A central issue in biology concerns the presence, timing and nature of phylotypic periods of development, but whether, when and why species exhibit conserved morphologies remains unresolved. Here, we construct a developmental morphospace to show that amniote faces share a period of reduced shape variance and convergent growth trajectories from prominence formation through fusion, after which phenotypic diversity sharply increases. We predict in silico the phenotypic outcomes of unoccupied morphospaces and experimentally validate in vivo that observed convergence is not due to developmental limits on variation but instead from selection against novel trajectories that result in maladaptive facial clefts. These results illustrate how epigenetic factors such as organismal geometry and shape impact facial morphogenesis and alter the locus of adaptive selection to variation in later developmental events.

SUMMARYCraniofacial anomalies can arise from both genetic and environmental factors, including prenatal hypoxia. Recent clinical evidence correlates hypoxia to craniofacial malformations. However, the mechanisms by which hypoxia mediates these defects are not yet understood. We examined the cellular mechanisms underlying malformations induced by hypoxia using a chicken (Gallus gallus) embryo model. Eggs were incubated in either hypoxic (7, 9, 11, 13, 15, 17 or 19% O2) or normoxic (21% O2) conditions. Embryos were photographed for morphological analysis at days 3–6. For analysis of skeletal development, 13-day embryos were cleared and stained with alcian blue and alizarin red for cartilage and bone, respectively. Quantitative analysis of facial shape variation was performed on images of embryos via geometric morphometrics. Early-stage embryos (day 2) were analyzed for apoptosis via whole-mount and section TUNEL staining and immunostaining for cleaved caspase-3, whereas later-stage embryos (days 4–6) were sectioned in paraffin for analysis of cell proliferation (BrdU), apoptosis (TUNEL) and metabolic stress (phospho-AMPK). Results demonstrate that survival is reduced in a dose-dependent manner. Hypoxic embryos displayed a spectrum of craniofacial anomalies, from mild asymmetry and eye defects to more severe frontonasal and cephalic anomalies. Skull bone development was delayed in hypoxic embryos, with some skeletal defects observed. Morphometric analysis showed facial shape variation relative to centroid size and age in hypoxic groups. Hypoxia disrupted cell proliferation and, in early-stage embryos, caused apoptosis of neural crest progenitor cells. Hypoxic embryos also displayed an increased metabolic stress response. These results indicate that hypoxia during early embryonic craniofacial development might induce cellular oxidative stress, leading to apoptosis of the neural crest progenitor cells that are crucial to normal craniofacial morphogenesis.

A well-known tenet of murine tooth development is that BMP4 and FGF8 antagonistically initiate odontogenesis, but whether this tenet is conserved across amniotes is largely unexplored. Moreover, changes in BMP4-signaling have previously been implicated in evolutionary tooth loss in Aves. Here we demonstrate that Bmp4, Msx1, and Msx2 expression is limited proximally in the red-eared slider turtle (Trachemys scripta) mandible at stages equivalent to those at which odontogenesis is initiated in mice, a similar finding to previously reported results in chicks. To address whether the limited domains in the turtle and the chicken indicate an evolutionary molecular parallelism, or whether the domains simply constitute an ancestral phenotype, we assessed gene expression in a toothed reptile (the American alligator, Alligator mississippiensis) and a toothed non-placental mammal (the gray short-tailed opossum, Monodelphis domestica). We demonstrate that the Bmp4 domain is limited proximally in M. domestica and that the Fgf8 domain is limited distally in A. mississippiensis just preceding odontogenesis. Additionally, we show that Msx1 and Msx2 expression patterns in these species differ from those found in mice. Our data suggest that a limited Bmp4 domain does not necessarily correlate with edentulism, and reveal that the initiation of odontogenesis in non-murine amniotes is more complex than previously imagined. Our data also suggest a partially conserved odontogenic program in T. scripta, as indicated by conserved Pitx2, Pax9, and Barx1 expression patterns and by the presence of a Shh-expressing palatal epithelium, which we hypothesize may represent potential dental rudiments based on the Testudinata fossil record.

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