Morphological diversity of integumentary traits in fowl domestication: Insights from disparity analysis and embryonic development.

The domestication of the fowl resulted in a large diversity of integumental structures in chicken breeds. Several integumental traits have been investigated from a developmental genetics perspective. However, their distribution among breeds and their developmental morphology remain unexplored. We constructed a discrete trait-breed matrix and conducted a disparity analysis to investigate the variation of these structures in chicken breeds; 20 integumental traits of 72 chicken breeds and the red junglefowl were assessed. The analyses resulted in slight groupings of breed types comparable to standard breed classification based on artificial selection and chicken type use. The red junglefowl groups together with bantams and European breeds. We provide new data on the red junglefowl and four chicken breeds, demonstrating where and when variation arises during embryonic development. We document variation in developmental timing of the egg tooth and feather formation, as well as other kinds of developmental patterning as in the anlagen of different type of combs. Changes in epithelial-mesenchymal signaling interactions may drive the highly diverse integument in chickens. Experimental and comparative work has revealed that the cranial neural crest mesenchyme mediates its interactions with the overlying epithelium and is the likely source of patterning that generates diversity in integumental structures.

FGF and TGFß signaling link form and function during jaw development and evolution.

How does form arise during development and change during evolution? How does form relate to function, and what enables embryonic structures 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. In connection with their specialized mode of feeding, duck develop a 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 the differential regulation of FGF and TGFß signaling. First, we perform transplants between chick and duck embryos and demonstrate that the ability of neural crest mesenchyme (NCM) to direct the species-specific insertion of muscle and the formation of secondary cartilage depends upon the amount and spatial distribution of NCM-derived connective tissues. Second, we quantify motility and build finite element models of the jaw complex in duck and quail, which reveals a link between species-specific jaw architecture and the predicted mechanical force environment. Third, we investigate the extent to which mechanical load mediates FGF and TGFß signaling in the duck jaw adductor insertion, and discover that both pathways are mechano-responsive and required for secondary cartilage formation. Additionally, we find that FGF and TGFß signaling can also induce secondary cartilage in the absence of mechanical force or in the adductor insertion of quail embryos. Thus, our results provide novel insights on molecular, cellular, and biomechanical mechanisms that couple musculoskeletal form and function during development and evolution.

The Society for Craniofacial Genetics and Developmental Biology 41st Annual Meeting.

The mission of the Society for Craniofacial Genetics and Developmental Biology (SCGDB) is to promote education, research, and communication about normal and abnormal development of the tissues and organs of the head. The SCGDB welcomes as members undergraduate students, graduate students, postdoctoral researchers, medical and dental practitioners, scientists, and academicians who possess an interest in craniofacial biology. Each year our members come together to share their novel findings, build upon, and challenge current knowledge of craniofacial biology.

Neural crest and the origin of species-specific pattern.

For well over half of the 150 years since the discovery of the neural crest, the special ability of these cells to function as a source of species-specific pattern has been clearly recognized. Initially, this observation arose in association with chimeric transplant experiments among differentially pigmented amphibians, where the neural crest origin for melanocytes had been duly noted. Shortly thereafter, the role of cranial neural crest cells in transmitting species-specific information on size and shape to the pharyngeal arch skeleton as well as in regulating the timing of its differentiation became readily apparent. Since then, what has emerged is a deeper understanding of how the neural crest accomplishes such a presumably difficult mission, and this includes a more complete picture of the molecular and cellular programs whereby neural crest shapes the face of each species. This review covers studies on a broad range of vertebrates and describes neural-crest-mediated mechanisms that endow the craniofacial complex with species-specific pattern. A major focus is on experiments in quail and duck embryos that reveal a hierarchy of cell-autonomous and non-autonomous signaling interactions through which neural crest generates species-specific pattern in the craniofacial integument, skeleton, and musculature. By controlling size and shape throughout the development of these systems, the neural crest underlies the structural and functional integration of the craniofacial complex during evolution.

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.

Embryonic bauplans and the developmental origins of facial diversity and constraint.

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.

Neural crest-mediated bone resorption is a determinant of species-specific jaw length.

Precise control of jaw length during development is crucial for proper form and function. Previously we have shown that in birds, neural crest mesenchyme (NCM) confers species-specific size and shape to the beak by regulating molecular and histological programs for the induction and deposition of cartilage and bone. Here we reveal that a hitherto unrecognized but similarly essential mechanism for establishing jaw length is the ability of NCM to mediate bone resorption. Osteoclasts are considered the predominant cells that resorb bone, although osteocytes have also been shown to participate in this process. In adults, bone resorption is tightly coupled to bone deposition as a means to maintain skeletal homeostasis. Yet, the role and regulation of bone resorption during growth of the embryonic skeleton have remained relatively unexplored. We compare jaw development in short-beaked quail versus long-billed duck and find that quail have substantially higher levels of enzymes expressed by bone-resorbing cells including tartrate-resistant acid phosphatase (TRAP), Matrix metalloproteinase 13 (Mmp13), and Mmp9. Then, we transplant NCM destined to form the jaw skeleton from quail to duck and generate chimeras in which osteocytes arise from quail donor NCM and osteoclasts come exclusively from the duck host. Chimeras develop quail-like jaw skeletons coincident with dramatically elevated expression of TRAP, Mmp13, and Mmp9. To test for a link between bone resorption and jaw length, we block resorption using a bisphosphonate, osteoprotegerin protein, or an MMP13 inhibitor, and this significantly lengthens the jaw. Conversely, activating resorption with RANKL protein shortens the jaw. Finally, we find that higher resorption in quail presages their relatively lower adult jaw bone mineral density (BMD) and that BMD is also NCM-mediated. Thus, our experiments suggest that NCM not only controls bone resorption by its own derivatives but also modulates the activity of mesoderm-derived osteoclasts, and in so doing enlists bone resorption as a key patterning mechanism underlying the functional morphology and evolution of the jaw.

A simple PCR-based strategy for estimating species-specific contributions in chimeras and xenografts.

Many tissue-engineering approaches for repair and regeneration involve transplants between species. Yet a challenge is distinguishing donor versus host effects on gene expression. This study provides a simple molecular strategy to quantify species-specific contributions in chimeras and xenografts. Species-specific primers for reverse transcription quantitative real-time PCR (RT-qPCR) were designed by identifying silent mutations in quail, duck, chicken, mouse and human ribosomal protein L19 (RPL19). cDNA from different pairs of species was mixed in a dilution series and species-specific RPL19 primers were used to generate standard curves. Then quail cells were transplanted into transgenic-GFP chick and resulting chimeras were analyzed with species-specific primers. Fluorescence-activated cell sorting (FACS) confirmed that donor- and host-specific levels of RPL19 expression represent actual proportions of cells. To apply the RPL19 strategy, we measured Runx2 expression in quail-duck chimeras. Elevated Runx2 levels correlated with higher percentages of donor cells. Finally, RPL19 primers also discriminated mouse from human and chick. Thus, this strategy enables chimeras and/or xenografts to be screened rapidly at the molecular level.

Divergence of craniofacial developmental trajectories among avian embryos.

BACKGROUND: Morphological divergence among related species involves changes to developmental processes. When such variation arises in development has garnered considerable theoretical interest relating to the broader issue of how development may constrain evolutionary change. The hourglass model holds that while early developmental events may be highly evolvable, there is a phylotypic stage when key developmental events are conserved. Thus, evolutionary divergence among related species should tend to arise after such a stage of reduced evolvability and, consequently, reduced variation among species. We test this prediction by comparing developmental trajectories among three avian species of varying relatedness (chick, quail, and duck) to locate their putative point of divergence. Three-dimensional geometric morphometrics and trajectory analyses were used to measure the significance of the facial shape variation observed among these species. RESULTS: Duck embryos, being more distantly related, differed from the more closely-related chick and quail embryos in the enlargement of their frontonasal prominences. Phenotypic trajectory analyses demonstrated divergence of the three species, most notably, duck. CONCLUSIONS: The results demonstrate that the two more closely related species share similar facial morphologies for a longer time during development, while ducks diverge. This suggests a surprising lability of craniofacial development during early face formation. Developmental Dynamics 244:1158-1167, 2015. © 2015 Wiley Periodicals, Inc.

Evolution of a developmental mechanism: Species-specific regulation of the cell cycle and the timing of events during craniofacial osteogenesis.

Neural crest mesenchyme (NCM) controls species-specific pattern in the craniofacial skeleton but how this cell population accomplishes such a complex task remains unclear. To elucidate mechanisms through which NCM directs skeletal development and evolution, we made chimeras from quail and duck embryos, which differ markedly in their craniofacial morphology and maturation rates. We show that quail NCM, when transplanted into duck, maintains its faster timetable for development and autonomously executes molecular and cellular programs for the induction, differentiation, and mineralization of bone, including premature expression of osteogenic genes such as Runx2 and Col1a1. In contrast, the duck host systemic environment appears to be relatively permissive and supports osteogenesis independently by providing circulating minerals and a vascular network. Further experiments reveal that NCM establishes the timing of osteogenesis by regulating cell cycle progression in a stage- and species-specific manner. Altering the time-course of D-type cyclin expression mimics chimeras by accelerating expression of Runx2 and Col1a1. We also discover higher endogenous expression of Runx2 in quail coincident with their smaller craniofacial skeletons, and by prematurely over-expressing Runx2 in chick embryos we reduce the overall size of the craniofacial skeleton. Thus, our work indicates that NCM establishes species-specific size in the craniofacial skeleton by controlling cell cycle, Runx2 expression, and the timing of key events during osteogenesis.

Molecular and cellular changes associated with the evolution of novel jaw muscles in parrots.

Vertebrates have achieved great evolutionary success due in large part to the anatomical diversification of their jaw complex, which allows them to inhabit almost every ecological niche. While many studies have focused on mechanisms that pattern the jaw skeleton, much remains to be understood about the origins of novelty and diversity in the closely associated musculature. To address this issue, we focused on parrots, which have acquired two anatomically unique jaw muscles: the ethmomandibular and the pseudomasseter. In parrot embryos, we observe distinct and highly derived expression patterns for Scx, Bmp4, Tgfß2 and Six2 in neural crest-derived mesenchyme destined to form jaw muscle connective tissues. Furthermore, immunohistochemical analysis reveals that cell proliferation is more active in the cells within the jaw muscle than in surrounding connective tissue cells. This biased and differentially regulated mode of cell proliferation in cranial musculoskeletal tissues may allow these unusual jaw muscles to extend towards their new attachment sites. We conclude that the alteration of neural crest-derived connective tissue distribution during development may underlie the spatial changes in jaw musculoskeletal architecture found only in parrots. Thus, parrots provide valuable insights into molecular and cellular mechanisms that may generate evolutionary novelties with functionally adaptive significance.

Crossing fibers may underlie the dynamic pulling forces of muscles that attach to cartilage at the tip of the nose.

The present study used microdissection, histology, and microcomputed tomography (micro-CT) with the aims of determining the prevalence and patterns of the depressor septi nasi (DSN) and orbicularis oris (OOr) muscles attached to the footplate of the medial crus (fMC) of the major alar cartilage, focusing on their crossing fibers. The DSN and OOr attached to the fMC of the major alar cartilage were investigated in 76 samples from 38 embalmed Korean adult cadavers (20 males, 18 females; mean age 70 years). The DSN, OOr, or both were attached to the fMC. When the DSN ran unilaterally or was absent, some OOr fibers ascended to attach to the fMC instead of the DSN in 20.6% of the samples. Crossing fibers of the DSN or OOr attached to the fMC were found in 82.4% of the samples. Bilateral and unilateral crossing fibers were found in 32.4% and 50.0%, respectively, and no crossing fibers were found in 17.6%. The DSN and OOr that attached to the fMC could be categorized into six types according to presence of the DSN and the crossing patterns of the DSN and OOr. Anatomical findings of the DSN and OOr that attached to the fMC were confirmed in histology and micro-CT images. These findings offer insights on anatomical mechanisms that may underlie the dynamic pulling forces generated by muscles that attach to the fMCs and on evolutionary variation observed in human facial expressions. They can also provide useful information for guiding rhinoplasty of the nasal tip.

Mesenchymal and mechanical mechanisms of secondary cartilage induction.

Secondary cartilage occurs at articulations, sutures, and muscle attachments, and facilitates proper kinetic movement of the skeleton. Secondary cartilage requires mechanical stimulation for its induction and maintenance, and accordingly, its evolutionary presence or absence reflects species-specific variation in functional anatomy. Avians illustrate this point well. In conjunction with their distinct adult mode of feeding via levered straining, duck develop a pronounced secondary cartilage at the insertion (i.e., enthesis) of the mandibular adductor muscles on the lower jaw skeleton. An equivalent cartilage is absent in quail, which peck at their food. We hypothesized that species-specific pattern and a concomitant dissimilarity in the local mechanical environment promote secondary chondrogenesis in the mandibular adductor enthesis of duck versus quail. To test our hypothesis we employed two experimental approaches. First, we transplanted neural crest mesenchyme (NCM) from quail into duck, which produced chimeric "quck" with a jaw complex resembling that of quail, including an absence of enthesis secondary cartilage. Second, we modified the mechanical environment in embryonic duck by paralyzing skeletal muscles, and by blocking the ability of NCM to support mechanotransduction through stretch-activated ion channels. Paralysis inhibited secondary cartilage, as evidenced by changes in histology and expression of genes that affect chondrogenesis, including members of the FGF and BMP pathways. Ion channel inhibition did not alter enthesis secondary cartilage but caused bone to form in place of secondary cartilage at articulations. Thus, our study reveals that enthesis secondary cartilage forms through mechanisms that are distinct from those regulating other secondary cartilage. We conclude that by directing the musculoskeletal patterning and integration of the jaw complex, NCM modulates the mechanical forces and molecular signals necessary to control secondary cartilage formation during development and evolution.

Structured coculture of mesenchymal stem cells and disc cells enhances differentiation and proliferation.

PURPOSE: During in vivo stem cell differentiation, mature cells often induce the differentiation of nearby stem cells. Accordingly, prior studies indicate that a randomly mixed coculture can help transform mesenchymal stem cells (MSC) into nucleus pulposus cells (NPC). However, because in vivo signaling typically occurs heterotopically between adjacent cell layers, we hypothesized that a structurally organized coculture between MSC and NPC will result in greater cell differentiation and proliferation over single cell-type controls and cocultures with random organization. METHODS: We developed a novel bilaminar cell pellet (BCP) system where a sphere of MSC is enclosed in a shell of NPC by successive centrifugation. Controls were made using single cell-type pellets and coculture pellets with random organization. The pellets were evaluated for DNA content, gene expression, and histology. RESULTS: A bilaminar 3D organization enhanced cell proliferation and differentiation. BCP showed significantly more cell proliferation than pellets with one cell type and those with random organization. Enhanced differentiation of MSC within the BCP pellet relative to single cell-type pellets was demonstrated by quantitative RT-PCR, histology, and in situ hybridization. CONCLUSIONS: The BCP culture system increases MSC proliferation and differentiation as compared to single cell type or randomly mixed coculture controls.

Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-ß and Runx2 in bone is required for hearing.

Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-ß (TGFß)-responsive pathway that controls osteoblast differentiation. Deregulated TGFß or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/⁻ mice, inhibition of TGFß signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.

Anatomical connections among the depressor supercilii, levator labii superioris alaeque nasi, and inferior fibers of orbicularis oculi: Implications for variation in human facial expressions.

The aim of this study was to determine how the depressor supercilii (DS) connects to the levator labii superioris alaeque nasi (LLSAN) and inferior fibers of the orbicularis oculi (OOc INF) in the human midface. While grimacing, contraction of the DS with fibers connecting to the LLSAN and OOc INF can assist in pulling the medial eyebrow downward more than when these connecting fibers are not present. Contraction of these distinct connecting fibers between the DS and the LLSAN can also slightly elevate the nasal ala and upper lip. The DS was examined in 44 specimens of embalmed adult Korean cadavers. We found that the DS connected to the LLSAN or the OOc INF by muscle fibers or thin aponeuroses in 33 (75.0%) of the 44 specimens. The DS was connected to both the LLSAN and OOc INF by muscle fibers or aponeuroses and had no connection to either in 5 (11.4%) and 11 (25.0%) specimens, respectively. The DS was connected to the LLSAN by the muscle fibers and thin aponeuroses in 6 (13.6%) and 4 (9.1%) specimens, respectively. The DS was connected to the OOc INF by the muscle fibers and thin aponeuroses in 5 (11.4%) and 23 (52.3%) specimens, respectively. Our findings regarding the anatomical connections of the glabellar region DS to the midface LLSAN and OOc INF provide insights on the dynamic balance between the brow depressors such as the DS and brow-elevating muscle and contribute to understanding the anatomical origins of individual variation in facial expressions. These results can also improve the safety, predictability, and aesthetics of treatments for the glabellar region with botulinum toxin type A and can be helpful when performing electromyography.

Species-specific sensitivity to TGFß signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution.

Precise developmental control of jaw length is critical for survival, but underlying molecular mechanisms remain poorly understood. The jaw skeleton arises from neural crest mesenchyme (NCM), and we previously demonstrated that these progenitor cells express more bone-resorbing enzymes including Matrix metalloproteinase 13 (Mmp13) when they generate shorter jaws in quail embryos versus longer jaws in duck. Moreover, if we inhibit bone resorption or Mmp13, we can increase jaw length. In the current study, we uncover mechanisms establishing species-specific levels of Mmp13 and bone resorption. Quail show greater activation of and sensitivity to transforming growth factor beta (TGFß) signaling than duck; where intracellular mediators like SMADs and targets like Runt-related transcription factor 2 (Runx2), which bind Mmp13, become elevated. Inhibiting TGFß signaling decreases bone resorption, and overexpressing Mmp13 in NCM shortens the duck lower jaw. To elucidate the basis for this differential regulation, we examine the Mmp13 promoter. We discover a SMAD-binding element and single nucleotide polymorphisms (SNPs) near a RUNX2-binding element that distinguish quail from duck. Altering the SMAD site and switching the SNPs abolish TGFß sensitivity in the quail Mmp13 promoter but make the duck promoter responsive. Thus, differential regulation of TGFß signaling and Mmp13 promoter structure underlie avian jaw development and evolution.

Timing of ossification in duck, quail, and zebra finch: intraspecific variation, heterochronies, and life history evolution.

Skeletogenic heterochronies have gained much attention in comparative developmental biology. The temporal appearance of mineralized individual bones in a species - the species ossification sequence - is an excellent marker in this kind of study. Several publications describe interspecific variation, but only very few detail intraspecific variation. In this study, we describe and analyze the temporal order of ossification of skeletal elements in the zebra finch, Taeniopygia guttata, the Japanese quail, Coturnix coturnix japonica, and the White Pekin duck, a domestic race of the mallard Anas platyrhynchos, and explore patterns of intraspecific variation in these events. The overall sequences were found to be conserved. In the duck, variability is present in the relative timing of ossification in the occipital, the basisphenoid and the otic regions of the skull and the phalanges in the postcranium. This variation appears generally in close temporal proximity. Comparison with previously published data shows differences in ossification sequence in the skull, the feet, and the pelvis in the duck, and especially the pelvis in the quail. This clearly documents variability among different breeds.

Developmental origins of species-specific muscle pattern.

Vertebrate jaw muscle anatomy is conspicuously diverse but developmental processes that generate such variation remain relatively obscure. To identify mechanisms that produce species-specific jaw muscle pattern we conducted transplant experiments using Japanese quail and White Pekin duck, which exhibit considerably different jaw morphologies in association with their particular modes of feeding. Previous work indicates that cranial muscle formation requires interactions with adjacent skeletal and muscular connective tissues, which arise from neural crest mesenchyme. We transplanted neural crest mesenchyme from quail to duck embryos, to test if quail donor-derived skeletal and muscular connective tissues could confer species-specific identity to duck host jaw muscles. Our results show that duck host jaw muscles acquire quail-like shape and attachment sites due to the presence of quail donor neural crest-derived skeletal and muscular connective tissues. Further, we find that these species-specific transformations are preceded by spatiotemporal changes in expression of genes within skeletal and muscular connective tissues including Sox9, Runx2, Scx, and Tcf4, but not by alterations to histogenic or molecular programs underlying muscle differentiation or specification. Thus, neural crest mesenchyme plays an essential role in generating species-specific jaw muscle pattern and in promoting structural and functional integration of the musculoskeletal system during evolution.

Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development.

Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.