HOXA5 protein expression and genetic fate mapping show lineage restriction in the developing musculoskeletal system

Abstract

HOX proteins act during development to regulate musculoskeletal morphology. HOXA5 patterns skeletal structures surrounding the cervical-thoracic transition including the vertebrae, ribs, sternum and forelimb girdle. However, the tissue types in which it acts to pattern the skeleton, and the ultimate fates of embryonic cells that activate Hoxa5 expression are unknown. A detailed characterization of HOXA5 expression by immunofluorescence was combined with Cre/LoxP genetic lineage tracing to map the fate of Hoxa5 expressing cells in axial musculoskeletal tissues and in their precursors, the somites and lateral plate mesoderm. HOXA5 protein expression is dynamic and spatially restricted in derivatives of both the lateral plate mesoderm and somites, including a subset of the lateral sclerotome, suggesting a local role in regulating early skeletal patterning. HOXA5 expression persists from somite stages through late development in differentiating skeletal and connective tissues, pointing to a continuous and direct role in skeletal patterning. In contrast, HOXA5 expression is excluded from the skeletal muscle and muscle satellite cell lineages. Furthermore, the descendants of Hoxa5-expressing cells, even after HOXA5 expression has extinguished, never contribute to these lineages. Together, these findings suggest cell autonomous roles for HOXA5 in skeletal development, as well as non-cell autonomous functions in muscle through expression in surrounding connective tissues. They also support the notion that different Hox genes display diverse tissue specificities and locations to achieve their patterning activity.

Figure 1.

HOXA5 protein expression in somites and LPM, and comparison to Hoxa5-Cre reporter expression. HOXA5 immunofluorescence is shown in green (A-E), while Hoxa5-Cre reporter staining is in red (F-J). Panels K-O show the overlay. (A-E) HOXA5 expression is dynamic and spatially restricted in the somites and LPM from E9.5 to E11.5. (F-K) Hoxa5-Cre reporter expression follows that of HOXA5 protein at E9.5 and E10.5. By E11.5, it is much broader than HOXA5 protein expression. Expression overlap between HOXA5 protein and Hoxa5-Cre reporter is indicated by the following arrows on panels K-O: LPM and forelimb bud (green arrowheads); foregut (yellow arrowheads); lateral and dorsal sclerotome (white arrowheads) including the cap of cells surrounding the VLL; dermomyotome (red arrowheads), phrenic motor column (blue arrowhead). The boundary between somites and LPM, the incipient somitic frontier, is marked by an arrow in Fig. 1A–D. The neural tube is outlined by a dotted line in the section shown in D, H, and M. All sections are transverse, dorsal is up, and lateral is to the left. cdm, central dermomyotome; csc, central sclerotome; d, dermis; dm, dermomyotome; dml; dorso-medial lip of the dermomyotome; dsc, dorsal sclerotome; fg, foregut; fl, forelimb; lpm, lateral plate mesoderm; lsc, lateral sclerotome; pmc, phrenic motor column; sc, sclerotome; vll, ventro-lateral lip of the dermomyotome. Hoxa5-Cre reporter line was either tdTOMATO (E9.5-E10.5) or nYFP (E11.5). Scalebars: 20μm (A-O).

Figure 2.

HOXA5 protein expression in somite and LPM derivatives from E12.5 to E16.5. HOXA5 immunofluorescence is shown in green and other markers including the Prx1-Cre reporter are in red. (A-B) Overview of the dorsal (A) and ventral (B) sides of an E12.5 embryo showing HOXA5 expression in somite-derived structures. Cartilage condensations in (B) are revealed by TENASCIN staining in (C). Boxed regions are magnified in D-G. (D-F) HOXA5 expression in TENASCIN-positive condensing cartilage is detected in proliferating chondrocytes (arrows), and in the perichondrium (arrowheads) of the vertebral body, rib and neural arch. (G) DIC optics confirm perichondrial cells by morphology. (H-K) Co-labeling with HOXA5 and tdTOMATO reporter in Tg^Prx1Cre+; Rosa26^tdTomato+ embryo (H-I) show HOXA5 expression in rib chondrocytes and abaxial muscle region into which the rib migrates. (J-K) show the somitic frontier between abaxial (Prx1-Cre-positive) and primaxial muscle (Prx1-Cre negative) regions. (L) Muscle MYOSIN staining reveals absence of HOXA5 expression in skeletal muscle cells. Instead, HOXA5-expressing cells are interspersed within the muscle (arrowheads). (M) HOXA5-expressing cells co-express TCF4, suggesting they are fated to become muscle connective tissue. (N) HOXA5 is expressed in dermis, labeled with Dermo1-Cre. At E13.5 (O-R), E14.5 (S-V), and E16.5 (W-Y), HOXA5 expression persists in cartilage elements including vertebral bodies, neural arches and ribs, and further is detected in the developing sternum by E13.5 and spinous process by E16.5. Expression in perichondrium is marked with white arrowheads, in chondrocytes with white arrows, in periosteium with yellow arrowheads, and in osteocytes with yellow arrows. (P) HOXA5 continues to be expressed in chondrocytes and perichondrium, and by E13.5 also becomes localized in the layer of cells surrounding the perichondrium (arrowheads). (Z) Expression of HOXA5 in muscle-embedded fibroblasts persists at E16.5 (arrowheads). All sections are transverse, dorsal is up. Cre reporters are tdTOMATO except for panels H-K, which are nYFP. ab, abaxial; bat, brown adipose tissue; ch, chondrocytes; d, dermis; lu, lung; mf; muscle fibroblast; na, neural arch; nc, notochord; ob, osteoblast; pc, perichondrium; po, periosteium; pmc, phrenic motor column; pri, primaxial; sb, sternal band; st, sternum; tr, trachea; vb, vertebral body. Scalebars: 20μm (A-E, W-X), 2μm (F-G, L-N, U-V,Y), 10μm (H-K), 50μm (X).

Figure 3.

Distribution of Hoxa5 descendants in compartmentalized somites and differentiating tissues. Hoxa5 descendants are labelled in red following Hoxa5-Cre activity and stained with cell markers by immunofluorescence. At E10.5, Hoxa5 descendants co-label with sclerotome marker SOX9 (A, arrowhead) and dermomyotome marker PAX7 (B, arrowhead) but not with PAX3, a marker of the muscle lineage. PAX3 muscle progenitors are detected in the dorso-medial and ventro-lateral myotome lips (C, arrowheads). (D) At E11.5, Hoxa5 descendants no longer co-label with PAX7 (arrowhead), which at this stage marks the muscle lineage almost exclusively. (E) Hoxa5 descendants also fail to co-label with muscle ACTIN. (F-G) There is also no contribution of Hoxa5 descendants to PAX3-positive muscle progenitors that migrate ventrally from the somites, and will give rise to forelimb and diaphragm musculature. Arrowheads show that PAX3 positive cells are not labelled with the Hoxa5 lineage label. The PAX3 channel is included (F) and excluded (G) to highlight absence of Hoxa5 descendants. (H) In E11.5 whole-mount embryo, somitic reporter expression is largely confined to the future C3-T2 region (between arrowheads). (I) Three days later, Hoxa5 descendants are broadly distributed along the DV and medio-lateral axes, and clear borders of somitic expression are not obvious in whole-mount. Arrows in H-I indicate the rostral border of expression in the neural tube, which is similar at these two stages. (J-K) In an E14.5 sagittal section, Hoxa5 descendants are broadly distributed along the dorsal ventral and medio-lateral axes within the domain from C3-T2 (arrowheads mark the dorsal and ventral sides of C3 and T2). Vertebrae and ribs are visualized by TENASCIN staining in perichondrium. Sections are transverse in A-G (dorsal is up) and sagittal in J-K (dorsal is to the left, anterior is up). Hoxa5-Cre activity is visualized with tdTOMATO reporter (A, C-K) or nYFP reporter (B). lu, lung; nt, neural tube. Scalebars: 20μm (A-E), 5μm (F-G), 50μm (J-K).

Figure 4.

Broad distribution of Hoxa5 descendants in skeletal elements at E14.5 (A-Q) and E16.5 (R-S). Hoxa5 descendants are labelled in red following Hoxa5-Cre activity and counterstained with cell markers by immunofluorescence. (A-C) show low power views of the dorsal body axis (A), ventral body wall (B), and forelimb (C). Dorsal is up (A-B), or to the left (C). Boxes of closeup views shown in D-O are indicated. Panels D,F,H,J,L,N,P show DIC to reveal skeletal by their morphology, and corresponding fluorescence panels (E,G,I,K,M,O) to show Hoxa5 descendants. They contribute to cartilage (arrows) and perichondrium (arrowheads) of: vertebral body (D-E, H-I), scapula (F-G), rib (J-K), neural arch (L-M), sternum (N-O). Labeled osteoblasts show brighter signal than cartilage and are evident even at low power magnification, indicated by hollow arrowheads in the scapula (A), humerus (B) and ulna (C). (P-Q) High magnification image of vertebral body perichondrium. Within perichondrium, Hoxa5 descendant cells contribute to all layers. (R-S) Hoxa5 descendants also co-label with chondrocyte marker SOX9 (R; arrows) and with osteoblast marker COL1A1 (S; arrows). Inset in panel S shows closer view of Hoxa5 descendants embedded in the COL1A1-containing extracellular matrix. All sections are transverse. ipc, inner perichondrium; na, neural arch; opc, outer perichondrium; st, sternum; vb, vertebral body. Scalebars: 50μm (A-C), 20μm (E-O,R,S), 10μm (inset in C), 5μm (inset in S), 2μm (P-Q).

Figure 5.

Distribution of Hoxa5 descendants in connective tissue, dermis, brown adipose tissue, but not skeletal muscle at 14.5-E16.5. Hoxa5 descendants are labelled in red following Hoxa5-Cre activity and counterstained with cell markers by immunofluorescence. (A) Low power view shows that Hoxa5 descendants contribute to tendons (grey arrowheads), dermis (white arrowhead), brown adipose tissue (blue arrowhead), and fibroblasts within abaxial and primaxial skeletal muscle (white arrow). (B-F) Hoxa5 descendants contribute to tendon and ligament, which co-labeled with Col1A1 and TENASCIN. These include tendons of the subscapularis (B-C), vertebral body (D), rib (E) and nuchal ligament (F). (G) Hoxa5 descendants also contribute to dermatocytes, which are co-labelled with PDGFRα. (H-I) Hoxa5 descendants were found in brown adipose tissue including adipocytes and preadipocytes, which can be recognized by round shape and/or expression of PPARγ. Hoxa5 descendants were also detected in stromal fibroblasts of brown fat (arrows) and in cells that appeared to be pre-adipocytes based on their expression of PPARγ and fibroblast-like morphology (I). (J) HOXA5 protein continues to be expressed in BAT cells at E14.5 (arrowheads). (K-M) Hoxa5 descendants are found in fibroblasts throughout skeletal muscle (arrowheads) but do not co-label with muscle actin. Sections are transverse except for panels E-F, which are sagittal. a, adipocyte; ab, abaxial region; bat, brown adipose tissue; d, dermis; epi, epidermis; f, stromal fibroblast of brown adipose tissue; l, ligamentocyte; mf, muscle-embedded fibroblast; na, neural arch; nl, nuchal ligament; nt, neural tube; pa, preadipocyte; pri, primaxial sc, scapula; t, tenocyte; vb, vertebral body. Scalebars: 50μm (A), 20μm (B-D, J-M), 10μm (E-F), 5μm (G-I).

Figure 6.

Distribution of Hoxa5 descendants within skeletal muscle bundles at E16.5. Hoxa5 descendants are labelled in red following Hoxa5-Cre activity and counterstained with cell markers by immunofluorescence. (A) Hoxa5 descendants are localized outside the basal lamina of muscle fiber bundles (arrowheads). (B) Hoxa5 descendants (white arrowheads) do not co-label with PAX7 (blue arrowheads) indicating that they do not contribute to the muscle satellite cell lineage. Note that autofluorescent blood cells are found in this section (grey arrowhead). (C) Hoxa5 descendant cells do co-label with PDGFRα, a marker of fibroblasts in muscle (arrowhead). (D) Some Hoxa5 descendants co-label with TCF4, a marker for muscle connective tissue (arrowhead), while other do not (arrow). Conversely, some TCF4 cells do not express the Hoxa5 lineage label (blue arrowhead). (E) HOXA5 immunofluorescence (green) shows that many Hoxa5 descendant muscle fibroblasts continue to express HOXA5 protein (arrowhead). Sections are transverse through muscle bundles. Scalebars: 5μm (A-E).