Meis2 controls skeletal formation in the hyoid region

Abstract

A vertebrate skull is composed of many skeletal elements which display enormous diversity of shapes. Cranial bone formation embodies a multitude of processes, i.e., epithelial-mesenchymal induction, mesenchymal condensation, and endochondral or intramembranous ossification. Molecular pathways determining complex architecture and growth of the cranial skeleton during embryogenesis are poorly understood. Here, we present a model of the hyoid apparatus development in Wnt1-Cre2-induced Meis2 conditional knock-out (cKO) mice. Meis2 cKO embryos develop an aberrant hyoid apparatus-a complete skeletal chain from the base of the neurocranium to lesser horns of the hyoid, resembling extreme human pathologies of the hyoid-larynx region. We examined key stages of hyoid skeletogenesis to obtain a complex image of the hyoid apparatus formation. Lack of Meis2 resulted in ectopic loci of mesenchymal condensations, ectopic cartilage and bone formation, disinhibition of skeletogenesis, and elevated proliferation of cartilage precursors. We presume that all these mechanisms contribute to formation of the aberrant skeletal chain in the hyoid region. Moreover, Meis2 cKO embryos exhibit severely reduced expression of PBX1 and HAND2 in the hyoid region. Altogether, MEIS2 in conjunction with PBX1 and HAND2 affects mesenchymal condensation, specification and proliferation of cartilage precursors to ensure development of the anatomically correct hyoid apparatus.

Keywords: Hand2; Meis2; PBX1; cartilage; hyoid bone; mesenchymal condensation.

FIGURE 1

The expression of Meis2 in the embryonic hyoid region. (A–C) Frontal paraffin sections of E12.5 hyoid regions. MEIS2 protein is abundant in non-skeletogenic mesenchyme of the hyoid region and in chondrocyte precursors in and around the hyoid primordium. MEIS2 signal is weaker in the hyoid body, while strong in the lesser horns, the greater horns and the styloid process. (D–F") MEIS1 and MEIS2 double immunofluorescence showing significantly weaker MEIS1 signal in the hyoid cartilage, surrounding mesenchyme and the tongue. (G–I") Higher magnification images showing MEIS1 and MEIS2 double immunofluorescence of boxed areas in (D–F). Abbreviations: hb, hyoid body; gh, greater horns; lh, lesser horns; mb, mandible; Mc, Meckel’s cartilage; oc, otic capsule; sp, styloid process; E, epiglottis; G, glottis; T, tongue. Scale bars in μm.

FIGURE 2

Craniofacial and hyoid phenotypes in control, Wnt1-Cre2; Meis2 ^f/f and Wnt1-Cre2; Meis1 ^f/f embryos. (A–A") Lateral view at skeletal preparations of heads of E17.5 control, Meis2 cKO and Meis1 cKO embryos. (B–B") Inferior view at skeletal preparations of heads of E17.5 control, Meis2 cKO and Meis1 cKO embryos. (C–C") Frontal view at skeletal preparations of hyoid-larynx complexes of E17.5 control, Meis2 cKO and Meis1 cKO embryos. In Meis2 cKO, observe an aberrant skeletal chain that stretches from the styloid process to the lesser horns of the hyoid (black arrowheads in A′,B′,C′). Arrows in A and A″ point at normal styloid process. Note an extra bony element close to the gonial bone, the malleus, and the tympanic ring (white arrowhead in B′). Abbreviations: hb, hyoid body; gh, greater horns; lh, lesser horns; sp, styloid process (arrows in A and A″"); tc, thyroid cartilage; cc, cricoid cartilage; bs, basisphenoid bone; tr, tympanic ring. Scale bars in μm.

FIGURE 3

The expression of Sox9 in the embryonic hyoid region. Frontal paraffin sections of hyoid regions in control and Wnt1-Cre2; Meis2 ^f/f embryos. (A, A′) See a clear SOX9⁺ outline of the hyoid primordium in E11.5 control (A, inset), whereas SOX9⁺ cells in E11.5 Wnt1-Cre2; Meis2 ^f/f did not form any distinctively recognizable shape (A′, inset). (B,B′) The cartilage primordium of the hyoid bone formed in its normal shape in E12.5 control, while the hyoid primordium in E12.5 Wnt1-Cre2; Meis2 ^f/f was still represented by persisting SOX9⁺ chondrogenic condensations. (C,C′) Persisting SOX9⁺ chondrogenic condensations were also observed in E13.5 Wnt1-Cre2; Meis2 ^f/f. Note an extra skeletal element close to the gonial bone, the malleus, and the tympanic ring (white arrowhead in C′). (D,D′) Col2a1 in situ hybridization showing the expression in the hyoid primordium and the aberrant skeletal chain (asterisk) in E14.5 control and Meis2 cKO. Abbreviations: hb, hyoid body; gh, greater horns; lh, lesser horns. Scale bars in μm.

FIGURE 4

The aberrant skeletal chain between the lesser horns and the styloid process in Wnt1-Cre2; Meis2 ^f/f. Frontal paraffin sections (A–C′) of the hyoid apparatuses in controls and Wnt1-Cre2; Meis2 ^f/f. (A,A′) In E14.5 Wnt1-Cre2; Meis2 ^f/f, SOX9-staining revealed the aberrant skeletal chain (asterisk) that was close to the lesser horns and was continuous with the styloid process. (B,B’) In E15.5 Wnt1-Cre2; Meis2 ^f/f, RUNX2-staining revealed an intensive staining of the aberrant skeletal chain (asterisk). (C,C′) In E16.5 Wnt1-Cre2; Meis2 ^f/f, SP7-staining revealed commitment of the aberrant skeletal chain (asterisk) to the osteoblast lineage. (D,D′) Frontal view at skeletal preparations of the hyoid-larynx complexes with temporal bones. E18.5 Wnt1-Cre2; Meis2 ^f/f specimens have developed the aberrant hyoid apparatus. A small caudal part of the aberrant skeletal chain is mineralized (asterisk in D′) and a cartilaginous nodule randomly formed along the chain (black arrowhead in D′). Abbreviations: hb, hyoid body; gh, greater horns; lh, lesser horns; sp, styloid process; tc, thyroid cartilage; cc, cricoid cartilage. Scale bars in μm.

FIGURE 5

Elevated proliferation of SOX9 cells in the cartilage primordium of the hyoid bone in Wnt1-Cre2; Meis2 ^f/f. (A–C′) Frontal frozen sections with orthogonal projections of hyoid regions in controls and Wnt1-Cre2; Meis2 ^f/f.. (D) Quantifications of the percentage of double positive EdU/SOX9 cells in controls and Wnt1-Cre2; Meis2 ^f/f (E12.5 control 27.88 ± 3.27); (E12.5 Wnt1-Cre2; Meis2 ^f/f 37.19 10.32); (E13.5 control 3.84 ± 0.70); (E12.5 Wnt1-Cre2; Meis2^f/f 22.20 ± 2.78); (E14.5 control 12.90 ± 2.41); (E14.5 Wnt1-Cre2; Meis2 ^f/f 25.24 ± 4.90) Statistical analysis was performed from biological triplicates for each genotype using unpaired two-tailed Student’s t-test. Scale bars in μm.

FIGURE 6

Unaltered proliferation of RUNX2 cells in Wnt1-Cre2; Meis2 ^f/f. (A–B′) Frontal frozen sections with orthogonal projections of hyoid regions in controls and Wnt1-Cre2; Meis2 ^f/f. (C) Quantification of the percentage of double-positive EdU/RUNX2 cells in controls and Wnt1-Cre2; Meis2 ^f/f. The results were presented as mean ± SD. (E13.5 control 24.99 ± 0.62); (E13.5 Wnt1-Cre2; Meis2^f/f 23.16 ± 0.12); (E14.5 control 18.15 ± 4.05); (E14.5 Wnt1-Cre2; Meis2^f/f 20.87 ± 4.11). Statistical analysis was performed from biological triplicates for each genotype using unpaired two-tailed Student’s t-test. Scale bars in μm.

FIGURE 7

Irregular spatial distribution of proliferating cartilage progenitors in Wnt1-Cre2; Meis2 ^f/f ectopic cartilage. (A–B′) Frontal frozen sections labelled with EdU (24-h pulse) and SOX9 antibody at stages E13.5, and E14.5 (C–D′). 2-Cell EdU⁺ clusters were quantified according to their orientation towards the longitudinal axis in three angles 90, 45, 0° (arrowheads in B and D). Note a higher number and irregular distribution of EdU⁺ dividing cartilage progenitors in the aberrant cartilage (white arrows). (E) Quantifications of EdU⁺ doublets were made separately in the hyoid body and hyoid horns together with the ectopic cartilage in mutants. Statistical analysis was performed from biological triplicates for each genotype using unpaired two-tailed Student’s t-test. Scale bars in μm.

FIGURE 8

Diminished expression of Hand2 and Pbx1 in hyoid regions of Wnt1-Cre2; Meis2 ^f/f. Frontal paraffin sections of hyoid regions in E12.5 controls and Wnt1-Cre2; Meis2 ^f/f. (A–C′) The expression domain of HAND2 was diminished in E12.5 Wnt1-Cre2; Meis2 ^f/f in the hyoid region, and the tongue. Sections were counterstained with nuclear fast red. (D–F′) The expression domain of PBX1 was diminished in the tongue, hyoid, and laryngeal regions in E12.5 Wnt1-Cre2; Meis2 ^f/f. Sections were counterstained with Alcian blue. Abbreviations: hb, hyoid body; gh, greater horns; lh, lesser horns; E, epiglottis; G, glottis; T, tongue; *—malformed hyoid primordium. Scale bars in μm.

FIGURE 9

Unaltered expression of molecular markers in hyoid arches of Wnt1-Cre; Meis2 ^f/f. (A–B′) Expression domain of Hoxa2 in E10.5 hyoid arch of control and Meis2 cKO, lateral (A,A′) and frontal (B,B′) views. (C–D′) Expression domain of Prrx1 in E10.5 pharyngeal arches in control and Meis2 cKO, lateral (C,C′) and frontal (D′-D) views. (E,E′) Expression domain of Dlx5 in E10.5 pharyngeal arches in control and Meis2 cKO, lateral views. Scale bars in μm. Abbreviations: PA1, pharyngeal arch 1 (mandibular), PA2, pharyngeal arch 2 (hyoid).

FIGURE 10

A proposed network of transcription factors converging to MEIS2. MEIS2-HAND2-PBX circuit controls skeletogenesis in mandibular and hyoid arches by restricting skeletogenic domains. On the other hand, MEIS2, HOXA2, and PRRX1 are not directly linked during the hyoid development. Created using Biotapestry (Longabaugh et al., 2005, 2009; Paquette et al., 2016).