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Developmental Cell
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HAND2 Targets Define a Network of Transcriptional Regulators that Compartmentalize the Early Limb Bud Mesenchyme

      Highlights

      • ChIP-seq identifies the CRMs bound by endogenous HAND2 in embryos and limb buds
      • HAND2 controls key transcriptional regulators acting upstream of SHH in limb buds
      • These transcriptional circuits define proximal, anterior, and posterior identities
      • HAND2 establishes anterior and posterior compartments by regulating Gli3 and Tbx3

      Summary

      The genetic networks that govern vertebrate development are well studied, but how the interactions of trans-acting factors with cis-regulatory modules (CRMs) are integrated into spatiotemporal regulation of gene expression is not clear. The transcriptional regulator HAND2 is required during limb, heart, and branchial arch development. Here, we identify the genomic regions enriched in HAND2 chromatin complexes from mouse embryos and limb buds. Then we analyze the HAND2 target CRMs in the genomic landscapes encoding transcriptional regulators required in early limb buds. HAND2 controls the expression of genes functioning in the proximal limb bud and orchestrates the establishment of anterior and posterior polarity of the nascent limb bud mesenchyme by impacting Gli3 and Tbx3 expression. TBX3 is required downstream of HAND2 to refine the posterior Gli3 expression boundary. Our analysis uncovers the transcriptional circuits that function in establishing distinct mesenchymal compartments downstream of HAND2 and upstream of SHH signaling.

      Graphical Abstract

      Introduction

      The limb bud is an excellent model to study the gene networks that govern growth and patterning during vertebrate organogenesis (
      • Zeller R.
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      Vertebrate limb bud development: moving towards integrative analysis of organogenesis.
      ). These gene regulatory networks impact the expression of target genes via large cis-regulatory landscapes that integrate different inputs in a spatiotemporally controlled manner (
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      Transcription factors: from enhancer binding to developmental control.
      ). For example, the expression of Shh in the posterior limb bud mesenchyme is controlled by a far upstream cis-regulatory module (CRM), termed ZRS (
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      ). Genetic ablation of the ZRS phenocopies the Shh loss of function in mouse limb buds, manifesting itself in loss of digits (
      • Sagai T.
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      Elimination of a long-rangecis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb.
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      ). Localized Shh expression depends on the interaction of different transcriptional regulators with the ZRS. In particular, the interaction with HOX, PBX, ETS, and HAND2 transcriptional complexes has been implicated in activation of Shh in the limb bud, whereas TWIST1, ETV, and GATA factors prevent anterior ectopic expression (
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      ). How the ZRS integrates these various inputs over time is unknown, but the resulting posterior restriction of SHH signaling is essential for proliferative expansion and anterior-posterior (AP) patterning of the distal limb bud mesenchyme that will form the skeletal elements of the zeugopod and autopod (
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      ).
      The mesenchymal progenitors giving rise to the most proximal skeletal structures (i.e., scapula and humerus in the forelimb) are likely specified prior to activation of SHH signaling (
      • Ahn S.
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      ,
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      ). It has been shown that proximal mesenchymal progenitors express several transcriptional regulators belonging to the Pbx, Meis, and Irx gene families, which participate in specification and/or morphogenesis of proximal skeletal elements (
      • Capdevila J.
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      Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1.
      ,
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      Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by Shh signaling.
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      • Leonardo E.
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      • Martínez-A C.
      • Ros M.A.
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      Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes.
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      Requirement for Pbx1 in skeletal patterning and programming chondrocyte proliferation and differentiation.
      ). Genetic evidence indicates that Raldh2, which is involved in retinoic acid synthesis, regulates the expression of several of these early genes, including the bHLH transcriptional regulator Hand2 (
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      Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse.
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      Retinoic acid promotes limb induction through effects on body axis extension but is unnecessary for limb patterning.
      ).
      Hand2 is genetically required for limb bud, branchial arch, and heart development and the lethality of Hand2-deficient mouse embryos around embryonic day E9.5 is caused by the severe heart malformations (
      • Srivastava D.
      • Thomas T.
      • Lin Q.
      • Kirby M.L.
      • Brown D.
      • Olson E.N.
      Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND.
      ,
      • Vincentz J.W.
      • Barnes R.M.
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      ). During early limb bud development, Hand2 is required for AP polarization of the nascent limb bud mesenchyme and activation of Shh expression as part of its genetic interactions with Gli3 (
      • Charité J.
      • McFadden D.G.
      • Olson E.N.
      The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development.
      ,
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ). In particular, the Hand2 and Gli3 genetic antagonism is required to establish AP asymmetry and pentadactyly, as limb buds deficient for both these transcriptional regulators lack discernible AP polarity, Shh expression, and are extremely polydactylous (
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ,
      • te Welscher P.
      • Fernandez-Teran M.
      • Ros M.A.
      • Zeller R.
      Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling.
      ). As inactivation of Hand2 after the onset of Shh expression does not severely alter limb bud development, Hand2 functions are required mostly upstream of activating SHH signaling (
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ). However, the molecular nature of the underlying transcriptional and cis-regulatory networks remained largely unknown, as Shh is the only known direct transcriptional target of HAND2 in limb buds.
      We have inserted a 3xFLAG epitope tag into the endogenous HAND2 protein to first determine the range of genomic regions enriched in endogenous HAND2 chromatin complexes. In a second step, we focused our in-depth analysis predominantly on HAND2 target genes that encode transcription factors expressed and/or required during early limb development. This analysis established that during the onset of limb bud outgrowth, HAND2 controls the expression of transcriptional regulators in the proximal mesenchyme that are involved in the formation of the most proximal forelimb skeletal elements. In addition, our study reveals the gene regulatory logic by which HAND2, in cooperation with GLI3 and TBX3, establishes AP axis polarity in the early limb bud mesenchyme. In summary, our analysis uncovers the HAND2-dependent molecular circuits that function in establishing proximal, anterior, and posterior compartments and activating Shh expression during the onset of limb bud development.

      Results

      A Hand23xFLAG Allele Generated by dRMCE Identifies the Genomic Regions Enriched in Endogenous HAND2 Chromatin Complexes

      A 3xFLAG epitope tag was inserted into the endogenous HAND2 protein using dual recombinase-mediated cassette exchange (dRMCE;
      • Osterwalder M.
      • Galli A.
      • Rosen B.
      • Skarnes W.C.
      • Zeller R.
      • Lopez-Rios J.
      Dual RMCE for efficient re-engineering of mouse mutant alleles.
      ) to replace the conditional allele with a Hand23xFLAG cassette in mouse embryonic stem cells (ES) (Figure 1A; Supplemental Experimental Procedures available online). Insertion of the 3xFLAG epitope tag into the N terminus does not alter HAND2 functions, as homozygous Hand23xFLAG (Hand23xF/3xF) mice develop normally and do not show any embryonic lethality (
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ,
      • Srivastava D.
      • Thomas T.
      • Lin Q.
      • Kirby M.L.
      • Brown D.
      • Olson E.N.
      Regulation of cardiac mesodermal and neural crest development by the bHLH transcription factor, dHAND.
      ; data not shown). The 3xFLAG epitope tag allows sensitive detection of the endogenous HAND2 protein isoforms in embryonic tissues (limb buds, heart, and branchial arches; Figures 1B and 1C). During the onset of forelimb bud outgrowth, HAND2-positive nuclei are detected in the posterior and proximal-anterior limb bud mesenchyme (green fluorescence; Figures 1D, S1A, and S1D). As HAND2 is required to activate Shh expression (
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ), we compared the distribution of HAND2-positive and Shh-expressing cells using the Shh-GFP allele (red fluorescence;
      • Harfe B.D.
      • Scherz P.J.
      • Nissim S.
      • Tian H.
      • McMahon A.P.
      • Tabin C.J.
      Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.
      ). The Shh-expressing cells define a subdomain within the posterior HAND2-positive limb bud mesenchyme (Figures 1D and S1D). We also colocalized HAND2 with the GLI3 repressor isoform (GLI3R) to determine their spatial distribution during activation of Shh expression. GLI3R proteins were detected by immunofluorescence using monoclonal GLI3 N-terminal antibodies (
      • Wen X.
      • Lai C.K.
      • Evangelista M.
      • Hongo J.A.
      • de Sauvage F.J.
      • Scales S.J.
      Kinetics of hedgehog-dependent full-length Gli3 accumulation in primary cilia and subsequent degradation.
      ) as they recognize the nuclear GLI3R isoform rather than the cytoplasmic full-length GLI3 and/or nuclear GLI3A activator isoforms in limb bud sections (Figures S1B and S1C). This analysis reveals the complementary distribution of the nuclear HAND2 (green) and GLI3R proteins (purple) in mouse forelimb bud mesenchymal cells (Figures 1D, S1B, and S1D).
      Figure thumbnail gr1
      Figure 1Insertion of a 3xFLAG Epitope Tag into the Endogenous HAND2 Protein Provides a Sensitive Tool to Detect HAND2 Protein Complexes
      (A) The Hand23xF allele was generated by dRMCE in mouse ES cells.
      (B) Left: Hand2-expressing tissues in mouse embryos at embryonic day E10.5. Right: immunoblot detection of the tagged HAND23xF protein isoforms (H2) in limb buds (LB), heart (HE), and branchial arches (BA). Midbrain (MB) and wild-type extracts are used as negative controls. An asterisk indicates a nonspecific band. FL, forelimb bud; HL, hindlimb bud.
      (C) Detection of HAND23xF proteins in expressing embryonic tissues by immunofluorescence (green) at E10.5. RV, right ventricle; RA, right atrium. 1st, mandibular arch; 2nd, hyoid arch.
      (D) Colocalization of HAND23xF proteins (green) and Shh transcripts (red) in limb buds (E9.75, 28–29 somites). The distribution of nuclear GLI3R proteins (magenta) is shown on an adjacent section. The right-most panel shows an artificial overlap of the two consecutive sections. Limb buds are always oriented with anterior to the top and posterior to the bottom. Nuclei are blue because of counterstaining with Hoechst. Scale bars, 50 μm.
      (E) ChIP-qPCR analysis shows the interaction of HAND23xF chromatin complexes with a specific region in the ZRS in developing limb buds (E10.5, E11.5, and E12.5). The Ebox core sequence (CATCTG) defined by in vitro analysis is indicated. The most relevant qPCR amplicons used are indicated as ZRS 1–ZRS 5. Fold enrichment is shown as mean ± SD (n = 3).
      See also .
      The only known direct target of HAND2 in limb buds is Shh, whose transcriptional activation is controlled by interaction of HAND2 with the ZRS cis-regulatory region located ∼800 kb upstream of the Shh locus (
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ,
      • Lettice L.A.
      • Heaney S.J.
      • Purdie L.A.
      • Li L.
      • de Beer P.
      • Oostra B.A.
      • Goode D.
      • Elgar G.
      • Hill R.E.
      • de Graaff E.
      A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly.
      ). Hand23xF/3xF limb buds were used for chromatin immunoprecipitation (ChIP) using anti-FLAG antibodies in combination with quantitative real-time PCR (ChIP-qPCR). While our previous analysis did not unambiguously identify the HAND2-binding region(s) within the ZRS (
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ), ChIP of the endogenous HAND23xF epitope-tagged protein resulted in identification of a specific region within the ZRS (Figure 1E). Amplicon-tiling of the ZRS core (∼1.1 kb;
      • Sagai T.
      • Hosoya M.
      • Mizushina Y.
      • Tamura M.
      • Shiroishi T.
      Elimination of a long-rangecis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb.
      ) showed that a region encoding an Ebox is most enriched in HAND2 chromatin complexes (ZRS2-4 in Figure 1E;
      • Dai Y.S.
      • Cserjesi P.
      The basic helix-loop-helix factor, HAND2, functions as a transcriptional activator by binding to E-boxes as a heterodimer.
      ). This interaction is specific, as another Ebox sequence is not enriched (ZRS5; Figure 1E). The enrichment of the ZRS2-4 region in HAND2 chromatin complexes is highest in early limb buds (E10.5; Figure 1E) in agreement with the early but transient genetic requirement of Hand2 for Shh activation (≤E10.5;
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ).
      To define the range of potential HAND2 targets during its early genetic requirement in mouse limb buds, we used chromatin immunoprecipitation sequencing (ChIP-seq). To this aim, the HAND2 binding profile in Hand2-expressing embryonic tissues at E10.5 (eT, limb buds and flank tissue, hearts, and branchial arches; Figure 1B) and dissected limb buds (Lb) was determined by HAND23xF ChIP-seq analysis (Figures 2A–2D and S2A). The primary data sets were analyzed using model-based analysis for ChIP-seq (MACS)-based peak calling, and regions enriched in wild-type eT controls were excluded as nonspecific (Tables S1 and S2 list all statistically validated regions enriched ≥2-fold). For both validated ChIP-seq data sets, the top 1,000 peaks were selected for the initial meta-analysis based on their fold enrichment over the input control and number of reads (Figures 2A–2D and S2A; Tables S3 and S4). The majority of the top 1,000 HAND2-binding peaks in both data sets map to conserved sequences located ≥10 kb away from the closest transcriptional start site (TSS; Figures 2A and 2B). De novo motif discovery showed that also the endogenous HAND2 chromatin complexes interact preferentially with the Ebox sequence defined in vitro (Figure 2C;
      • Dai Y.S.
      • Cserjesi P.
      The basic helix-loop-helix factor, HAND2, functions as a transcriptional activator by binding to E-boxes as a heterodimer.
      ).
      Figure thumbnail gr2
      Figure 2ChIP-Seq Analysis Identifies a Set of HAND2 Target Regions in Mouse Limb Buds
      (A and B) The top 1,000 HAND23xF target regions enriched in E10.5 limb buds (Lb) and Hand2-expressing embryonic tissues (eT) are mostly evolutionarily conserved (A) and map generally ≥10kb away from the closest transcriptional start site (TSS; B).
      (C) De novo motif discovery analysis of the top 1,000 HAND23xF bound regions reveals enrichment in Ebox consensus sequences.
      (D) Gene ontology (GO) analysis reveals the most prominent biological processes associated with HAND23xF binding regions that are represented in both top 1,000 Lb and eT data sets.
      (E) UCSC Genome Browser window shows the Ets1 and Ets2 regions enriched in HAND2 chromatin complexes from limb buds (Lb) and expressing tissues (eT). Distances to the Ets1 and Ets2 TSS are indicated in kilobases. The profiles of DNaseI HS and H3K27ac marks in limb buds (E11.5) are shown in black (
      • Cotney J.
      • Leng J.
      • Oh S.
      • DeMare L.E.
      • Reilly S.K.
      • Gerstein M.B.
      • Noonan J.P.
      Chromatin state signatures associated with tissue-specific gene expression and enhancer activity in the embryonic limb.
      ). The placental mammal conservation (Cons) plot (PhyloP) is shown below. Green bars represent the peaks identified by MACS analysis. The blue bar indicates the hs1516 enhancer element assayed by LacZ transgenesis (Vista Enhancer Browser). The ChIP-seq panels in all figures are organized the same.
      (F) ChIP-qPCR statistically verifies the HAND23xF binding regions in the Ets1 and Ets2 genomic landscapes in limb buds (n = 3; E10.5). Mean ± SD is shown.
      (G) Ets1 and Ets2 transcript distribution in wild-type and Hand2Δ/Δc forelimb buds (≤E10.25).
      (H) The activity of the Ets2 +146kb human ortholog (hs1516-LacZ reporter, Vista Enhancer Browser) is compared with the endogenous Ets2 and Hand2 expression in forelimb buds (E11.5). Scale bars, 100 μm.
      See also and , , , , and .
      Intersection of the top 1,000 eT and Lb data sets shows that 259 of them are shared (Figure S2A; Table S5). GREAT analysis showed that the shared HAND2-binding regions are most often associated with genes known to function in limb bud and/or skeletal development (Figures 2D and S2B). Hence, this set of shared regions and associated genes likely corresponds to HAND2 target genes in limb buds. However, it is important to consider that the sequence coverage was overall significantly lower for the Lb than for the eT ChIP-seq data set. This is a likely consequence of the much lower amounts of tissue and HAND2 chromatin complexes recovered from the ∼1,000 fine-dissected limb buds used for ChIP-seq analysis (Supplemental Experimental Procedures). Furthermore, the ZRS, which is an established HAND2 target region (Figure 1E), was moderately enriched only in the eT ChIP-seq data set (Table S4). This could reflect the fact that the HAND2-ZRS interaction occurs only in a small fraction of posterior mesenchymal cells in early limb buds, and ChIP-qPCR analysis relying on specific oligos is more sensitive (Figures 1D and 1E). The limited sensitivity of the Lb ChIP-seq data set, in particular, indicates that not all relevant HAND2 binding peaks have been detected and represented in the top 1,000 peaks used for the comparative analysis. This is likely a consequence of a significant fraction of HAND2 target genes being restricted very proximally in early limb buds and extending into the flank mesenchyme (see below). However, the flank mesenchyme was only included in the eT, but not in the Lb ChIP-seq sample. Therefore, all potential HAND2 target regions selected for further analysis were first verified in early limb buds (E10.5), using the more sensitive HAND2 ChIP-qPCR analysis (n = 3; Experimental Procedures).
      As we mainly wanted to get insight into the transcriptional circuitry controlled by HAND2 during prepatterning of early limb buds (
      • te Welscher P.
      • Fernandez-Teran M.
      • Ros M.A.
      • Zeller R.
      Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling.
      ), the current study focused on the genomic regions associated with transcription factors functioning and/or expressed upstream of activating Shh expression. To assess the expression of the associated genes in mutant mouse limb buds, Hand2 was conditionally deleted during limb bud initiation using the Hoxb6-Cre transgene (
      • Lowe L.A.
      • Yamada S.
      • Kuehn M.R.
      HoxB6-Cre transgenic mice express Cre recombinase in extra-embryonic mesoderm, in lateral plate and limb mesoderm and at the midbrain/hindbrain junction.
      ). This inactivates Hand2 in the posterior two-thirds of the forelimb bud mesenchyme (Figures S2C and S2D), resulting in almost complete penetrance of the Hand2 loss-of-function limb bud phenotype (n = 9/10). Our initial analysis of the curated list of genes expressed and/or functioning in early limb buds showed that most often their expression is altered in the posterior-proximal mesenchyme of Hand2Δ/Δc forelimb buds (≤E10.25; n = 17/29 genes analyzed; Table 1). Their expression is either significantly reduced or lost from the posterior mesenchyme (n = 10/17; including Ets1, Ets2, Gsc, Tbx2, and Tbx3; Table 1; Figure S2E) or expands posteriorly (n = 7/17; including Gli3, Tbx18, Irx3, and Irx5; Table 1; Figure S2F). This initial analysis pointed to the existence of a network of transcriptional regulators that are direct targets of HAND2 during the onset of limb bud development (Table 1; data not shown).
      Table 1Summary of the RNA In Situ Hybridization Analysis of a Select Set of HAND2 Target Genes in Hand2-Deficient Limb Buds
      Genes
      UpregulatedAlx4,
      Genes analyzed by Galli et al. (2010).
      Gas1, Gli3, Irx3, Irx5, Msx2,
      Genes analyzed by Galli et al. (2010).
      Tbx18
      DownregulatedEts1, Ets2, Furin, Gsc, Shh,
      Only enriched in the eT ChIP-seq data set.
      Slit3, Tbx2, Tbx3, Unc5c, Zfp503
      Not changedBmp4,
      Genes analyzed by Galli et al. (2010).
      Bmp7, Cyp26b1, Fgfr1, Gli2, Lmx1b, Meis2, Msx1, Mycn, Snai1, Tbx4, Tbx5
      Validated ChIP-seq peaks associated with genes encoding transcriptional regulators and/or essential roles during the onset of mouse limb bud development were selected. This table summarizes the alterations in their expression in Hand2-deficient mouse forelimb buds (≤E10.5). Genomic landscapes associated with genes functioning during progression of limb bud development and genes expressed either in the limb bud ectoderm or during advanced stages (e.g., chondrogenesis) were not considered for this study.
      a Genes analyzed by
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      .
      b Only enriched in the eT ChIP-seq data set.
      For example, two candidate CRMs located 23 kb upstream of the Ets1 and 146 kb downstream of the Ets2 TSS are significantly enriched in HAND23xF chromatin complexes as confirmed by ChIP-qPCR in early limb buds (Figures 2E and 2F). Indeed, the expression of both Ets1 and Ets2 is reduced in Hand2-deficient limb buds (Figure 2G). The Ets2 +146kb CRM likely encodes an enhancer as the orthologous human region is active in the posterior limb bud mesenchyme of transgenic mouse embryos (Figure 2H, left panel; Vista enhancer hs1516;
      • Visel A.
      • Minovitsky S.
      • Dubchak I.
      • Pennacchio L.A.
      VISTA Enhancer Browser—a database of tissue-specific human enhancers.
      ). The domain of the LacZ reporter not only recapitulates major aspects of Ets2 expression (Figure 2H, middle panel) but also overlaps the endogenous Hand2 domain (Figure 2H, right panel). These results establish the Ets1 and Ets2 genes as bona fide HAND2 target genes in early limb buds. As the ETS binding sites in the ZRS are essential for Shh expression in the posterior limb bud (
      • Lettice L.A.
      • Williamson I.
      • Wiltshire J.H.
      • Peluso S.
      • Devenney P.S.
      • Hill A.E.
      • Essafi A.
      • Hagman J.
      • Mort R.
      • Grimes G.
      • et al.
      Opposing functions of the ETS factor family define Shh spatial expression in limb buds and underlie polydactyly.
      ,
      • Lettice L.A.
      • Williamson I.
      • Devenney P.S.
      • Kilanowski F.
      • Dorin J.
      • Hill R.E.
      Development of five digits is controlled by a bipartite long-rangecis-regulator.
      ), HAND2-mediated upregulation of the Ets transcription factors likely helps to reinforce Shh expression.

      HAND2 Target Genes Are Required in the Proximal Limb Bud for Morphogenesis of the Scapula and Humerus

      As part of the initial analysis, HAND2 peaks were detected in the genomic landscapes of several transcription factors expressed in the proximal limb bud mesenchyme (Figure 3; Table 1) and that function in morphogenesis of proximal limb skeletal elements (scapula and humerus;
      • Belo J.A.
      • Leyns L.
      • Yamada G.
      • De Robertis E.M.
      The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants.
      ,

      Farin, H. (2009). Function and regulation of the murine T-box genes Tbx15 and Tbx18. PhD thesis (Hannover: University of Hannover).

      ,
      • Li D.
      • Sakuma R.
      • Vakili N.A.
      • Mo R.
      • Puviindran V.
      • Deimling S.
      • Zhang X.
      • Hopyan S.
      • Hui C.C.
      Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by Shh signaling.
      ). In particular, two prominent HAND2 peaks, whose enrichment in early limb buds was confirmed by ChIP-qPCR, map to the Goosecoid (Gsc) genomic landscape (at positions +33 and −133 kb; Figure 3A). Gsc is expressed in the proximal and flank mesenchyme during early limb bud development and is required for scapular head development in the mouse (
      • Belo J.A.
      • Leyns L.
      • Yamada G.
      • De Robertis E.M.
      The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants.
      ). In agreement with Gsc being a direct transcriptional target of HAND2, its expression is lost from the posterior mesenchyme of Hand2Δ/Δc forelimb buds (Figure 3A). Genetic analysis underscores the functional importance of Gsc regulation by HAND2, as Hand2Δ/Δc and Gsc-deficient limbs display strikingly similar scapular defects affecting the glenoid cavity and coracoid process (n = 4/8; Figures 3B and S3A; compare to
      • Belo J.A.
      • Leyns L.
      • Yamada G.
      • De Robertis E.M.
      The prechordal midline of the chondrocranium is defective in Goosecoid-1 mouse mutants.
      ). In limb buds, HAND2 chromatin complexes also interact with a specific region in the Tbx18 genomic landscape (at position −338 kb; middle panel, Figure 3C). Indeed, Tbx18 expression expands posteriorly in Hand2Δ/Δc forelimb buds (Figure 3C), and previous genetic analysis established that Tbx18 also functions during scapula and humerus development (

      Farin, H. (2009). Function and regulation of the murine T-box genes Tbx15 and Tbx18. PhD thesis (Hannover: University of Hannover).

      ).
      Figure thumbnail gr3
      Figure 3HAND2 Directly Regulates Genes Participating in Proximal Limb Bud Development
      (A) Left: HAND23xF ChIP profiles in the genomic landscape encoding the Gsc transcriptional regulator (left panel). Green bars: HAND2 ChIP-seq peaks identified by MACS analysis. Red bar: genomic region interacting with GLI3R in limb buds (
      • Vokes S.A.
      • Ji H.
      • Wong W.H.
      • McMahon A.P.
      A genome-scale analysis of thecis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb.
      ). Middle panel: HAND2 ChIP-qPCR analysis establishes the significant enrichment of the Gsc +33 kb and Gsc −133 kb regions in limb buds (E10.5). Mean ± SD (n=3). Right: Gsc expression in wild-type and Hand2Δ/Δc forelimb buds (E10.0, 30–31 somites). White arrowhead points to loss of Gsc expression from the posterior mesenchyme. Scale bar, 100 μm.
      (B) Forelimb skeletons (FL) at E16.5 with cartilage in blue and bone in red. Arrowhead points to the malformed scapular head. s, scapula; h, humerus; r, radius; u, ulna; d, digits. Scale bar, 500 μm.
      (C) Left: HAND23xF ChIP profiles in the genomic landscape encoding the Tbx18 transcriptional regulator (left panel). Middle panel: HAND2 ChIP-qPCR analysis establishes the significant enrichment of the Tbx18 −338 kb region in limb buds (E10.5). Mean ± SD (n=3). Right: Tbx18 expression expands to the posterior in Hand2Δ/Δc forelimb buds (black arrowhead, E9.75, 28–29 somites). Scale bar, 100 μm.
      (D) Left: HAND23xF ChIP profiles in the genomic landscape encoding the Irx3 and Irx5 transcriptional regulators. Blue bar: genomic region tested for enhancer activity in mouse transgenic embryos. Right: HAND2 ChIP-qPCR analysis establishes the significant enrichment of the Irx3 +85 kb region in limb buds (E10.5). Mean ± SD (n=3).
      (E) The expression of Irx3 and Irx5 expands posteriorly in Hand2Δ/Δc forelimb buds (black arrowhead, E9.75, 28–29 somites). Scale bar, 100 μm.
      (F) Expression of the LacZ reporter under control of the Irx3 +85 kb CRM in a representative transgenic mouse embryo (E10.5) FL, forelimb bud. Scale bars, 200 μm.
      See also .
      Recently, it has been shown that the Irx3 and Irx5 transcriptional regulators, which are part of the same gene cluster (Figure 3D), are required to specify the progenitors of proximal and anterior limb skeletal elements during limb bud initiation (
      • Li D.
      • Sakuma R.
      • Vakili N.A.
      • Mo R.
      • Puviindran V.
      • Deimling S.
      • Zhang X.
      • Hopyan S.
      • Hui C.C.
      Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by Shh signaling.
      ). Interestingly, HAND2 chromatin complexes bind to a region located 85 kb downstream of the Irx3 TSS and adjacent to a GLI3R binding region (red bar in Figure 3D;
      • Vokes S.A.
      • Ji H.
      • Wong W.H.
      • McMahon A.P.
      A genome-scale analysis of thecis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb.
      ). Irx3 and Irx5 are likely part of the same cis-regulatory landscape as they are coexpressed in the anterior-proximal limb bud mesenchyme and their expression expands posteriorly in Hand2Δ/Δc forelimb buds (Figure 3E). As this region encompasses binding regions for both HAND2 and GLI3R chromatin complexes, its enhancer potential (blue bar in Figure 3D) was assessed in transgenic mouse embryos (Figure 3F). Indeed, this region functions as a CRM in limb buds, but the mesenchymal LacZ reporter activity is rather uniform in contrast to the proximal-anterior restriction of the endogenous Irx3 and Irx5 expression domains (n = 10/10; Figure 3F). Furthermore, as the GLI3R binding regions in limb buds (
      • Vokes S.A.
      • Ji H.
      • Wong W.H.
      • McMahon A.P.
      A genome-scale analysis of thecis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb.
      ) often map close to the ones for HAND2 (red bars in Figures 3A, 3C, and 3D; see also Figure S3B), the expression of these proximal genes was also analyzed in limb buds lacking both Hand2 and Gli3 (Figures S3C and S3D). While Tbx18 expression is not significantly altered, the expression of Gsc and Irx3 is much more reduced in double than in wild-type or in single mutant limb buds (Figures S3C and S3D; data not shown). These alterations in gene expression are paralleled by a more severely dysplastic scapula and humerus in Hand2Δ/ΔcGli3Δ/Δ limbs (Figure S3E). Together, these results indicate that HAND2 acts in concert with GLI3R to control a transcriptional network that functions in morphogenesis of proximal limb skeletal elements.

      Direct Cross-Regulation between HAND2 and GLI3R Underlies Molecular Establishment of an Anterior and Posterior Limb Bud Compartment

      Genetic analysis has shown that Hand2 and Gli3 are mutually antagonistic, and this has been proposed to prepattern the limb bud mesenchyme along its AP axis prior to activation of SHH signaling (
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ,
      • te Welscher P.
      • Fernandez-Teran M.
      • Ros M.A.
      • Zeller R.
      Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling.
      ). Hand2 is initially expressed uniformly throughout the limb field mesenchyme, but its expression becomes rapidly posteriorly restricted as Gli3 is activated in the anterior margin (
      • Charité J.
      • McFadden D.G.
      • Olson E.N.
      The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development.
      ,
      • te Welscher P.
      • Fernandez-Teran M.
      • Ros M.A.
      • Zeller R.
      Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling.
      ). At this early stage, the full-length cytoplasmic GLI3 protein is constitutively processed to GLI3R, which translocates to the nucleus in the absence of SHH signaling (Figure S4;
      • Wang B.
      • Fallon J.F.
      • Beachy P.A.
      Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb.
      ). To gain insight into the underlying molecular and cellular interactions, we colocalized the endogenous HAND23xF and GLI3R proteins during forelimb bud formation (E9.25; Figure 4A). HAND2 protein levels are highest in posterior and proximal-anterior nuclei (green fluorescence; Figure 4A), whereas GLI3R is most abundant in anterior nuclei (red fluorescence, Figure 4A). In spite of their opposing distribution, most mesenchymal cells coexpress nuclear HAND2 and GLI3R proteins during forelimb bud formation (yellow overlap; n = 4/4 at E9.25; Figures 4A and S5A). Subsequently, HAND2 becomes undetectable in anterior cells, which retain GLI3R. Conversely, GLI3R is lost from posterior cell nuclei, which retain HAND2 (n = 3/3 at E9.5-9.75; Figures 4B and S5B). Cells at the interface of the two domains continue to coexpress both proteins (Figures 4B and S5B). These spatial dynamics reveal how the populations of GLI3R-positive anterior and HAND2-positive posterior cells segregate, likely concurrently with molecular establishment of AP axis polarity in forelimb buds. The SHH-independent nature of these interactions is revealed by the fact that the HAND2 and GLI3R domains are initially normal in Shh-deficient limb buds (E9.5–9.75; Figure S5C). During progression of forelimb bud outgrowth, the HAND2 and GLI3R domains are increasingly separated by cells expressing neither of these proteins (n = 3/3 at E10.25; Figure 4B), which likely reflects the inhibition of GLI3R formation by SHH signaling. As the two proteins are initially coexpressed, direct cross-regulation could underlie establishment of the mutually exclusive HAND2 and GLI3R domains. Indeed, GLI3R interacts with two CRMs in the Hand2 genomic landscape that function in repressing Hand2 from the anterior limb bud mesenchyme (
      • Vokes S.A.
      • Ji H.
      • Wong W.H.
      • McMahon A.P.
      A genome-scale analysis of thecis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb.
      ). The CRM at position +13 kb is associated with one overlapping and a close-by region (at +11 kb) enriched in HAND2 chromatin complexes from limb buds (Figures S5D and S5E). This indicates that in addition to mediating repression by GLI3R, this CRM could also participate in autoregulation of Hand2 expression.
      Figure thumbnail gr4
      Figure 4The Dynamics of the HAND2 and GLI3R Distributions Reveal the Establishment of a Posterior and Anterior Limb Bud Compartment
      (A and B) Coimmunolocalization of HAND23xF (green) and GLI3R (red) in wild-type forelimb buds at E9.25 (22–23 somites), E9.5 (25–26 somites), E9.75 (28–29 somites), and E10.25 (32–33 somites). Scale bars, 50 μm.
      (C) HAND23xF binding regions in the Gli3 genomic landscape revealed by ChIP-seq analysis (green). Blue bar demarcates the Gli3 −120 kb region chosen for LacZ reporter analysis. The right panel shows the temporal occupancy of the Gli3 −120 kb region by HAND2 complexes as revealed by ChIP-qPCR analysis of limb buds from E10.5–E12.5. Mean ± SD is indicated (n = 3).
      (D) Expression of the LacZ reporter under control of the Gli3 −120 kb HAND2 binding region in a transgenic embryo (E10.5) FL, forelimb bud. Scale bars, 200 μm.
      See also and .
      As Gli3 expression expands posteriorly in Hand2-deficient mouse limb buds (
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ,
      • te Welscher P.
      • Fernandez-Teran M.
      • Ros M.A.
      • Zeller R.
      Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling.
      ), we screened also the Gli3 genomic landscape for HAND2-interacting regions (Figure 4C). Indeed, a region located 120 kb upstream of the Gli3 transcription unit is prominently enriched in early limb buds (right panel, Figure 4C). Transgenic analysis showed that this CRM is indeed active both in fore- and hindlimb buds (Figure 4D). In particular, this CRM is able to drive LacZ expression in a spatial pattern reminiscent of the endogenous Gli3 transcript distribution, with LacZ being excluded from the most posterior mesenchyme (n = 6/7; Figure 4D; data not shown).

      Tbx3 Is a HAND2 Target Gene that Positions the Posterior Gli3 Expression Boundary

      HAND2 chromatin complexes are also enriched in two specific regions of the Tbx3 genomic landscape that is required for expression in limb buds (Figures 5A and 5B ;
      • Horsthuis T.
      • Buermans H.P.
      • Brons J.F.
      • Verkerk A.O.
      • Bakker M.L.
      • Wakker V.
      • Clout D.E.
      • Moorman A.F.
      • ’t Hoen P.A.
      • Christoffels V.M.
      Gene expression profiling of the forming atrioventricular node using a novel tbx3-based node-specific transgenic reporter.
      ). While the eT and Lb HAND2 ChIP-seq data sets do not unequivocally identify the two regions for the reasons stated above, HAND2 ChIP-qPCR using early limb buds established that the region located 58 kb upstream of the Tbx3 transcription unit is ∼3-fold more enriched than the one located at −19 kb (left panel, Figure 5B). The enrichment of the Tbx3 −58 kb region is most prominent in early limb buds (right panel, Figure 5B), pointing to an involvement of HAND2 in regulating the early Tbx3 expression. This was corroborated by transgenic analysis, which established that the Tbx3 −58 kb CRM drives LacZ expression in the posterior-proximal forelimb bud and flank mesenchyme in a pattern strikingly similar to the endogenous Tbx3 expression (n = 8/10; Figures 5C and S6A). In contrast, the Tbx3 −19 kb region is not active in mouse limb buds (n = 0/7; Figure S6A). The Tbx3 −58 kb CRM regulates Tbx3 expression during early limb bud development, as the LacZ expression domain fails to expand distally as limb bud development progresses (Figure S6A). As neither of the two HAND2 binding regions is active in hindlimb buds (Figures 5C and S6A; data not shown), the expression of Tbx3 in hindlimb buds must be controlled by different cis-regulatory interactions.
      Figure thumbnail gr5
      Figure 5HAND2 Controls Tbx3 Expression by Interacting with a CRM that Is Active Early in the Posterior Forelimb Bud Mesenchyme
      (A) HAND23xF binding regions in the Tbx3 locus as defined by ChIP-seq analysis.
      (B) Left: ChIP-qPCR reveals the significance of the interactions of HAND23xF chromatin complexes with the Tbx3 −58 kb and Tbx3 −19 kb regions in limb buds (E10.5). Mean ± SD (n = 3). Right: temporal dynamics of the occupancy of the Tbx3 −58 kb region by HAND23xF complexes in limb buds.
      (C) Upper: expression of LacZ under control of the Tbx3 −58 kb HAND2 binding region in transgenic embryos at E9.75 and E10.25. Lower: endogenous Tbx3 transcript distribution in wild-type embryos. FL, forelimb bud; HL, hindlimb bud. Scale bars, 100 μm.
      (D) Tbx3 expression in wild-type and Hand2Δ/Δc fore- (FL) and hindlimb (HL) buds at E9.75. White arrowhead, loss of Tbx3 in the posterior forelimb bud mesenchyme. Scale bar, 100 μm.
      (E) Colocalization of the nuclear HAND23xF (green) and TBX3 (red) proteins in wild-type and Shh-deficient forelimb buds (E9.5, 25–26 somites; E10.0, 30–31 somites). Lower panels show enlargements of the posterior TBX3 protein domains.
      (F) TBX3 protein distribution in Hand2Δ/Δc and Hand2Δ/Δc Gli3Δ/Δ forelimb buds (E10.0, 30–31 somites).
      See also .
      Indeed, Tbx3 expression is lost from the posterior mesenchyme of Hand2-deficient forelimb buds, while it remains comparable to wild-types in mutant hindlimb buds (Figure 5D;
      • Galli A.
      • Robay D.
      • Osterwalder M.
      • Bao X.
      • Bénazet J.D.
      • Tariq M.
      • Paro R.
      • Mackem S.
      • Zeller R.
      Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development.
      ). Furthermore, the expression of Tbx5, which is located ∼163 kb downstream of Tbx3 as part of the same gene cluster, is not affected in Hand2-deficient forelimb buds (Figure S6B). Several HAND2 ChIP peaks were also detected in the Tbx2-Tbx4 genomic landscape (Figures S6C and S6D). Similar to the Tbx3-Tbx5 genomic landscape, Tbx2, but not Tbx4, expression is reduced in Hand2-deficient limb buds (Figures S6E and S6F). These apparent similarities in cis-regulation are likely related to the emergence of these gene clusters by duplication of an ancestral complex (
      • Agulnik S.I.
      • Garvey N.
      • Hancock S.
      • Ruvinsky I.
      • Chapman D.L.
      • Agulnik I.
      • Bollag R.
      • Papaioannou V.
      • Silver L.M.
      Evolution of mouse T-box genes by tandem duplication and cluster dispersion.
      ). Genetic and overexpression analysis has previously implicated Tbx3 in early forelimb development, together with Hand2 and Gli3 (
      • Davenport T.G.
      • Jerome-Majewska L.A.
      • Papaioannou V.E.
      Mammary gland, limb and yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary syndrome.
      ,
      • Rallis C.
      • Del Buono J.
      • Logan M.P.
      Tbx3 can alter limb position along the rostrocaudal axis of the developing embryo.
      ), whereas Tbx2 is essential only during termination of limb bud outgrowth (
      • Farin H.F.
      • Lüdtke T.H.
      • Schmidt M.K.
      • Placzko S.
      • Schuster-Gossler K.
      • Petry M.
      • Christoffels V.M.
      • Kispert A.
      Tbx2 terminates shh/fgf signaling in the developing mouse limb bud by direct repression of gremlin1.
      ). Therefore, we focused our analysis on the HAND2-TBX3 interactions during the onset of forelimb bud development. HAND2 and TBX3 proteins are initially coexpressed (Figure S6G). However, TBX3 becomes rapidly more restricted than HAND2 such that the double-positive cells hallmark the proximal-posterior forelimb bud and posterior flank (Figure 5E). In Shh-deficient forelimb buds, both protein domains are more restricted but are otherwise maintained (right panels, Figure 5E). In contrast, TBX3 is absent from Hand2Δ/Δc and Hand2Δ/ΔcGli3Δ/Δ forelimb buds, with the exception of few positive cells at the proximal border (right panels, Figure 5F). Taken together, these results provide compelling evidence that Tbx3 is a direct transcriptional target of HAND2 in the posterior and flank mesenchyme during the onset of limb bud development.
      To gain insight into the potential involvement of TBX3 in AP patterning of the early limb bud mesenchyme, the spatial distribution of the mesenchymal cells actively transcribing Gli3 was determined in relation to HAND2 and TBX3 proteins (Figures 6A and 6B ). To achieve cellular resolution, the Gli3 allele expressing EGFP under control of the endogenous locus (
      • Lopez-Rios J.
      • Speziale D.
      • Robay D.
      • Scotti M.
      • Osterwalder M.
      • Nusspaumer G.
      • Galli A.
      • Holländer G.A.
      • Kmita M.
      • Zeller R.
      GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis.
      ) was used in combination with GFP antibodies (red fluorescence; Figures 6A and 6B). While the GLI3R and HAND2 protein domains are increasingly separated (Figure 4B), a significant fraction of boundary cells continue to coexpress HAND2 protein and Gli3 transcripts in wild-type limb buds (yellow fluorescence, left panels, Figure 6A). In contrast, far fewer cells coexpress TBX3 and Gli3 in the boundary region (right panels, Figure 6A). This difference is even more striking in Gli3Δ/Δ forelimb buds, which continue to express the mutant Gli3 transcripts (Figure 6B). There is a large population of cells coexpressing HAND2 and Gli3 due to the anterior expansion of the Hand2 expression domain in Gli3-deficient limb buds (left panels, Figure 6B;
      • te Welscher P.
      • Fernandez-Teran M.
      • Ros M.A.
      • Zeller R.
      Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling.
      ). However, TBX3 remains posteriorly restricted in Gli3-deficient limb buds, and similar to wild-types, only few cells coexpress TBX3 proteins and Gli3 transcripts (right panels, Figure 6B). This boundary is also retained in Shh-deficient forelimb buds in spite of the posterior expansion of Gli3 (Figures S7A and S7B). These results show that HAND2 is not sufficient to establish a sharp boundary but rather that TBX3 defines the limit between the anterior Gli3-positive and posterior Gli3-negative mesenchymal cells. Indeed, the comparative analysis of wild-type, Shh-deficient, and Tbx3-deficient forelimb buds reveals that Tbx3 is required to establish this boundary (Figures 6C and 6D). In Shh-deficient limb buds, Gli3 transcription expands posteriorly with more restricted HAND2 and TBX3 domains but fails to reach the posterior margin (right panel, Figures 6C, 5E, and S7A). Rather strikingly, Gli3 expression expands into the posterior flank mesenchyme in Tbx3-deficient forelimb buds as in Hand2-deficient limb buds (Figure 6D). Hand2-deficient forelimb buds lack Tbx3 (Figures 5D and 5F) in their posterior mesenchyme, whereas Hand2 transcripts remain in Tbx3-deficient limb buds albeit at reduced levels (Figure S7C). Together, these results corroborate the proposal that the HAND2 transcriptional target Tbx3 is required to establish the posterior Gli3 expression boundary by inhibiting its expression in the posterior-most mesenchyme (Figures 6 and 7).
      Figure thumbnail gr6
      Figure 6The TBX3 Transcriptional Repressor Participates in Excluding Gli3 from the Posterior Limb Bud Mesenchyme
      (A and B) Left: colocalization of HAND23xF proteins (green) with Gli3 transcripts (red) in Gli3ΔGFP/+ (E10.5, 35–36 somites) and Gli3-deficient (Gli3ΔGFP/ΔGFP; E10.25, 32–33 somites) forelimb buds. Right: colocalization of TBX3 proteins (green) with Gli3 transcripts (red) in forelimb buds of the same genotypes. White marks indicate the enlargements. Cells coexpressing HAND23xF or TBX3 proteins with Gli3 transcripts appear yellow. Scale bar, 50 μm.
      (C and D) Gli3 expression in wild-type, Shh-deficient, Hand2Δ/Δc, and Tbx3Δ/Δ forelimb buds (E10.25; 32–33 somites). Arrowheads point to expanded Gli3 expression in the posterior flank mesenchyme. Scale bar, 100 μm.
      See also .
      Figure thumbnail gr7
      Figure 7The Major Transcriptional Interactions and Networks Governed by HAND2 in the Forelimb Bud Mesenchyme Upstream of SHH
      HAND2 is at the core of the transcriptional networks that control establishment of a proximal, anterior, and posterior compartment in early forelimb buds. In addition to directly impacting on Shh and Gli3 expression, HAND2 re-enforces their activation and repression via Ets1/2 and Tbx3, respectively. Solid lines indicate direct interactions, whereas dashed lines indicate interactions deduced from genetic analysis. Note that the activating or repressive nature of the interactions has mostly been deduced from genetic analysis. (+) indicates CRMs interacting with HAND2 chromatin complexes that have been identified in this study and drive LacZ reporter expression in the forelimb bud mesenchyme.

      Discussion

      There is ample evidence that tissue-specific CRMs orchestrate the dynamics of gene expression during embryonic development and fine-tune morphogenesis as a consequence of their often highly dynamic regulation (
      • Attanasio C.
      • Nord A.S.
      • Zhu Y.
      • Blow M.J.
      • Li Z.
      • Liberton D.K.
      • Morrison H.
      • Plajzer-Frick I.
      • Holt A.
      • Hosseini R.
      • et al.
      Fine tuning of craniofacial morphology by distant-acting enhancers.
      ,
      • Spitz F.
      • Furlong E.E.
      Transcription factors: from enhancer binding to developmental control.
      ). Functional modification of CRMs in an increasing number of genomic landscapes has been causally linked to both adaptive evolution and human congenital malformations (
      • Anderson E.
      • Peluso S.
      • Lettice L.A.
      • Hill R.E.
      Human limb abnormalities caused by disruption of hedgehog signaling.
      ,
      • Lopez-Rios J.
      • Duchesne A.
      • Speziale D.
      • Andrey G.
      • Peterson K.A.
      • Germann P.
      • Unal E.
      • Liu J.
      • Floriot S.
      • Barbey S.
      • et al.
      Attenuated sensing of SHH by Ptch1 underlies evolution of bovine limbs.
      ). Therefore, it is important to identify the trans-acting factors and complexes that regulate these CRMs in a tissue and stage-specific manner. To this aim, we have inserted a 3xFLAG-tag into the endogenous HAND2 protein, which permitted us to uncover genomic regions enriched in HAND2-containing chromatin complexes in mouse embryos. The current study focuses on an in-depth functional analysis of HAND2-CRMs in genomic landscapes of transcriptional regulators that are required during early mouse forelimb bud development. This analysis uncovers the architecture of a network of HAND2 target transcription factors that function in early determinative events that set up the proximal, anterior, and posterior domains during the onset of forelimb bud outgrowth (Figure 7). An essential role of HAND2 in proximal skeletal development was revealed by the fact that its early Hoxb6-Cre mediated conditional inactivation disrupts scapular head morphogenesis. Our study provides good evidence that this is a consequence of HAND2 directly regulating the expression of transcription factors that function in the development of the proximal limb skeletal elements, such as Gsc, Irx3, Irx5, and Tbx18 (Figure 7). In particular, the HAND2 ChIP analysis identifies a CRM located upstream of Irx3 that is active in the limb bud mesenchyme. In turn, IRX3 regulates Gli3 expression by directly interacting with a limb bud mesenchymal CRM located in the Gli3 genomic landscape (
      • Abbasi A.A.
      • Paparidis Z.
      • Malik S.
      • Bangs F.
      • Schmidt A.
      • Koch S.
      • Lopez-Rios J.
      • Grzeschik K.H.
      Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development.
      ,
      • Li D.
      • Sakuma R.
      • Vakili N.A.
      • Mo R.
      • Puviindran V.
      • Deimling S.
      • Zhang X.
      • Hopyan S.
      • Hui C.C.
      Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by Shh signaling.
      ). These direct interactions begin to reveal the underlying complexity of the cis-regulatory circuitry operating during the onset of limb bud development (Figure 7). In summary, our genetic analysis supports the proposal that the HAND2-regulated gene networks are required to pattern the early mesenchymal territory that gives rise to the proximal limb skeletal elements in a Shh-independent manner (
      • Ahn S.
      • Joyner A.L.
      Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning.
      ,
      • Chiang C.
      • Litingtung Y.
      • Harris M.P.
      • Simandl B.K.
      • Li Y.
      • Beachy P.A.
      • Fallon J.F.
      Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function.
      ).
      It is unclear at which stage AP axis polarity is established in mouse limb buds, but
      • Tanaka M.
      • Cohn M.J.
      • Ashby P.
      • Davey M.
      • Martin P.
      • Tickle C.
      Distribution of polarizing activity and potential for limb formation in mouse and chick embryos and possible relationships to polydactyly.
      showed that the competence to activate Shh expression is widespread and without posterior bias in the forelimb field of early mouse embryos (E9.0). Together with other studies (see Introduction), this suggests that the limb bud mesenchyme might only be polarized along the AP axis during the onset of limb bud development in mouse embryos. The present study shows that the nuclear HAND2 and GLI3R proteins are initially coexpressed by the mesenchymal progenitors that give rise to the forelimb bud. However, during initiation of limb bud development, the spatial distributions of the two transcriptional regulators rapidly segregate into a distinct GLI3R-positive anterior and HAND2-positive posterior compartment. In addition to establishing this dynamic segregation with cellular resolution, we identify the HAND2-dependent cis-regulatory interactions and transcription factor networks that establish these anterior and posterior compartments (Figure 7). The loss of Hand2 transcripts and proteins from the anterior limb bud mesenchyme (
      • te Welscher P.
      • Fernandez-Teran M.
      • Ros M.A.
      • Zeller R.
      Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling.
      ; this study) is the result of GLI3R-mediated direct repression (
      • Vokes S.A.
      • Ji H.
      • Wong W.H.
      • McMahon A.P.
      A genome-scale analysis of thecis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb.
      ). The HAND2 ChIP analysis identifies one CRM in the Gli3 genomic landscape that is active in limb buds, with exception of the most posterior mesenchyme. The transcriptional regulation of Gli3 in early limb buds is complex and likely controlled by several CRMs with similar activities, one of which is bound by IRX3, a direct transcriptional target of Hand2 (
      • Abbasi A.A.
      • Paparidis Z.
      • Malik S.
      • Bangs F.
      • Schmidt A.
      • Koch S.
      • Lopez-Rios J.
      • Grzeschik K.H.
      Human intronic enhancers control distinct sub-domains of Gli3 expression during mouse CNS and limb development.
      ,
      • Li D.
      • Sakuma R.
      • Vakili N.A.
      • Mo R.
      • Puviindran V.
      • Deimling S.
      • Zhang X.
      • Hopyan S.
      • Hui C.C.
      Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by Shh signaling.
      ,
      • Visel A.
      • Minovitsky S.
      • Dubchak I.
      • Pennacchio L.A.
      VISTA Enhancer Browser—a database of tissue-specific human enhancers.
      ; this study). While the genetic analysis shows that Hand2 is required to repress the expression of Gli3 and other direct targets in the posterior mesenchyme, our transgenic studies of HAND2-interacting CRMs rather reveals their ability to activate LacZ expression in the limb bud mesenchyme. HAND2 was first described as a transcriptional activator (
      • Dai Y.S.
      • Cserjesi P.
      The basic helix-loop-helix factor, HAND2, functions as a transcriptional activator by binding to E-boxes as a heterodimer.
      ), but more recently its transcriptional repressing activity has been established in the context of its interaction with a specific CRM that regulates the Dlx5/6 gene cluster (
      • Barron F.
      • Woods C.
      • Kuhn K.
      • Bishop J.
      • Howard M.J.
      • Clouthier D.E.
      Downregulation of Dlx5 and Dlx6 expression by Hand2 is essential for initiation of tongue morphogenesis.
      ). Furthermore, HAND2 forms heterodimers with transcriptional repressors, such as TWIST1 and RUNX2 (
      • Firulli B.A.
      • Krawchuk D.
      • Centonze V.E.
      • Vargesson N.
      • Virshup D.M.
      • Conway S.J.
      • Cserjesi P.
      • Laufer E.
      • Firulli A.B.
      Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities.
      ,
      • Funato N.
      • Chapman S.L.
      • McKee M.D.
      • Funato H.
      • Morris J.A.
      • Shelton J.M.
      • Richardson J.A.
      • Yanagisawa H.
      Hand2 controls osteoblast differentiation in the branchial arch by inhibiting DNA binding of Runx2.
      ), which could determine the repressive activity of HAND2 chromatin complexes interacting with CRMs in, e.g., the Gli3 genomic landscape. Alternatively, the repressive effect of HAND2 on Gli3 could be mediated via the transcriptional target Tbx3, which is required to repress Gli3 in the posterior mesenchyme. In fact, TBX3 has an essential role in positioning the Gli3 expression boundary (Figure 7), and its overexpression in forelimb buds of chicken embryos inhibits Gli3 expression (
      • Rallis C.
      • Del Buono J.
      • Logan M.P.
      Tbx3 can alter limb position along the rostrocaudal axis of the developing embryo.
      ). Finally, HAND2 controls the activation of Shh expression by directly binding to the ZRS and indirectly via its transcriptional targets Ets1 and Ets2 that also regulate Shh (
      • Lettice L.A.
      • Williamson I.
      • Wiltshire J.H.
      • Peluso S.
      • Devenney P.S.
      • Hill A.E.
      • Essafi A.
      • Hagman J.
      • Mort R.
      • Grimes G.
      • et al.
      Opposing functions of the ETS factor family define Shh spatial expression in limb buds and underlie polydactyly.
      ,
      • Lettice L.A.
      • Williamson I.
      • Devenney P.S.
      • Kilanowski F.
      • Dorin J.
      • Hill R.E.
      Development of five digits is controlled by a bipartite long-rangecis-regulator.
      ). Such feed-forward loops are important motifs within transcriptional networks as they contribute to their stability and robustness (
      • Alon U.
      Network motifs: theory and experimental approaches.
      ).
      Previous studies had provided genetic and experimental evidence that both proximodistal and AP identities are specified early during limb bud development (
      • Dudley A.T.
      • Ros M.A.
      • Tabin C.J.
      A re-examination of proximodistal patterning during vertebrate limb development.
      ,
      • Zhu J.
      • Nakamura E.
      • Nguyen M.T.
      • Bao X.
      • Akiyama H.
      • Mackem S.
      Uncoupling Sonic hedgehog control of pattern and expansion of the developing limb bud.
      ). Our study now uncovers distinct transcriptional networks interlinked by HAND2 that are required to set-up the initial proximal, posterior, and anterior mesenchymal compartments prior to the onset of SHH signaling. These networks define proximal fates and the AP boundaries with cellular precision through selective activation and/or repression of downstream transcriptional regulators. The direct cross-regulation among these transcriptional regulators not only defines the limb bud mesenchymal compartments but also enables activation of SHH signaling, which then elaborates these compartments during distal progression of limb bud outgrowth.

      Experimental Procedures

      For generation of the Hand23xF allele and details on methodology, see the Supplemental Experimental Procedures.

      Mouse Strains and Embryos

      Experiments involving mice were performed with strict adherence to Swiss law, the 3R principles, and the Basel Declaration. All animal studies were approved by the cantonal animal welfare and ethics committees. The procedures for generating transgenic mice at the Lawrence Berkeley National Laboratory (LBNL) were reviewed and approved by the LBNL Animal Welfare and Research Committee. The Hand23xF and Gli3C3xF alleles were maintained in an NMRI background. The Hand2f (floxed), Hand2Δ, and Gli3ΔGFP alleles were maintained in mixed backgrounds. The Gli3C3xF allele was constructed by inserting a 3XFLAG epitope tag in frame at the carboxy terminus of the endogenous GLI3 protein. Western blotting detects the 190 kD full-length GLI3 protein. Mice and embryos homozygous for this allele are phenotypically wild-type and will be described elsewhere (J.L.-R. and R.Z., unpublished data). The ShhGFPCre allele (
      • Harfe B.D.
      • Scherz P.J.
      • Nissim S.
      • Tian H.
      • McMahon A.P.
      • Tabin C.J.
      Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities.
      ) and the Hoxb6-Cre transgene (
      • Lowe L.A.
      • Yamada S.
      • Kuehn M.R.
      HoxB6-Cre transgenic mice express Cre recombinase in extra-embryonic mesoderm, in lateral plate and limb mesoderm and at the midbrain/hindbrain junction.
      ) were maintained in a C57BL/6J background. The Tbx3Venus allele used to generate Tbx3-deficient embryos recapitulates all known Tbx3 loss-of-function phenotypes and will be described elsewhere (R.M. and V.M.C., unpublished data).

      Immunofluorescence

      Limb buds were fixed in 4% paraformaldehyde for 2–3 hr at 4°C, and proteins were detected on 10 μm cryosections. Primary antibodies against the FLAG epitope (M2; 1:500; Sigma), GLI3 (3.6 ng/ml; clone 6F5;
      • Wen X.
      • Lai C.K.
      • Evangelista M.
      • Hongo J.A.
      • de Sauvage F.J.
      • Scales S.J.
      Kinetics of hedgehog-dependent full-length Gli3 accumulation in primary cilia and subsequent degradation.
      ), TBX3 (1:300; E-20; Santa Cruz), and GFP (1:1,000; Life Technologies) were used. Goat anti-mouse (FLAG/GLI3), goat anti-rabbit (GFP), and rabbit/donkey anti-goat (TBX3) secondary antibodies conjugated to Alexa Fluor 488 or 594 (1:1,000; Life Technologies) were used for detection. For colocalization of HAND2 and GLI3R, anti-FLAG (M2; F3165, Sigma) antibodies were labeled with Alexa Fluor 488 using the APEX antibody labeling kit (Life Technologies). Sequential treatment with rabbit anti-Alexa Fluor 488 (Life Technologies) and goat anti-rabbit 488 Alexa Fluor enhanced the signal. Nuclei were counterstained with Hoechst-33258. The ShhGFPCre and Gli3ΔGFP null alleles were used in combination with immunodetection of GFP to visualize cells expressing Shh or Gli3, respectively. Autofluorescence, e.g., of blood cells, was detected equally in all channels. Therefore, it was captured utilizing the 633 nm laser of the confocal microscope and digitally removed using the subtraction mode in Photoshop CS5 (Adobe) as shown in Figure S1A. Images were acquired using a Leica SP5 confocal microscope.

      ChIP-qPCR and ChIP-Seq Analysis

      Fore- and hindlimb buds dissected from ∼50 Hand23xF/3xF embryos at E10.5 were processed for ChIP as described (
      • Lopez-Rios J.
      • Speziale D.
      • Robay D.
      • Scotti M.
      • Osterwalder M.
      • Nusspaumer G.
      • Galli A.
      • Holländer G.A.
      • Kmita M.
      • Zeller R.
      GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis.
      ,
      • Vokes S.A.
      • Ji H.
      • Wong W.H.
      • McMahon A.P.
      A genome-scale analysis of thecis-regulatory circuitry underlying sonic hedgehog-mediated patterning of the mammalian limb.
      ) using M2 anti-FLAG antibody (F1804; Sigma). For time course experiments, 20 fore- and hindlimbs of E11.5 or E12.5 Hand23xF/3xF embryos were processed in an identical manner. Chromatin was fragmented for 15 (E10.5) or 20 min (E11.5, E12.5) using a S220 Covaris Ultrasonicator, which yielded average fragment sizes in the range of 200–300 bp. All results (mean ± SD) represent three independent biological replicates. The details of the ChIP-seq analysis are given in the Supplemental Experimental Procedures.

      Acknowledgments

      We wish to thank the mouse transgenic core facility of the University of Basel for generation of chimeric mice and A. Offinger and her team for excellent animal care. Frédéric Laurent is thanked for performing the OPT analysis. We are grateful to Jennifer Akiyama for generation of constructs, Ina Nissen (D-BSSE) for the preparation of the ChIP-seq library, and Isabelle Ginez for preparing the tissue sections. This research was supported by Swiss National Science Foundation grants ( 31003A_130803/146248 ) and both cantons of Basel (to R.Z.), an EU Reintegration grant ( PERG-GA-2009-246576 ) to J.L.-R.), a Netherlands Heart Foundation grant ( 2010B205 ) (to V.M.C.), and NIH grants ( R01HG003988 and U01DE020060 ) (to A.V.). A.V.’s research was conducted at the E.O. Lawrence Berkeley National Laboratory and performed under a Department of Energy Contract ( DE-AC02-05CH11231 ), University of California . X.W. and S.J.S. are employees of Genentech, a member of the Roche group.

      Accession Numbers

      The GEO database accession number for all data sets reported in this paper is GSE55707.

      Supplemental Information

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