Pattern and polarity in the development and evolution of the gnathostome jaw: both conservation and heterotopy in the branchial arches of the shark, Scyliorhinus canicula.

The acquisition of jaws constitutes a landmark event in vertebrate evolution, one that in large part potentiated their success and diversification. Jaw development and patterning involves an intricate spatiotemporal series of reciprocal inductive and responsive interactions between the cephalic epithelia and the cranial neural crest (CNC) and cephalic mesodermal mesenchyme. The coordinated regulation of these interactions is critical for both the ontogenetic registration of the jaws and the evolutionary elaboration of variable jaw morphologies and designs. Current models of jaw development and evolution have been built on molecular and cellular evidence gathered mostly in amniotes such as mice, chicks and humans, and augmented by a much smaller body of work on the zebrafish. These have been partnered by essential work attempting to understand the origins of jaws that has focused on the jawless lamprey. Chondrichthyans (cartilaginous fish) are the most distant group to amniotes within extant gnathostomes, and comprise the crucial clade uniting amniotes and agnathans; yet despite their critical phylogenetic position, evidence of the molecular and cellular underpinnings of jaw development in chondrichthyans is still lacking. Recent advances in genome and molecular developmental biology of the lesser spotted dogfish shark, Scyliorhinus canicula, make it ideal for the molecular study of chondrichthyan jaw development. Here, following the 'Hinge and Caps' model of jaw development, we have investigated evidence of heterotopic (relative changes in position) and heterochronic (relative changes in timing) shifts in gene expression, relative to amniotes, in the jaw primordia of S. canicula embryos. We demonstrate the presence of clear proximo-distal polarity in gene expression patterns in the shark embryo, thus establishing a baseline molecular baüplan for branchial arch-derived jaw development and further validating the utility of the 'Hinge and Caps' model in comparative studies of jaw development and evolution. Moreover, we correlate gene expression patterns with the absence of a lambdoidal junction (formed where the maxillary first arch meets the frontonasal processes) in chondrichthyans, further highlighting the importance of this region for the development and evolution of jaw structure in advanced gnathostomes.

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.

Pax6 regulates craniofacial form through its control of an essential cephalic ectodermal patterning center.

Normal patterning and morphogenesis of the complex skeletal structures of the skull requires an exquisite, reciprocal cross-talk between the embryonic cephalic epithelia and mesenchyme. The mesenchyme associated with the jaws and the optic and olfactory capsules is derived from a Hox-negative cranial neural crest (CNC) population that acts much as an equivalence group in its interactions with specific local cephalic epithelial signals. Craniofacial pattern and morphogenesis is therefore controlled in large part through the regulation of these local cephalic epithelial signals. Here, we demonstrate that Pax6 is essential to the formation and maturation of the complex cephalic ectodermal patterning centers that govern the development and morphogenesis of the upper jaws and associated nasal capsules. Previous examinations of the craniofacial skeletal defects associated with Pax6 mutations have suggested that they arise from an optic-associated blockage in the migration of a specific subpopulation of midbrain CNC to the lateral frontonasal processes. We have addressed an alternative explanation for the craniofacial skeletal defects. We show that in Pax6(SeyN/SeyN) mutants regional CNC is present by E9.25 while there is already specific disruption in the early ontogenetic elaboration of cephalic ectodermal expression, associated with the nascent lambdoidal junction, of secreted signaling factors (including Fgf8 and Bmp4) and transcription factors (including Six1 and Dlx5) essential for upper jaw and/or nasal capsular development. Pax6 therefore regulates craniofacial form, at stages when CNC has just arrived in the frontonasal region, through its control of surface cephalic ectodermal competence to form an essential craniofacial patterning center.

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.

Programmed Cell Death Not as Sledgehammer but as Chisel: Apoptosis in Normal and Abnormal Craniofacial Patterning and Development.

Coordination of craniofacial development involves an complex, intricate, genetically controlled and tightly regulated spatiotemporal series of reciprocal inductive and responsive interactions among the embryonic cephalic epithelia (both endodermal and ectodermal) and the cephalic mesenchyme - particularly the cranial neural crest (CNC). The coordinated regulation of these interactions is critical both ontogenetically and evolutionarily, and the clinical importance and mechanistic sensitivity to perturbation of this developmental system is reflected by the fact that one-third of all human congenital malformations affect the head and face. Here, we focus on one element of this elaborate process, apoptotic cell death, and its role in normal and abnormal craniofacial development. We highlight four themes in the temporospatial elaboration of craniofacial apoptosis during development, namely its occurrence at (1) positions of epithelial-epithelial apposition, (2) within intra-epithelial morphogenesis, (3) during epithelial compartmentalization, and (4) with CNC metameric organization. Using the genetic perturbation of Satb2, Pbx1/2, Fgf8, and Foxg1 as exemplars, we examine the role of apoptosis in the elaboration of jaw modules, the evolution and elaboration of the lambdoidal junction, the developmental integration at the mandibular arch hinge, and the control of upper jaw identity, patterning and development. Lastly, we posit that apoptosis uniquely acts during craniofacial development to control patterning cues emanating from core organizing centres.

Tweaking the hinge and caps: testing a model of the organization of jaws.

Historically, examinations of gnathostome skulls have indicated that for essentially the entirety of their existence, jaws have been characterized by a high degree of fidelity to an initial basic structural design that will then go on to manifest an amazing array of end-point phenotypes. These two traits-bauplan fidelity and elaboration of design-are inter-connected and striking, and beg a number of questions, including: Are all jaws made in the same manner and if not how not? To begin to tackle such questions, we herein operationally define jaws as two appositional, hinged cranial units for which polarity and potential modularity are characteristics, and then address what is necessary for them to form, including delineating both the sources of cells and tissues that will formally yield the jaws as well as what informs their ontogeny (e.g., sources of positional information and factors directing the interpretation of developmental cues). Following on this, we briefly describe a predictive, testable model of jaw development (the "Hinge and Caps" model) and present evidence that the Satb2+cell population in the developing jaw primordia of mice defines a developmentally and evolutionarily significant jaw module such as would be predicted by the model.

Suture neontology and paleontology: the bases for where, when and how boundaries between bones have been established and have evolved.

Much of what has been written about sutures has either focused on the genetic and biologic etiologies of specific sutural development, maintenance, and pathogenesis or on the utilization of sutures as character states in vertebrate cladistic analyses. There is a much more modest literature explicitly concerned with the evolution of sutures. We provide a small bridge of these literatures by presenting a discussion of the evolutionary biologic bases for the patterns of where, when, and how sutural boundaries between skeletal and dental elements have been established and have evolved. As sutural boundaries do not exist in the absence of the nucleation events that initiate the generation of skeletal elements, we explore historic models seeking to identify the inductive events dictating the specific times and places where a cranial skeletal element forms, the elaboration of its sutural boundaries, and the mechanisms whereby subsequent phyletic changes may be manifested and recognized.

21st century neontology and the comparative development of the vertebrate skull.

Classic neontology (comparative embryology and anatomy), through the application of the concept of homology, has demonstrated that the development of the gnathostome (jawed vertebrate) skull is characterized both by a fidelity to the gnathostome bauplan and the exquisite elaboration of final structural design. Just as homology is an old concept amended for modern purposes, so are many of the questions regarding the development of the skull. With due deference to Geoffroy-St. Hilaire, Cuvier, Owen, Lankester et al., we are still asking: How are bauplan fidelity and elaboration of design maintained, coordinated, and modified to generate the amazing diversity seen in cranial morphologies? What establishes and maintains pattern in the skull? Are there universal developmental mechanisms underlying gnathostome autapomorphic structural traits? Can we detect and identify the etiologies of heterotopic (change in the topology of a developmental event), heterochronic (change in the timing of a developmental event), and heterofacient (change in the active capacetence, or the elaboration of capacity, of a developmental event) changes in craniofacial development within and between taxa? To address whether jaws are all made in a like manner (and if not, then how not), one needs a starting point for the sake of comparison. To this end, we present here a "hinge and caps" model that places the articulation, and subsequently the polarity and modularity, of the upper and lower jaws in the context of cranial neural crest competence to respond to positionally located epithelial signals. This model expands on an evolving model of polarity within the mandibular arch and seeks to explain a developmental patterning system that apparently keeps gnathostome jaws in functional registration yet tractable to potential changes in functional demands over time. It relies upon a system for the establishment of positional information where pattern and placement of the "hinge" is driven by factors common to the junction of the maxillary and mandibular branches of the first arch and of the "caps" by the signals emanating from the distal-most first arch midline and the lamboidal junction (where the maxillary branch meets the frontonasal processes). In this particular model, the functional registration of jaws is achieved by the integration of "hinge" and "caps" signaling, with the "caps" sharing at some critical level a developmental history that potentiates their own coordination. We examine the evidential foundation for this model in mice, examine the robustness with which it can be applied to other taxa, and examine potential proximate sources of the signaling centers. Lastly, as developmental biologists have long held that the anterior-most mesendoderm (anterior archenteron roof or prechordal plate) is in some way integral to the normal formation of the head, including the cranial skeletal midlines, we review evidence that the seminal patterning influences on the early anterior ectoderm extend well beyond the neural plate and are just as important to establishing pattern within the cephalic ectoderm, in particular for the "caps" that will yield medial signaling centers known to coordinate jaw development.

Satb2 haploinsufficiency phenocopies 2q32-q33 deletions, whereas loss suggests a fundamental role in the coordination of jaw development.

The recent identification of SATB2 as a candidate gene responsible for the craniofacial dysmorphologies associated with deletions and translocations at 2q32-q33, one of only three regions of the genome for which haploinsufficiency has been significantly associated with isolated cleft palate, led us to investigate the in vivo functions of murine Satb2. We find that, similar to the way in which SATB2 is perceived to act in humans, craniofacial defects due to haploinsufficiency of Satb2, including cleft palate (in approximately 25% of cases), phenocopy those seen with 2q32-q33 deletions and translocations in humans. Full functional loss of Satb2 results in amplification of these defects and leads both to increased apoptosis in the craniofacial mesenchyme where Satb2 is usually expressed and to changes in the pattern of expression of three genes implicated in the regulation of craniofacial development in humans and mice: Pax9, Alx4, and Msx1. The Satb2-dosage sensitivity in craniofacial development is conspicuous--along with its control of cell survival, pattern of expression, and reversible functional modification by SUMOylation, it suggests that Satb2/SATB2 function in craniofacial development may prove to be more profound than has been anticipated previously. Because jaw development is Satb2-dosage sensitive, the regulators of Satb2 expression and posttranslational modification become of critical importance both ontogenetically and evolutionarily, especially since such regulators plausibly play undetected roles in jaw and palate development and in the etiology of craniofacial malformations.

Reassessing the Dlx code: the genetic regulation of branchial arch skeletal pattern and development.

The branchial arches are meristic vertebrate structures, being metameric both between each other within the rostrocaudal series along the ventrocephalic surface of the embryonic head and within each individual arch: thus, just as each branchial arch must acquire a unique identity along the rostrocaudal axis, each structure within the proximodistal axis of an arch must also acquire a unique identity. It is believed that regional specification of metameric structures is controlled by the nested expression of related genes resulting in a regional code, a principal that is though to be demonstrated by the regulation of rostrocaudal axis development in animals exerted by the nested HOM-C/Hox homeobox genes. The nested expression pattern of the Dlx genes within the murine branchial arch ectomesenchyme has more recently led to the proposal of a Dlx code for the regional specification along the proximodistal axis of the branchial arches (i.e. it establishes intra-arch identity). This review re-examines this hypothesis, and presents new work on an allelic series of Dlx loss-of-function mouse mutants that includes various combinations of Dlx1, Dlx2, Dlx3, Dlx5 and Dlx6. Although we confirm fundamental aspects of the hypothesis, we further report a number of novel findings. First, contrary to initial reports, Dlx1, Dlx2 and Dlx1/2 heterozygotes exhibit alterations of branchial arch structures and Dlx2-/- and Dlx1/2-/- mutants have slight alterations of structures derived from the distal portions of their branchial arches. Second, we present evidence for a role for murine Dlx3 in the development of the branchial arches. Third, analysis of compound Dlx mutants reveals four grades of mandibular arch transformations and that the genetic interactions of cis first-order (e.g. Dlx5 and Dlx6), trans second-order (e.g. Dlx5 and Dlx2) and trans third-order paralogues (e.g. Dlx5 and Dlx1) result in significant and distinct morphological differences in mandibular arch development. We conclude by integrating functions of the Dlx genes within the context of a hypothesized general mechanism for the establishment of pattern and polarity in the first branchial arch of gnathostomes that includes regionally secreted growth factors such as Fgf8 and Bmp and other transcription factors such as Msx1, and is consistent both with the structure of the conserved gnathostome jaw bauplan and the elaboration of this bauplan to meet organismal end-point designs.

Specification of jaw subdivisions by Dlx genes.

The success of vertebrates was due in part to the acquisition and modification of jaws. Jaws are principally derived from the branchial arches, embryonic structures that exhibit proximodistal polarity. To investigate the mechanisms that specify the identity of skeletal elements within the arches, we examined mice lacking expression of Dlx5 and Dlx6, linked homeobox genes expressed distally but not proximally within the arches. Dlx5/6-/- mutants exhibit a homeotic transformation of lower jaws to upper jaws. We suggest that nested Dlx expression in the arches patterns their proximodistal axes. Evolutionary acquisition and subsequent refinement of jaws may have been dependent on modification of Dlx expression.