HAND2 targets define a network of transcriptional regulators that compartmentalize the early limb bud mesenchyme

Abstract

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.

Figure 1. Insertion of a 3xFLAG epitope tag into the endogenous HAND2 protein provides a sensitive tool to detect HAND2 protein complexes

(A) The Hand2^3xF allele was generated by dRMCE in mouse ES cells. (B) Left panel: Hand2 expressing tissues in mouse embryos at embryonic dayE10.5. Right panels: immunoblot detection of the tagged HAND2^3xF protein isoforms (H2) in limb buds (LB), heart (HE) and branchial arches (BA). Midbrain (MB) and wild-type extracts are used as negative controls. Anasterisk indicates a non-specific band. FL: forelimb bud. HL: hindlimb bud. (C) Detection of HAND2^3xF proteins in expressing embryonic tissues by immunofluorescence (green) at E10.5. RV: right ventricle; RA: right atrium.1st: mandibular arch. 2nd: hyoid arch. (D) Co-localization of HAND2^3xF 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 due to counterstaining with Hoechst. Scale bars: 50μm. (E) ChIP-qPCR analysis shows the interaction of HAND2^3xF 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 Figure S1.

Figure 2. ChIP-Seq analysis identifies a set of HAND2 target regions in mouse limb buds

(A, B) The top 1000 HAND2^3xF target regions enriched in E10.5 limb buds (Lb) and Hand2 expressing embryonic tissues (eT) are mostly evolutionarily conserved (panel A) and map generally ≥10kb away from the closest transcriptional start site (TSS, panel B). (C) De novo motif discovery analysis of the top 1000 HAND2^3xF bound regions reveals enrichment in Ebox consensus sequences. (D) Gene ontology (GO) analysis reveals the most prominent biological processes associated with HAND2^3xF binding regions that are represented in both top 1000 Lb and eT datasets. (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 kb. The profiles of DNaseI HS and H3K27ac marks in limb buds (E11.5) are shown in black (Cotney et al., 2012). 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 HAND2^3xF 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 Hand2^Δ/Δc forelimb buds (≤E10.25). (H) The activity of the Ets2 +146kb human orthologue (hs1516-LacZ reporter, Vista Enhancer Browser) is compared with the endogenous Ets2 and Hand2 expression in forelimb buds (E11.5). Scale bars: 100μm. See also Figure S2 and Tables S1-S5.

Figure 3. HAND2 directly regulates genes participating in proximal limb bud development

(A, C, D) HAND2^3xF ChIP profiles in the genomic landscapes encoding the Gsc (A), Tbx18 (C) and Irx3 (D) transcriptional regulators (left panels). Green bars: HAND2 ChIP-Seq peaks identified by MACS analysis. Red bars: genomic regions interacting with GLI3R in limb buds (Vokes et al., 2008). Blue bar: genomic region tested for enhancer activity in mouse transgenic embryos. Middle panels: ChIP-qPCR analysis to establish the significant enrichment of particular HAND2^3xF binding peaks in limb buds (E10.5). Mean ±SD (n=3). Right panels in (A) and (C): RNA in situ hybridization analysis of wild-type and Hand2^Δ/Δc forelimb buds (E9.75, 28-29 somites; E10.0 30-31 somites). Black arrowheads: expanded expression domains. White arrowhead: reduction/loss of expression. Scale bars: 100μm. (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: 500μm. (E) Irx3 and Irx5 expression in wild-type and Hand2^Δ/Δc forelimb buds (E9.75,28-29 somites) (F) Expression of the LacZ reporter under control of the Irx3 +85kb CRM in a transgenic embryos (E10.5) FL: forelimb bud. Scale bars: 200μm. See also Figure S3.

Figure 4. The dynamics of the HAND2 and GLI3R distributions reveal the establishment of a posterior and anterior limb bud compartment

(A, B) Co-immunolocalization of HAND2^3xF (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: 50μm. (C) HAND2^3xF binding regions in the Gli3 genomic landscape revealed by ChIP-Seq analysis (green). Blue bar demarcates the Gli3 -120kb region chosen for LacZ reporter analysis. The right panel shows the temporal occupancy of the Gli3 -120kb 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 -120kb HAND2 binding region in a transgenic embryo (E10.5) FL: forelimb bud. Scale bars: 200μm. See also Figures S4 and S5.

Figure 5. HAND2 controls Tbx3 expression by interacting with a CRM that is active early in the posterior forelimb bud mesenchyme

(A) HAND2^3xF binding regions in the Tbx3 locus as defined by ChIP-Seq analysis. (B) Left panel: ChIP-qPCR reveals the significance of the interactions of HAND2^3xF chromatin complexes with the Tbx3 -58kb and Tbx3 -19kb regions in limb buds (E10.5). Mean ±SD (n=3). Right panel: temporal dynamics of the occupancy of the Tbx3 -58kb region by HAND2^3xF complexes in limb buds. (C) Upper panels: Expression of LacZ under control of the Tbx3 -58kb HAND2 binding region in transgenic embryos at E9.75 and E10.25. Lower panels: endogenous Tbx3 transcript distribution in wild-type embryos. FL: forelimb bud, HL: hindlimb bud. Scale bars: 100μm. (D) Tbx3 expression in wild-type and Hand2^Δ/Δc fore- (FL) and hindlimb (HL) buds at E9.75. White arrowhead: loss of Tbx3 in the posterior forelimb bud mesenchyme. Scale bar: 100μm. (E) Co-localization of the nuclear HAND2^3xF (green) and TBX3 (red) proteins in wild-type and Shh-deficient forelimb buds (E9.5, 25-26 somites and E10.0,30-31 somites). Lower panels show enlargements of the posterior TBX3 protein domains. (F) TBX3 protein distribution in Hand2^Δ/Δc and Hand2^Δ/Δc Gli3^Δ/Δ forelimb buds(E10.0, 30-31 somites). See also Figure S6.

Figure 6. The TBX3 transcriptional repressor participates in excluding Gli3 from the posterior limb bud mesenchyme

(A, B) Left panels: co-localization of HAND2^3xF proteins (green) with Gli3 transcripts (red) in Gli3^ΔGFP/+ (E10.5, 35-36 somites) and Gli3-deficient (Gli3^ΔGFP/ΔGFP; E10.25, 32-33 somites) forelimb buds. Right panels: co-localization of TBX3 proteins (green) with Gli3 transcripts (red) in forelimb buds of the same genotypes. White marks indicate the enlargements. Cells co-expressing HAND2^3xF or TBX3 proteins with Gli3 transcripts appear yellow. Scale bar: 50μm. (C, D) Gli3 transcript distribution in wild-type, Shh-deficient, Hand2^Δ/Δc and Tbx3^ΔΔ forelimb buds (E10.25; 32-33 somites). Arrowheads point to expanded Gli3 expression in the posterior flank mesenchyme. Scale bar: 100μm. See also Figure S7.

Figure 7. The 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, while 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.