch, M. Ros, and R. Toftgård for reagents. This work was supported by the Swedish Research Council (33X-06555; Developmental Biology for Regenerative Medicine); The Royal Swedish Academy of Sciences by donation from the Wallenberg Foundation; the Swedish Foundation for Strategic Research (Center of Excellence in Developmental Biology, SRL10-0030); The Knut and Alice Wallenberg Foundation (KAW2011.0161); research funds of Karolinska Institutet; and Wenner-Gren Foundation and European Union Marie Curie MEIF-CT-2006-025416 (to T.O.).

Supporting Information

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 110 | No. 18
April 30, 2013
PubMed: 23589857

Classifications

Submission history

Published online: April 15, 2013
Published in issue: April 30, 2013

Keywords

  1. Sox3
  2. Gli
  3. Sox2
  4. positional information

Acknowledgments

We thank J. Briscoe, M. Goulding, B. Novitch, M. Ros, and R. Toftgård for reagents. This work was supported by the Swedish Research Council (33X-06555; Developmental Biology for Regenerative Medicine); The Royal Swedish Academy of Sciences by donation from the Wallenberg Foundation; the Swedish Foundation for Strategic Research (Center of Excellence in Developmental Biology, SRL10-0030); The Knut and Alice Wallenberg Foundation (KAW2011.0161); research funds of Karolinska Institutet; and Wenner-Gren Foundation and European Union Marie Curie MEIF-CT-2006-025416 (to T.O.).

Notes

*This Direct Submission article had a prearranged editor.

Authors

Affiliations

Tony Oosterveen1
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and
Sanja Kurdija1
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and
Mats Ensterö
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and
Ludwig Institute for Cancer Research, 171 77, Stockholm, Sweden
Christopher W. Uhde
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and
Maria Bergsland
Ludwig Institute for Cancer Research, 171 77, Stockholm, Sweden
Magnus Sandberg
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and
Ludwig Institute for Cancer Research, 171 77, Stockholm, Sweden
Rickard Sandberg
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and
Ludwig Institute for Cancer Research, 171 77, Stockholm, Sweden
Jonas Muhr
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and
Ludwig Institute for Cancer Research, 171 77, Stockholm, Sweden
Johan Ericson2 [email protected]
Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden; and

Notes

2
To whom correspondence should be addressed. E-mail: [email protected].
Author contributions: T.O., S.K., M.E., M.B., M.S., R.S., J.M., and J.E. designed research; T.O., S.K., M.E., C.W.U., M.B., and M.S. performed research; M.E. contributed new reagents/analytic tools; T.O., S.K., M.E., C.W.U., M.B., R.S., J.M., and J.E. analyzed data; and T.O., C.W.U., R.S., and J.E. wrote the paper.
1
T.O. and S.K. contributed equally to this work.

Competing Interests

The authors declare no conflict of interest.

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    SoxB1-driven transcriptional network underlies neural-specific interpretation of morphogen signals
    Proceedings of the National Academy of Sciences
    • Vol. 110
    • No. 18
    • pp. 7099-7528

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