The transcriptional regulator MEIS2 sets up the ground state for palatal osteogenesis in mice

Abstract

Haploinsufficiency of Meis homeobox 2 (MEIS2), encoding a transcriptional regulator, is associated with human cleft palate, and Meis2 inactivation leads to abnormal palate development in mice, implicating MEIS2 functions in palate development. However, its functional mechanisms remain unknown. Here we observed widespread MEIS2 expression in the developing palate in mice. Wnt1^Cre -mediated Meis2 inactivation in cranial neural crest cells led to a secondary palate cleft. Importantly, about half of the Wnt1^Cre ;Meis2^f/f mice exhibited a submucous cleft, providing a model for studying palatal bone formation and patterning. Consistent with complete absence of palatal bones, the results from integrative analyses of MEIS2 by ChIP sequencing, RNA-Seq, and an assay for transposase-accessible chromatin sequencing identified key osteogenic genes regulated directly by MEIS2, indicating that it plays a fundamental role in palatal osteogenesis. De novo motif analysis uncovered that the MEIS2-bound regions are highly enriched in binding motifs for several key osteogenic transcription factors, particularly short stature homeobox 2 (SHOX2). Comparative ChIP sequencing analyses revealed genome-wide co-occupancy of MEIS2 and SHOX2 in addition to their colocalization in the developing palate and physical interaction, suggesting that SHOX2 and MEIS2 functionally interact. However, although SHOX2 was required for proper palatal bone formation and was a direct downstream target of MEIS2, Shox2 overexpression failed to rescue the palatal bone defects in a Meis2-mutant background. These results, together with the fact that Meis2 expression is associated with high osteogenic potential and required for chromatin accessibility of osteogenic genes, support a vital function of MEIS2 in setting up a ground state for palatal osteogenesis.

Keywords: Meis homeobox 2 (MEIS2); bone; chromatin accessibility; craniofacial development; gene knockout; gene regulation; osteogenesis; palatal development; short stature homeobox 2 (SHOX2); transcription; transcription factor; transgenic mice.

Figure 1.

MEIS2 expression in developing palatal shelves. A–E”, immunostaining using anti-MEIS2 antibody shows strong expression of MEIS2 in the palatal epithelium and mesenchyme throughout the A-P axis of the developing palate. The section levels indicate the domains along the A-P axis: Anterior, the palatine process of the maxilla; Middle, the palatine; Posterior, the soft palate. Two unique MEIS2-negative domains are located at either end of the nasal passage just dorsal to the palate in D' (white arrows). Scale bars = 100 μm (A–C”) and 200 μm (D–E”). T, tongue; PS, palatal shelf; P, palate.

Figure 2.

Conditional deletion of Meis2 in CNC cells leads to submucous cleft. A and A', oral view showing disorganized rugae (black arrows) and cleft soft palate in Wnt1^Cre;Meis2^f/f mice at P0 compared with the control. B and B', skeletal staining shows complete loss of palatal bones, including the palatine process of the maxilla (black triangle in the control) and the palatine bone as well as abnormal lamina obturans and pterygoid in Wnt1^Cre;Meis2^f/f mice at P0 compared with the control. The vomer and presphenoid in B' are not visible in B because they are under the maxilla and palatine. C–J', histological examination shows the histology of the developing palatal shelves in WT and Wnt1^Cre;Meis2^f/f mice from E13.5 to P0. Generally, although the anterior palatal shelves appeared to be slightly malformed in Wnt1^Cre;Meis2^f/f mice, they elevated, met, and eventually fused in the midline. However, the posterior palatal shelves exhibited dramatically reduced size, never met and failed to fuse, causing cleft soft palate (asterisks in A', H', F', J'). In addition, mutants also showed a deformed tongue compared with the control. Scale bars = 1 mm (A--B') and 200 μm (C–J'). T, tongue; PS, palatal shelf; P, palate; Max, maxilla; PL, palatine; LO, lamina obturans; Vo, vomer; PSp, presphenoid; Pt, pterygoid; BS, basisphenoid.

Figure 3.

Meis2 inactivation down-regulates expression of osteogenic genes in the developing palate. A--F', immunostaining shows absence of the osteogenic markers RUNX2 and SP7 in the palate of Wnt1^Cre;Meis2^f/f mice at E14.5–E16.5 compared with the WT. At the either end of the nasal passage just dorsal to the palate, where MEIS2 was not expressed, the expression of RUNX2 and SP7 remained (white arrows in B', D', and F'). G, GO analysis of RNA-Seq results reveals that the down-regulated genes in the palatal mesenchyme of E12.5 Wnt1^Cre;Meis2^f/f mice are highly associated with osteogenesis. H, RT-qPCR analysis validates down-regulation of selected osteogenic genes in E12.5 Wnt1^Cre;Meis2^f/f mice as compared with controls. I--K', immunostaining and in situ hybridization further confirm down-regulation of SHOX2 (I'), PBX1 (J'), and Bmp2 (K', black arrows) in E12.5 Wnt1^Cre;Meis2^f/f anterior palatal shelves compared with the control. Scale bars = 100 μm (A–F' and I–K'). *, p < 0.05. PP of Max, palatine process of the maxilla; T, tongue; PS, palatal shelf; P, palate.

Figure 4.

MEIS2 binds to osteogenic gene loci directly and arranges chromatin accessibility. A, the majority of MEIS2-bound regions have great distance to its nearest TSS. B, GO analysis of MEIS2 ChIP-Seq datasets indicates that genes near MEIS2-bound sites are associated with ossification and osteoblast differentiation. C, Venn diagram shows the overlapping genes identified by MEIS2 ChIP-Seq, ATAC-Seq, and RNA-Seq. D, volcano plots show the regions that lose chromatin accessibility in E12.5 Wnt1^Cre;Meis2^f/f mice are linked to key osteogenic genes.

Figure 5.

MEIS2 and SHOX2 exhibit co-occupancy on osteogenic gene loci in the palate. A, de novo motif analysis unravels highly enriched binding motifs of osteogenic factors, including SHOX2, RUNX2, and PBX1 in the MEIS2-bound regions. B, a Venn diagram shows extensively overlapped genes bound by MEIS2 and SHOX2 ChIP-Seq to the E12.5 anterior palatal mesenchyme. C, a GO analysis reveals the same functional categories of the MEIS2-bound sites (blue bars) and SHOX2-bound sites (red bars). The x axis values correspond to the gene ratio of each term. D, MEIS2 directly binds to the distal regions near the Shox2, Runx2, Pbx1, and Bmp2 loci, which are also occupied by SHOX2 and are enriched in H3K27ac (boxes). ATAC-Seq analysis further reveals dramatically reduced chromatin accessibility within these regions in the E12.5 Wnt1^Cre; Meis2^f/f palate.

Figure 6.

Meis2 is closely associated with osteogenic potential of Shox2-expressing cells in developing palatal shelves. A, single-cell RNA-Seq analysis shows that E13.5 Shox2-positive palatal mesenchymal cells are clustered into five groups, with cluster 2 cells expressing lowest level of Meis2 (defined as Meis2-negative cells) compared with other clusters. B, GO analysis reveals that genes with low expression levels in cluster 2 are associated with roof of mouth development, regulation of ossification, and ossification. C, expression heatmap confirms the significantly low expression levels of osteogenic genes in cluster 2 (Meis2-negative cells). D, a Venn diagram shows that more than half of the ossification-associated genes with lower expression levels in cluster 2 are also down-regulated in Wnt1^Cre;Meis2^f/f palatal shelves.