Diane Hu

A zone of frontonasal ectoderm regulates patterning and growth in the face.

A fundamental set of patterning genes may define the global organization of the craniofacial region. One of our goals has been to identify these basic patterning genes and understand how they regulate outgrowth of the frontonasal process, which gives rise to the mid and upper face. We identified a molecular boundary in the frontonasal process ectoderm, defined by the juxtaposed domains of Fibroblast growth factor 8 and Sonic hedgehog, which presaged the initial site of frontonasal process outgrowth. Fate maps confirmed that this boundary region later demarcated the dorsoventral axis of the upper beak. Ectopic transplantation of the ectodermal boundary region activated a cascade of molecular events that reprogrammed the developmental fate of neural crest-derived mesenchyme, which resulted in duplications of upper and lower beak structures. We discuss these data in the context of boundary/morphogen models of patterning, and in view of the recent controversy regarding neural crest pre-patterning versus neural crest plasticity.

A model for intramembranous ossification during fracture healing.

We have developed a method to study the molecular basis of intramembranous fracture healing. Unlike intramedullary rods that permit rotation of the fractured bone segments, our murine model relies on an external fixation device to provide stabilization. In this study we compare stabilized fracture callus tissues with callus tissues from non‐stabilized fractures during the inflammatory, soft callus, hard callus, and remodeling stages of healing. Histological analyses indicate that stabilized fractures heal with virtually no evidence of cartilage whereas non‐stabilized fractures produce abundant cartilage at the fracture site. Expression patterns of collagen type IIa (colIIa) and osteocalcin (oc) reveal that mesenchymal cells at the fracture site commit to either a chondrogenic or an osteogenic lineage during the earliest stages of healing. The mechanical environment influences this cell fate decision, since mesenchymal cells in a stabilized fracture express oc and fail to express colIIa. Future studies will use this murine model of intramembranous fracture healing to explore, at a molecular level, how the mechanical environment exerts its influence on healing of a fracture.

Molecular aspects of healing in stabilized and non-stabilized fractures

Bone formation is a continuous process that is initiated during fetal development and persists in adults in the form of bone regeneration and remodeling. These latter two aspects of bone formation are clearly influenced by the mechanical environment. In this study we tested the hypothesis that alterations in the mechanical environment regulate the program of mesenchymal cell differentiation, and thus the formation of a cartilage or bony callus, at the site of injury. As a first step in testing this hypothesis we produced stabilized and non‐stabilized tibial fractures in a mouse model, then used molecular and cellular methods to examine the stage of healing. Using the “molecular map” of the fracture callus, we divided our analyzes into three phases of fracture healing: the inflammatory or initial phase of healing, the soft callus or intermediate stage, and the hard callus stage. Our results show that indian hedgehog(ihh), which regulates aspects of chondrocyte maturation during fetal and early postnatal skeletogenesis, was expressed earlier in an non‐stabilized fracture callus as compared to a stabilized callus, ihh persisted in the non‐stabilized fracture whereas its expression was down‐regulated in the stabilized bone. IHH exerts its effects on chondrocyte maturation through a feedback loop that may involve bone morphogenetic protein 6 [bmp6; (S. Pathi, J.B. Rutenberg, R.L. Johnson, A. Vortkamp, Developmental Biology 209 (1999) 239–253)] and the transcription factor gli3, bmp6 and gli3 were re‐induced in domain adjacent to the ihh‐positive cells during the soft and hard callus stages of healing. Thus, stabilizing the fracture, which circumvents or decreases the cartilaginous phase of bone repair, correlates with a decrease in ihh signaling in the fracture callus. Collectively, our results illustrate that the ihh signaling pathway participates in fracture repair, and that the mechanical environment affects the temporal induction of ihh, bmp6 and gli3. These data support the hypothesis that mechanical influences affect mesenchymal cell differentiation to bone.

Temporal perturbations in sonic hedgehog signaling elicit the spectrum of holoprosencephaly phenotypes

One of the most perplexing questions in clinical genetics is why patients with identical gene mutations oftentimes exhibit radically different clinical features. This inconsistency between genotype and phenotype is illustrated in the malformation spectrum of holoprosencephaly (HPE). Family members carrying identical mutations in sonic hedgehog (SHH) can exhibit a variety of facial features ranging from cyclopia to subtle midline asymmetries. Such intrafamilial variability may arise from environmental factors acting in conjunction with gene mutations that collectively reduce SHH activity below a critical threshold. We undertook a series of experiments to test the hypothesis that modifying the activity of the SHH signaling pathway at discrete periods of embryonic development could account for the phenotypic spectrum of HPE. Exposing avian embryos to cyclopamine during critical periods of craniofacial development recreated a continuum of HPE-related defects. The craniofacial malformations included hypotelorism, midfacial hypoplasia, and facial clefting and were not the result of excessive crest cell apoptosis. Rather, they resulted from molecular reprogramming of an organizing center whose activity controls outgrowth and patterning of the mid and upper face. Collectively, these data reveal one mechanism by which the variable expressivity of a disorder such as HPE can be produced through temporal disruption of a single molecular pathway.

Common Molecular Pathways in Skeletal Morphogenesis and Repair

ABSTRACT: The formation of bone is a continual process in vertebrate development, initiated during fetal development and persisting in adulthood in the form of remodeling and repair. The remarkable capacity of skeletal tissues to regenerate has led to the hypothesis that the molecular signaling pathways regulating skeletogenesis are shared during fetal development and adult wound healing. A number of key regulatory pathways that are required for endochondral ossification during fetal development are described, and their reintroduction in fracture repair demonstrated. Secreted proteins such as Sonic and Indian hedgehog exert their effect on pattern formation and chondrogenesis in the appendicular skeleton, partly through regulation of molecules such as bone morphogenic proteins (Bmps) and parathyroid hormone‐related peptide (PTHrP). Once chondrocytes have matured and hypertrophied, they undergo apoptosis and are replaced by bone; the transcription factor Cbfal plays a critical role in this process of chondrocyte differentiation and ossification. Analyses of the expression patterns of these genes during fracture healing strongly suggest that they play equivalent roles in adult wound repair. Knowledge acquired through the study of fetal skeletogenesis will undoubtedly contribute to an understanding of fracture repair, and subsequently guide the development of biologically based therapeutic interventions.

A SHH-responsive signaling center in the forebrain regulates craniofacial morphogenesis via the facial ectoderm

Interactions among the forebrain, neural crest and facial ectoderm regulate development of the upper jaw. To examine these interactions, we activated the Sonic hedgehog (SHH) pathway in the brain. Beginning 72 hours after activation of the SHH pathway, growth within the avian frontonasal process (FNP) was exaggerated in lateral regions and impaired in medial regions. This growth pattern is similar to that in mice and superimposed a mammalian-like morphology on the upper jaw. Jaw growth is controlled by signals from the frontonasal ectodermal zone (FEZ), and the divergent morphologies that characterize birds and mammals are accompanied by changes in the FEZ. In chicks there is a single FEZ spanning the FNP, but in mice both median nasal processes have a FEZ. In treated chicks, the FEZ was split into right and left domains that resembled the pattern present in mice. Additionally, we observed that, in the brain, fibroblast growth factor 8 (Fgf8) was downregulated, and signals in or near the nasal pit were altered. Raldh2 expression was expanded, whereas Fgf8, Wnt4, Wnt6 and Zfhx1b were downregulated. However, Wnt9b, and activation of the canonical WNT pathway, were unaltered in treated embryos. At later time points the upper beak was shortened owing to hypoplasia of the skeleton, and this phenotype was reproduced when we blocked the FGF pathway. Thus, the brain establishes multiple signaling centers within the developing upper jaw. Changes in organization of the brain that occur during evolution or as a result of disease can alter these centers and thereby generate morphological variation.

Quantitative analyses link modulation of sonic hedgehog signaling to continuous variation in facial growth and shape

Variation is an intrinsic feature of biological systems, yet developmental biology does not frequently address population-level phenomena. Sonic hedgehog (SHH) signaling activity in the vertebrate forebrain and face is thought to contribute to continuous variation in the morphology of the upper jaw, but despite its potential explanatory power, this idea has never been quantitatively assessed. Here, we test this hypothesis with an experimental design that is explicitly focused on the generation and measurement of variation in multivariate shape, tissue growth, cellular behavior and gene expression. We show that the majority of upper jaw shape variation can be explained by progressive changes in the spatial organization and mitotic activity of midfacial growth zones controlled by SHH signaling. In addition, nonlinearity between our treatment doses and phenotypic outcomes suggests that threshold effects in SHH signaling may play a role in variability in midfacial malformations such as holoprosencephaly (HPE). Together, these results provide novel insight into the generation of facial morphology, and demonstrate the value of quantifying variation for our understanding of development and disease.

From head to toe: conservation of molecular signals regulating limb and craniofacial morphogenesis.

Abstract Recent evidence indicates that many molecules involved in generating and patterning the limbs also play a role during craniofacial morphogenesis. On the surface, this is an unexpected finding given that these regions of the body have separate evolutionary origins, are composed of different embryonic tissues, and are quite dissimilar in their anatomy. Results from several experiments involving Sonic hedgehog and retinoic acid point to a remarkable conservation of the signaling pathways mediated by these morphogens across multiple organ systems. Moreover, mutants such as the extra-toes and doublefoot mouse, and the talpid chicken also provide insights on common developmental processes that underlie the formation of the limbs and face. The identification of highly conserved aspects of morphogenesis is important for understanding fundamental mechanisms of development, as well as for revealing the common denominator of countless birth defects and providing new strategies for their prevention and cure.

Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development.

A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh-/- chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh-/- skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh-/- cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh-/- cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh-/- cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment, the milieu surrounding the cartilage - comprising osteoblast precursors and endothelial cells - as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh-/- embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.

Mechanisms that underlie co-variation of the brain and face.

The effect of the brain on the morphology of the face has long been recognized in both evolutionary biology and clinical medicine. In this work, we describe factors that are active between the development of the brain and face and how these might impact craniofacial variation. First, there is the physical influence of the brain, which contributes to overall growth and morphology of the face through direct structural interactions. Second, there is the molecular influence of the brain, which signals to facial tissues to establish signaling centers that regulate patterned growth. Importantly, subtle alterations to these physical or molecular interactions may contribute to both normal and abnormal variation. These interactions are therefore critical to our understanding of how a diversity of facial morphologies can be generated both within species and across evolutionary time. genesis 49:177–189, 2011.