Hoxa5 Activity Across the Lateral Somitic Frontier Regulates Development of the Mouse Sternum

Abstract

The skeletal system derives from multiple embryonic sources whose derivatives must develop in coordination to produce an integrated whole. In particular, interactions across the lateral somitic frontier, where derivatives of the somites and lateral plate mesoderm come into contact, are important for proper development. Many questions remain about genetic control of this coordination, and embryological information is incomplete for some structures that incorporate the frontier, including the sternum. Hox genes act in both tissues as regulators of skeletal pattern. Here, we used conditional deletion to characterize the tissue-specific contributions of Hoxa5 to skeletal patterning. We found that most aspects of the Hoxa5 skeletal phenotype are attributable to its activity in one or the other tissue, indicating largely additive roles. However, multiple roles are identified at the junction of the T1 ribs and the anterior portion of the sternum, or presternum. The embryology of the presternum has not been well described in mouse. We present a model for presternum development, and show that it arises from multiple, paired LPM-derived primordia. We show evidence that HOXA5 expression marks the embryonic precursor of a recently identified lateral presternum structure that is variably present in therians.

Keywords: Hoxa5; lateral plate mesoderm; lateral somitic frontier; skeletal patterning; somites; sternum.

FIGURE 1

Hoxa5 expression in somites and LPM. (A) HOXA5 protein expression and (B) domain of cells with an expression history of Hoxa5 at E11.5, prior to migration of axial skeletal progenitors across the lateral somatic frontier. Compare to the location of cells derived from (C) somites, labelled with Meox1-Cre, and (D) LPM, labelled with Prx1-Cre. White arrows mark the lateral (left) and medial (right) borders of the somites. White arrowheads indicate the axillary region known to contain sternal bar progenitors. Grey arrowhead indicates additional HOXA5 expression in the limb bud, and asterisk marks the myotome (see text). (E,F) Summary of skeletal phenotypes following conditional Hoxa5 deletion. Frequency of Hoxa5 associated phenotypes in the (E) C6-T1 vertebrae and (F) sternum at E18.5. *p < 0.05, **p < 0.01, ****p < 0.0001, Fisher’s exact test. Complete genotypes of control and experimental groups are given in Supplementary Table S1.

FIGURE 2

Vertebral phenotypes associated with conditional Hoxa5 deletion at E18.5. Alcian blue and Alizarin red staining of vertebrae from control (A), null (B), somite deleted (C), and LPM-deleted (D) embryos. Arrowheads indicate tuberculum anterior (TA) on C6, ribs on C7 and the position of the dorsal process on T1 (absence of a dorsal process indicated by a tilted arrowhead). Note that the T1 ribs have been cut within the cartilaginous, distal portion for photography. Scale bar 1 mm (top four rows) or 0.47 mm (bottom row).

FIGURE 3

Sternum phenotypes associated with conditional Hoxa5 deletion at E18.5. (A) Control sternum at E18.5. Arrowheads indicate the positions at which measurements were taken for data shown in panels (E–H). (B–D) Examples of anterior sternum phenotypes. In all panels, filled black arrowheads indicate T1 ribs, black arrows indicate T2 ribs, and grey arrowheads indicate C7 ribs. White arrowheads indicate Y-shaped cartilage reduction, and white arrow in (Biii) indicates ectopic ossification of the Y-shaped cartilage. Grey arrows in (Di,ii) indicate an elongated sternum region between the Y-shaped cartilage and T1 attachment position, which was sometimes associated with an ectopic ossification. Scale bar: 500 µm. (E–H) Comparative lengths of sternal regions between arroweads indicated in (A). In each panel, null crosses are graphed on the left, somite-specific deletion in the middle and LPM-specific deletion on the right. Bars and brackets indicate the mean and standard deviation, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant, Welch’s t-test. (I,J) Adult sternum with T1 and T2 attachments shown in side (I) or front (J) views. (K) Schematic of E14.5 sternum and ribs attachments. (L) Schematic of a cross-sections through an E14.5 embryo at the axial level indicated by the blue line in (K).

FIGURE 4

Development of the presternum is disrupted in Hoxa5 null mutants. (A–M′) SOX9 expression, shown with (A–M) and without (A′–M′) DAPI labelling, in serial sections from the anterior presternum through T1–T3 attachments of an E14.5 WT embryo. (N,N′–Z,Z′) SOX9 expression in serial sections through the presternum and T1–T3 attachment of a Hoxa5^−/− null littermate. (A–H) and (N–U) Blue arrows mark dorsal IC/ICC; pink arrows indicate ventral ICC; white arrows point to the site of pectoral muscle attachment. Red arrows in G′ and T′ indicate first contact of T1 with the sternum. Blue arrow in J indicates the beginning of the second contact of T1 with the sternum. Scale bar: 200 μM. C, clavicle; IC, interclavicular; ICC, intercostoclavicular; P, pectoral muscle.

FIGURE 5

HOXA5 is specifically expressed in ventral elements of the presternum. SOX9 (A–M′) and HOXA5 (N–Z′) expression in alternate sections of a WT E14.5, from the anterior presternum through T1–T3 attachments, shown with (A–M, N–Z) and without (A′–M′, N′–Z′) DAPI. C, clavicle. Pink arrow in (O′–T′) identifies expression of HOXA5 in the paired ventral elements of the ICC mesenchyme. Orange arrows in P′ and Q′ mark HOXA5 expression lateral to, but continuous with, the presternum. Red arrow in V′ indicates HOXA5 expression within the sternum as it shifts to a more rounded morphology. Red arrows in X′ and Y′ point to a shift in Hoxa5 expression more dorsally, likely marking the connective tissue of an infrahyoid muscles. HOXA5 can also be seen in the perichondrium of ribs 2 and 3 (yellow arrows in Y′ and Z′), and in the body wall mesothelium (red arrow in Z′). Scale bar: 200 μM. C, clavicle; IC, interclavicular; ICC, intercostoclavicular; T1, thoracic rib 1.

FIGURE 6

At E13.5, HOXA5 and SOX9 co-expression identifies a component of the presternum that is continuous with the sternal bars, and is the site of rib 1 attachment. (A–I‴) E13.5 Meox1-Cre-RFP embryo co-labelled for HOXA5 (green (A′,A″–I′), SOX9 (blue, (A′,A‴–I′,I‴)), and RFP (red (A–I)). As observed at E14.5, HOXA5, and SOX9 are co-expressed (cyan cells, (A,A′–I,I′) in the ventral ICC element of the presternum—with HOXA5 expression extending more broadly ventral to SOX9 (C′–E′; L–N). (J–R) At higher magnification (corresponding to yellow square in (A′)), co-expression is more clearly visualized. SOX9, but not HOXA5, is detected at lower levels in the dorsal IC and ICC (A′–E′), while both are co-expressed in ribs 1 and 2 (F′–I′) and spanning the site of rib 1-to-sternum fusion (F′–H′; O–Q). HOXA5 is additionally found in the perichondrium of ribs 1 and 2 (G′–I′), as well as in the connective tissue of the infrahyoid muscles, dorsal to the sternum (I″). Red arrow in (D′) indicates exclusion of HOXA5 from the most medial region of the ventral ICC mesenchyme. Orange arrows in (C′,D′) point to portion of HOXA5 lateral domain that does not co-express with SOX9. Red arrow in (G) indicates site of rib 1 attachment. Scale bars: 200 μM T1, thoracic rib 1; T2, thoracic rib 2; T3, thoracic rib 3.

FIGURE 7

At E12.5, HOXA5 occupies a lateral domain as well as marking the site of rib 1 attachment to the sternum. E12.5 Meox1-Cre-RFP embryo in which alternate sections were labelled for SOX9 (A–H′′) or HOXA5 (I–P′′). Whole cross-sections are shown in (A) and (I), with remaining images from the region indicated by blue rectangle in (A). Images are shown with RFP and either SOX9 (A–H) or HOXA5 (I–P); RFP alone (A′–H′; I′–P′), or SOX9 (A″–H″) or HOXA5 (I″–P″) alone. Pink arrows in (C–F) and (L–N) indicate ventral ICC. Blue arrows in (C–F) point to dorsal IC/ICC. Orange arrow in (M″) indicates lateral HOXA5 expression domain. Red arrows in (M″,N″) point to exclusion of HOXA5 from the medial-most region of the ventral ICC mesenchyme. Yellow arrow in (G,H) marks triangle of SOX9 expression contacting the rib. White arrow in (H) marks stream of cells crossing the midline. Scale bar: 100 μM. C, clavicle; T1, thoracic rib 1; T2, thoracic rib 2.

FIGURE 8

LPM-derived HOXA5 expression can be seen laterally as early as E11.5 and E12. (A–J″) E12.5 Prx1-Cre-RFP embryo in which alternate sections were labelled for SOX9 (A–E″) or HOXA5 (F–J″). Images are shown with RFP and either SOX9 (A–E) or HOXA5 (F–J); RFP alone (A′–E′; F′–J′), or SOX9 (A″–E″) or HOXA5 (F″–J″) alone. Pink arrows in (A,B) indicate SOX9 expression associated with developing clavicles and IC elements. (K) Whole mount in situ hybridization for Hoxa5 in an E11.5 embryo. Black arrow indicates LPM expression anterior to the forelimb; red arrow points to LPM expression adjacent to the forelimb; green arrow marks additional HOXA5 expression that remains untranslated. (L,P) RFP and HOXA5 expression in sections through an E11.5 Prx1-Cre RFP embryo at the level indicated in (K) by black arrow (L), or red arrow (P). (M,N) expression of Hoxa5 (M) or EBF3 and HOXA5 (N) in the area indicated by blue square in (L). (O) SOX9 expression in an alternate section of same embryo shown in (L). (Q,R) expression of HOXA5 (Q) or EBF3 and HOXA5 (R) in area indicated by blue square in (P). (S) SOX9 expression in an alternate section of same embryo shown in (P). Yellow arrow in (N,R) indicates region of HOXA5 and EBF3 co-expression. Yellow arrow in (O,S) shows corresponding region of SOX9 expression. Inset in (O) shows SOX9 expression in the context of Prx1-Cre-RFP. Scale bar: (A–J), 200 μM; (K), 25 mm; (L–S), 100 μM. SB, sternal bar; T1, thoracic rib 1.

FIGURE 9

The mammalian presternum is a composite structure with dorsal-ventral organization and molecularly distinct domains. (A) Schematic of sternum development in an E12.5 embryo. The clavicles (orange) and dorsal IC/ICC mesenchyme form as paired condensations that express low levels of SOX9. The ventral ICC of the presternum appears as a region of high SOX9 expression that is continuous with the posterior sternal bars (SB). HOXA5 is expressed in both the ventral ICC and rib 1, marking the site of rib 1 attachment to the sternum. Rib 2 also expresses high levels of HOXA5. SOX9 expression can be seen in a stream of cells that cross the midline between the sternal bars. (B) Schematic of sternum development in an E14.5 embryo. By E14.5 sternal bar fusion is mostly complete and the presternum can be seen to have a morphology distinct from the rest of the sternum. The clavicles articulate with the dorsal IC, while the first rib contacts the HOXA5-expressing ventral ICC in two places. The pectoral muscles attach along the ventral midline of the sternum. (C) Side view of an E14.5 sternum showing dorsal ventral regionalization. The dorsal IC and ICC (blue) form a distinct element, while the ventral ICC appears continuous with the sternal bars. HOXA5 specifically marks the ventral ICC. (D) Key for coloring in (A–C).