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The vertebrate transcription factor protein Runx2 is regarded as a "master regulator" of bone formation due to the dramatic loss of the osseous skeleton in the mouse homozygous knockout. However, Runx2 mRNA also is expressed in the pre-hypertrophic cartilaginous skeleton of the mouse and chicken, where its developmental function is largely unknown. Several tiers of Runx2 regulation exist in the mouse, any of which may account for its seeming biological inactivity during early stages of skeletogenesis. Unlike mouse and chicken, zebrafish require Runx2 function in early cartilage differentiation. The present study reveals that the earlier functional role of Runx2 in cartilage differentiation is shared between zebrafish and Xenopus. A combination of morpholino oligonucleotide injections and neural crest transplants indicate that Runx2 is involved in differentiation of the cartilaginous hyobranchial skeleton in the frog, Xenopus laevis. Additionally, in situ hybridizations show runx2 mRNA expression in mesenchymal precursors of the cartilaginous skull, which reveals the earliest pre-patterning of these cartilages described to date. The early distribution of runx2 resolves the homology of the larval suprarostral plate, which is one of the oldest controversies of anuran skull development. Together these data reveal a shift in Runx2 function protein during vertebrate evolution towards its exclusive roles in cartilage hypertrophy and bone differentiation within the amniote lineage.
Figure 2. In situ hybridizations of runx2 and sox9 in the mandibular neural crest stream. A: Anterior views of the head showing the distribution of both transcripts over several developmental stages. Runx2 mRNA expression is first apparent in the pre-chondrogenic suprarostral plate (SP) by stage 31. Additional expression appears in the cranial trabeculae (CT) by stage 35/36 (see Fig. 3). This expression is in a subset of the mandibular arch crest (MA), which expresses sox9. The expression of sox9 mRNA is progressively restricted to co-localize with runx2. By stage 39, both transcripts overlap in the presumptive suprarostal plate (SP). The expression of runx2 decreases in the suprarostral plate by stage 41, although some expression persists in the palatoquadrate anlagen (PQ). Sox9 expression in the suprarostral plate is fused in the midline by stage 41, with continued expression in the palatoquadrate and new expression in the infrarostral (IR) and Meckel's cartilages (MC). B: Ventral views of stage 39 embryos stained with runx2 and sox9. The cement gland has been removed with a scalpel. Both transcripts are expressed in the future ceratohyal (CH) and palatoquadrate cartilages. The latter is clearly divided into a posterior sub-ocular arc (SA) and anterior quadratocranial commissure (QC), which connects to the suprarostral plate dorsally. There is a small flange of staining off of the palatoquadrate in both preparations that reveals the future Meckel's cartilage (asterisk).
Figure 3. In situ hybridizations of runx2, sox9, collagen2a1, and cbfb. All images are lateral views, anterior to the right. Runx2 expression begins in the presumptive suprarostral plate (SP). It expands to the anlage of the cranial trabeculae (CT) and a faint portion of the hyoid arch crest (HA) by stage 35/36. Runx2 staining includes the future ceratobranchial (CB) and ceratohyal (CH) cartilages by stage 39, and is additionally found in the otic capsule (OC) by stage 41. The expression of runx2 in the ceratohyal decreases during stage 41 as this element begins to chondrify. The distribution of sox9 is more extensive than that of runx2 by stage 33/34, with strong expression in the mandibular (MA), hyoid (HA), and branchial (BA) arch neural crest streams. Its expression in the crest becomes restricted by stage 35/36 with a clear distribution in the precursors of the future palatoquadrate and continued expression in the hyoid and branchial arches. By stage 39, both runx2 and sox9 are co-expressed in the future cartilaginous elements. Sox9 expression in the ceratohyal is also diminished by stage 41 with the onset of chondrogenesis. Col2a1 is distributed through the notochord and otic capsule along with a subset of the branchial arches (BA) and presumptive suprarostral plate by stage 33/34. Col2a1 is expressed widely throughout the head during the stages examined with intensifying staining in the presumptive cranial cartilages by stages 39 and 41. The runx co-factor cbfb is expressed throughout the head during development, with stronger expression in the presumptive ceratohyal and palatoquadrate by stage 39.
runx2 (RUNX family transcription factor 2) gene expression in Xenopus laevis embryo, assayed via in sitiu hybridization, NF stage 39 embryo, ventral view, anterior up. (note the cement gland has been removed). Transcripts are expressed in the future ceratohyal (CH) and palatoquadrate cartilages ( which form the upper jaw). The latter is clearly divided into a posterior sub-ocular arc (SA) and anterior quadratocranial commissure (QC), which connects to the suprarostral plate dorsally. There is a small flange of staining off of the palatoquadrate in both preparations that reveals the future Meckel's cartilage (asterisk).
Anti-runx2 morpholino injections do not affect cranial neural crest cell migration. A and C are lateral views of live stage-33/34 hosts that have received neural crest transplants. B and D are the stage-44 hatchlings of the same individuals as A and C, which have been stained with the anti-collagen II antibody (red in B, white in D). A: Live image of a wild-type host that has received neural crest from a morpholino-injected donor. Portions of the presumptive hyoid and branchial neural crest streams have been replaced with morpholino-injected cells (green), which are migrat- ing normally. B: Same individual as A at stage 44, stained with anti-collagen II to reveal cartilage formation (red). The ceratohyalcartilage is truncated on the transplanted side, with a population of donor cells derived from the hyoid stream concentrated on the distal end (arrow). C,D: Neural crest transplanted from a wild-type donor into a morpholino-injected host can rescue cartilage loss. C: Live image of a morpholino-injected host that has received wild-type neural crest. The rhodamine labeled wild-type crest (red) migrates normally in the morpholino-injected host (green). D: Same individual as C at stage 44, stained with anti-collagen II (white). The ceratohyal is partially rescued on the morpholino-injected side (arrow). The rhodamine-label of the wild-type crest did not persist through antibody staining. E,F: Sox9 in situ hybridization in a single stage-32 embryo unilaterally injected with both runx2-I and runx2-II isoform-specific morpholinos. E: Wild-type side of the embryo, showing normal neural crest morphology revealed by the distribution of the sox9 probe. F: Anti-runx2-morpholino-injected side showing the same sox9 staining pattern as the wild-type side. The fluorescein label did not persist through the whole mount in situ hybridization. Abbrevi- ations as in Figures 2 and 4.
In situ hybridizations of runx2 in the mandibular neural crest stream. A: Anterior views of the head showing the distribution of both transcripts over several developmental stages. Runx2 mRNA expression is first apparent in the pre-chondrogenic suprarostral plate (SP) by stage 31. Additional expression appears in the cranial trabeculae (CT) by stage 35/36 . By stage 39, [expression is seen] in the presumptive suprarostal plate (SP). The expression of runx2 decreases in the suprarostral plate by stage 41, although some expression persists in the palatoquadrate anlagen (PQ).
col2a1(collagen, type II, alpha 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, head region, lateral view, anteriorright, dorsal up.
col2a1(collagen, type II, alpha 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, head region, lateral view, anteriorright, dorsal up.