Avian species display a remarkable diversity of facial morphologies, from the small, pointed, insect-catching beak of the common sparrow, to the long, narrow, nectar-sipping beak of a hummingbird. Using fate-mapping studies, we know that all facial skeletal elements are derived from neural crest mesenchyme (NCM), a multipotent embryonic cell population. From transplant experiments, we know that NCM plays an instructive role in patterning and growth in the face (i.e., when we transplant quail NCM into a duck host, the chimera forms a quail-like face and beak). What remains to be understood is how NCM carries out the components of what is undoubtedly a very complex task - to pattern beaks with great precision for function in established niches, but also to allow plasticity for evolution in response to changes in the natural environment.

Thus, one of the questions I address during my dissertation research is what are developmental, cellular, and molecular mechanisms underlying evolvability in avian faces? I have previously been intrigued by the rapid rate of generation of novel beak morphologies as historically described in Darwin's finches and other models. To begin to understand these phenomena, I investigated the function, regulation, and evolution of one transcription factor, Runx2, as a model for understanding processes that can modulate the generation of heritable, selectable, phenotypic variation.

Runx2 is often considered a master regulator of osteogenesis. However, mechanisms by which Runx2 might regulate timing of osteogenesis in vivo have not been previously described. Here, by using a unique avian chimeric experimental system, we identify Runx2 as a critical player in both NCM-dependent timing of osteogenesis and developmental growth and patterning of the craniofacial complex. Specifically, we find that NCM controls stage- and species-specific cell cycle progression and Runx2 expression in highly interwoven processes. Further, Runx2 expression levels affect mandible size and correlate to species-specific sequence variation at a highly evolvable Runx2 regulatory region. Taken together, these data suggest that NCM may be able generate a range of skeletal element sizes and morphologies in part by temporally regulating cell cycle in conjunction with cell differentiation through highly regulated, mutation-labile transcription factors such as Runx2.

How does form arise during development and change during evolution? How does form relate to function, and what enables structures of embryos to presage their later use in adults? To address these questions, we leverage the distinct functional morphology of the jaw in duck, chick, and quail. Duck develop secondary cartilage at the tendon insertion of their jaw adductor muscle on the mandible. An equivalent cartilage is absent in chick and quail. We hypothesize that species-specific jaw architecture and mechanical forces promote secondary cartilage in duck through differential regulation of FGF and TGFβ signaling. First, we examine the role of neural crest mesenchyme (NCM), which produces all jaw skeletal and connective tissues, in establishing species-specific pattern by transplanting NCM from chick to duck. Second, we investigate links between jaw architecture and mechanical forces by examining motility and by using finite element modeling. Third, we utilize loss-of-function approaches to determine whether candidate signaling mechanisms like voltage-gated ion channels and FGF or TGFβ signaling are required for secondary cartilage induction. Fourth, we perform gain-of-function experiments to determine whether FGF and TGFβ signaling are sufficient to induce chondrogenesis. Fifth, we quantified FGF and TGFβ pathway member expression in paralyzed and control samples to pinpoint potential mechanically regulated target genes. Our results provide insights on mechanisms linking musculoskeletal form and function during development, disease, and evolution.

Mesenchymal Regulation of Jaw Bone Length

Erin Lynn Ealba

With the goal of devising new therapies for skeletal diseases, injuries, and birth defects, there is keen interest to discover mechanisms through which mesenchymal cells make bone. Many studies have pointed to molecular factors that induce the osteogenic differentiation of mesenchyme in vitro, but since a clinical objective is to engineer mesenchyme for applications in vivo, more work is required to identify critical interactions between osteogenic mesenchyme and surrounding tissues, such as osteoclasts. In the developing jaws and face, all bone comes from neural crest mesenchyme (NCM) whereas osteoclasts arise from mesoderm. We take advantage of these distinct embryonic origins and transplant faster developing quail NCM into slower developing duck embryos. Previously, using this quail-duck chimeric system, we have shown that NCM makes bone by executing autonomous molecular and histogenic programs. Here, I assess the effects of NCM and their effects on host-derived osteoclasts by using histology and immunochemistry (Milligan's Trichrome, Q¢PN, Tartrate-resistant acid phosphatase (TRAP)), whole-mount staining analysis (TRAP, alizarin red), gene expression (in situs, RNA isolation, cDNA synthesis, reverse transcription quantitative real-time PCR (RT-qPCR)), Cloning (PCR, pGEM-T Easy), Fluorescence-Activated Cell Sorting (FACS), proliferation analysis (BrdU, ImageJ), Inhibiting and activating resorption (Alendronate, matrix metalloproteinase 13 (MMP13) inhibitor, recombinant osteoprotogerin (rOPG), recombinant receptor activator of nuclear factor κ β (rRANKL)), and morphometric analysis. We find that the influence of NCM on osteoclast activity has direct implications for differences in craniofacial elements. We conclude that the timing of matrix remodeling plays an important role in determining jaw length.