Jennifer L. Fish

Satb2, modularity, and the evolvability of the vertebrate jaw

Modularity is a key mechanism bridging development and evolution and is fundamental to evolvability. Herein, we investigate modularity of the Vertebrate jaw with the aim of understanding mechanisms of its morphological evolution. Conservation of the basic structural bauplan of Vertebrate jaws led to a Hinge and Caps model, in which polarity in the patterning system of developing jaws predicts modularity. We have tested the hypothesis that the Satb2+ cell population delineates a developmental module within the mandibular jaw. Satb2 is expressed in the mesenchyme of the jaw primordia that gives rise to distal elements of both the upper and lower jaws. Loss of Satb2 specifically affects structural elements of the distal (incisor) domain, reflecting the integration of these elements as well as their independence from other mandibular domains. Reducing Satb2 dosage leads to an increase in variation in mandibular length, providing insight into the developmental potential to generate variation. Inter‐taxa comparisons reveal that the Satb2 domain is conserved within gnathostomes. We complement previous loss of function studies in mice with gene knock‐down experiments in Xenopus, providing evidence for functional conservation of Satb2 in regulating size. Finally, we present evidence that the relative size of the amniote mandibular Satb2+ domain varies in relation to epithelial Fgf8 expression, suggesting a mechanism for evolutionary change in this domain. Taken together, our data support the Hinge and Caps model and provide evidence that Satb2 regulates coordinated distal jaw modules that are subject to evolutionary modification by signals emanating from the Hinge.

Fgf8 dosage determines midfacial integration and polarity within the nasal and optic capsules

Craniofacial development requires an exquisitely timed and positioned cross-talk between the embryonic cephalic epithelia and mesenchyme. This cross-talk underlies the precise translation of patterning processes and information into distinct, appropriate skeletal morphologies. The molecular and cellular dialogue includes communication via secreted signaling molecules, including Fgf8, and effectors of their interpretation. Herein, we use genetic attenuation of Fgf8 in mice and perform gain-of-function mouse-chick chimeric experiments to demonstrate that significant character states of the frontonasal and optic skeletons are dependent on Fgf8. Notably, we show that the normal orientation and polarity of the nasal capsules and their developing primordia are dependent on Fgf8. We further demonstrate that Fgf8 is required for midfacial integration, and provide evidence for a role for Fgf8 in optic capsular development. Taken together, our data highlight Fgf8 signaling in craniofacial development as a plausible target for evolutionary selective pressures.

Multiple developmental mechanisms regulate species-specific jaw size.

Variation in jaw size during evolution has been crucial for the adaptive radiation of vertebrates, yet variation in jaw size during development is often associated with disease. To test the hypothesis that early developmental events regulating neural crest (NC) progenitors contribute to species-specific differences in size, we investigated mechanisms through which two avian species, duck and quail, achieve their remarkably different jaw size. At early stages, duck exhibit an anterior shift in brain regionalization yielding a shorter, broader, midbrain. We find no significant difference in the total number of pre-migratory NC; however, duck concentrate their pre-migratory NC in the midbrain, which contributes to an increase in size of the post-migratory NC population allocated to the mandibular arch. Subsequent differences in proliferation lead to a progressive increase in size of the duck mandibular arch relative to that of quail. To test the role of pre-migratory NC progenitor number in regulating jaw size, we reduced and augmented NC progenitors. In contrast to previous reports of regeneration by NC precursors, we find that neural fold extirpation results in a loss of NC precursors. Despite this reduction in their numbers, post-migratory NC progenitors compensate, producing a symmetric and normal-sized jaw. Our results suggest that evolutionary modification of multiple aspects of NC cell biology, including NC allocation within the jaw primordia and NC-mediated proliferation, have been important to the evolution of jaw size. Furthermore, our finding of NC post-migratory compensatory mechanisms potentially extends the developmental time frame for treatments of disease or injury associated with NC progenitor loss.

miR-302 Is Required for Timing of Neural Differentiation, Neural Tube Closure, and Embryonic Viability

SUMMARY The evolutionarily conserved miR-302 family of microRNAs is expressed during early mammalian embryonic development. Here, we report that deletion of miR-302a-d in mice results in a fully penetrant late embryonic lethal phenotype. Knockout embryos have an anterior neural tube closure defect associated with a thickened neuroepithelium. The neuroepithelium shows increased progenitor proliferation, decreased cell death, and precocious neuronal differentiation. mRNA profiling at multiple time points during neurulation uncovers a complex pattern of changing targets over time. Overexpression of one of these targets, Fgf15, in the neuroepithelium of the chick embryo induces precocious neuronal differentiation. Compound mutants between mir-302 and the related mir-290 locus have a synthetic lethal phenotype prior to neurulation. Our results show that mir-302 helps regulate neurulation by suppressing neural progenitor expansion and precocious differentiation. Furthermore, these results uncover redundant roles for mir-290 and mir-302 early in development.

Tfap2a-dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

Failure of facial prominence fusion causes cleft lip and palate (CL/P), a common human birth defect. Several potential mechanisms can be envisioned that would result in CL/P, including failure of prominence growth and/or alignment as well as a failure of fusion of the juxtaposed epithelial seams. Here, using geometric morphometrics, we analyzed facial outgrowth and shape change over time in a novel mouse model exhibiting fully penetrant bilateral CL/P. This robust model is based upon mutations in Tfap2a, the gene encoding transcription factor AP-2α, which has been implicated in both syndromic and non-syndromic human CL/P. Our findings indicate that aberrant morphology and subsequent misalignment of the facial prominences underlies the inability of the mutant prominences to fuse. Exencephaly also occured in some of the Tfap2a mutants and we observed additional morphometric differences that indicate an influence of neural tube closure defects on facial shape. Molecular analysis of the CL/P model indicates that Fgf signaling is misregulated in the face, and that reducing Fgf8 gene dosage can attenuate the clefting pathology by generating compensatory changes. Furthermore, mutations in either Tfap2a or Fgf8 increase variance in facial shape, but the combination of these mutations restores variance to normal levels. The alterations in variance provide a potential mechanistic link between clefting and the evolution and diversity of facial morphology. Overall, our findings suggest that CL/P can result from small gene-expression changes that alter the shape of the facial prominences and uncouple their coordinated morphogenesis, which is necessary for normal fusion.

Further supporting evidence for the SATB2‐associated syndrome found through whole exome sequencing

The SATB2‐associated syndrome (SAS) was recently proposed as a clinically recognizable syndrome that results from deleterious alterations of the SATB2 gene in humans. Although interstitial deletions at 2q33 encompassing SATB2, either alone or contiguously with other genes, have been reported before, there is limited literature regarding intragenic mutations of this gene and the resulting phenotype. We describe five patients in whom whole exome sequencing identified five unique de novo mutations in the SATB2 gene (one splice site, one frameshift, and three nonsense mutations). The five patients had overlapping features that support the characteristic features of the SAS: intellectual disability with limited speech development and craniofacial abnormalities including cleft palate, dysmorphic features, and dental abnormalities. Furthermore, Patient 1 also had features not previously described that represent an expansion of the phenotype. Osteopenia was seen in two of the patients, suggesting that this finding could be added to the list of distinctive findings. We provide supporting evidence that analysis for deletions or point mutations in SATB2 should be considered in children with intellectual disability and severely impaired speech, cleft or high palate, teeth abnormalities, and osteopenia.

Selection, Morphological Integration, and Strepsirrhine Locomotor Adaptations

Clades with taxa that have multiple locomotor adaptations represent a direct way to test the relationship between adaptation and integration. If integration is influenced by functional requirements, integration should be most apparent where selection is strongest and less evident where selection has been relaxed. If integration is primarily regulated by genetic constraints, integration should be present irrespective of selection pressures. Here we use patterns of integration in the strepsirrhine fore- and hind limbs as a test case. Strepsirrhine locomotion is relatively well-studied, and there are multiple clades that share different locomotor modes. We found that quadrupeds have greater limb integration than vertical leapers. These results suggest that variation can be expressed if selection for integration is relaxed. However, an unexpected pattern was revealed, in which there appears to be some broader regulatory mechanism controlling overall limb integration. Our tests identified a strong correlation between integration of the forelimb and integration of the hind limb. This broader mechanism may be evidence of the primitive genetic control of limb integration.

SATB2‐associated syndrome: Mechanisms, phenotype, and practical recommendations

The SATB2‐associated syndrome is a recently described syndrome characterized by developmental delay/intellectual disability with absent or limited speech development, craniofacial abnormalities, behavioral problems, dysmorphic features, and palatal and dental abnormalities. Alterations of the SATB2 gene can result from a variety of different mechanisms that include contiguous deletions, intragenic deletions and duplications, translocations with secondary gene disruption, and point mutations. The multisystemic nature of this syndrome demands a multisystemic approach and we propose evaluation and management guidelines. The SATB2‐associated syndrome registry has now been started and that will allow gathering further clinical information and refining the provided surveillance recommendations.

Craniofacial modularity, character analysis, and the evolution of the premaxilla in early African hominins

Phylogenetic analyses require evolutionarily independent characters, but there is no consensus, nor has there been a clear methodology presented on how to define character independence in a phylogenetic context, particularly within a complex morphological structure such as the skull. Following from studies of craniofacial development, we hypothesize that the premaxilla is an independent evolutionary module with two integrated characters that have traditionally been treated as independent. We test this hypothesis on a large sample of primate skulls and find evidence supporting the premaxilla as an independent module within the larger module of the palate. Additionally, our data indicate that the convexity of the nasoalveolar clivus and the contour of the alveolus are integrated within the premaxilla. We show that the palate itself is composed of two distinct modules: the FNP-derived premaxillae and the mxBA1-derived maxillae and palatines. Application of our data to early African hominin facial morphology suggests that at least three separate transitions contributed to robust facial morphology: 1) an increase in the size of the post-canine dentition housed within the maxillae and palatines, 2) modification of the premaxilla generating a concave clivus and reduced incisor alveolus, and 3) modification of the zygomatic, shifting the zygomatic root and lateral face anteriorly. These data lend support to the monophyly of Paranthropus boisei and Paranthropus robustus, and provide mounting evidence in favor of a Paranthropus clade. This study also highlights the utility of applying developmental evidence to studies of morphological evolution.

Neural Crest-Mediated Tissue Interactions During Craniofacial Development: The Origins of Species-Specific Pattern

The vertebrate craniofacial complex is both highly conserved in its general anatomical organization and remarkably diversified in its size and shape. Individual components within the craniofacial complex can change rapidly over time while maintaining indispensable internal relationships that are necessary to meet functional demands. This chapter discusses molecular and cellular regulation of species-specific pattern in the craniofacial complex. We explore how developmental programs can be modified internally to provide the variation essential for evolution while simultaneously sustaining those signaling interactions that facilitate structural and functional integration. We review studies involving a broad range of model and non-model organisms and provide a synthesis of mechanisms that pattern the craniofacial complex across vertebrates. We also describe experiments in quail and duck embryos that demonstrate the importance of the neural crest in generating species-specific pattern in the craniofacial skeleton, musculature, and integument and which reveal those neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate size and shape.