Retinoic Acid Deficiency Underlies the Etiology of Midfacial Defects.

Embryonic craniofacial development depends on the coordinated outgrowth and fusion of multiple facial primordia, which are populated with cranial neural crest cells and covered by the facial ectoderm. Any disturbance in these developmental events, their progenitor tissues, or signaling pathways can result in craniofacial deformities such as orofacial clefts, which are among the most common birth defects in humans. In the present study, we show that Rdh10 loss of function leads to a substantial reduction in retinoic acid (RA) signaling in the developing frontonasal process during early embryogenesis, which results in a variety of craniofacial anomalies, including midfacial cleft and ectopic chondrogenic nodules. Elevated apoptosis and perturbed cell proliferation in postmigratory cranial neural crest cells and a substantial reduction in Alx1 and Alx3 transcription in the developing frontonasal process were associated with midfacial cleft in Rdh10-deficient mice. More important, expanded Shh signaling in the ventral forebrain, as well as partial abrogation of midfacial defects in Rdh10 mutants via inhibition of Hh signaling, indicates that misregulation of Shh signaling underlies the pathogenesis of reduced RA signaling-associated midfacial defects. Taken together, these data illustrate the precise spatiotemporal function of Rdh10 and RA signaling during early embryogenesis and their importance in orchestrating molecular and cellular events essential for normal midfacial development.

Med23 Regulates Sox9 Expression during Craniofacial Development.

The etiology and pathogenesis of craniofacial birth defects are multifactorial and include both genetic and environmental factors. Despite the identification of numerous genes associated with congenital craniofacial anomalies, our understanding of their etiology remains incomplete, and many affected individuals have an unknown genetic diagnosis. Here, we show that conditional loss of a Mediator complex subunit protein, Med23 in mouse neural crest cells (Med23fx/fx;Wnt1-Cre), results in micrognathia, glossoptosis, and cleft palate, mimicking the phenotype of Pierre Robin sequence. Sox9 messenger RNA and protein levels are both upregulated in neural crest cell-derived mesenchyme surrounding Meckel's cartilage and in the palatal shelves in Med23fx/fx;Wnt1-Cre mutant embryos compared to controls. Consistent with these observations, we demonstrate that Med23 binds to the promoter region of Sox9 and represses Sox9 expression in vitro. Interestingly, Sox9 binding to ß-catenin is enhanced in Med23fx/fx;Wnt1-Cre mutant embryos, which, together with downregulation of Col2a1 and Wnt signaling target genes, results in decreased proliferation and altered jaw skeletal differentiation and cleft palate. Altogether, our data support a cell-autonomous requirement for Med23 in neural crest cells, potentially linking the global transcription machinery through Med23 to the etiology and pathogenesis of craniofacial anomalies such as micrognathia and cleft palate.

Dynamic sex chromosomes in Old World chameleons (Squamata: Chamaeleonidae).

Much of our current state of knowledge concerning sex chromosome evolution is based on a handful of 'exceptional' taxa with heteromorphic sex chromosomes. However, classifying the sex chromosome systems of additional species lacking easily identifiable, heteromorphic sex chromosomes is indispensable if we wish to fully understand the genesis, degeneration and turnover of vertebrate sex chromosomes. Squamate reptiles (lizards and snakes) are a potential model clade for studying sex chromosome evolution as they exhibit a suite of sex-determining modes yet most species lack heteromorphic sex chromosomes. Only three (of 203) chameleon species have identified sex chromosome systems (all with female heterogamety, ZZ/ZW). This study uses a recently developed method to identify sex-specific genetic markers from restriction site-associated DNA sequence (RADseq) data, which enables the identification of sex chromosome systems in species lacking heteromorphic sex chromosomes. We used RADseq and subsequent PCR validation to identify an XX/XY sex chromosome system in the veiled chameleon (Chamaeleo calyptratus), revealing a novel transition in sex chromosome systems within the Chamaeleonidae. The sex-specific genetic markers identified here will be essential in research focused on sex-specific, comparative, functional and developmental evolutionary questions, further promoting C. calyptratus' utility as an emerging model organism.

Craniofacial malformations: intrinsic vs extrinsic neural crest cell defects in Treacher Collins and 22q11 deletion syndromes.

The craniofacial complex is anatomically the most sophisticated part of the body. It houses all the major sensory organ systems and its origins are synonymous with vertebrate evolution. Of fundamental importance to craniofacial development is a specialized population of stem and progenitor cells, known as the neural crest, which generate the majority of the bone, cartilage, connective and peripheral nerve tissue in the head. Approximately one third of all congenital abnormalities exhibit craniofacial malformations and consequently, most craniofacial anomalies are considered to arise through primary defects in neural crest cell development. Recent advances however, have challenged this classical dogma, underscoring the influence of tissues with which the neural crest cells interact as the primary origin of patterning defects in craniofacial morphogenesis. In this review we discuss these neural crest cell interactions with mesoderm, endoderm and ectoderm in the head in the context of a better understanding of craniofacial malformations such as in Treacher Collins and 22q11 deletion syndromes.

Hox genes, neural crest cells and branchial arch patterning.

Proper craniofacial development requires the orchestrated integration of multiple specialized tissue interactions. Recent analyses suggest that craniofacial development is not dependent upon neural crest pre-programming as previously thought but is regulated by a more complex integration of cell and tissue interactions. In the absence of neural crest cells it is still possible to obtain normal arch patterning indicating that neural crest is not responsible for patterning all of arch development. The mesoderm, endoderm and surface ectoderm tissues play a role in the patterning of the branchial arches, and there is now strong evidence that Hoxa2 acts as a selector gene for the pathways that govern second arch structures.

Synergy between Hoxa1 and Hoxb1: the relationship between arch patterning and the generation of cranial neural crest.

Hoxa1 and Hoxb1 have overlapping synergistic roles in patterning the hindbrain and cranial neural crest cells. The combination of an ectoderm-specific regulatory mutation in the Hoxb1 locus and the Hoxa1 mutant genetic background results in an ectoderm-specific double mutation, leaving the other germ layers impaired only in Hoxa1 function. This has allowed us to examine neural crest and arch patterning defects that originate exclusively from the neuroepithelium as a result of the simultaneous loss of Hoxa1 and Hoxb1 in this tissue. Using molecular and lineage analysis in this double mutant background we demonstrate that presumptive rhombomere 4, the major site of origin of the second pharyngeal arch neural crest, is reduced in size and has lost the ability to generate neural crest cells. Grafting experiments using wild-type cells in cultured normal or double mutant mouse embryos demonstrate that this is a cell-autonomous defect, suggesting that the formation or generation of cranial neural crest has been uncoupled from segmental identity in these mutants. Furthermore, we show that loss of the second arch neural crest population does not have any adverse consequences on early patterning of the second arch. Signalling molecules are expressed correctly and pharyngeal pouch and epibranchial placode formation are unaffected. There are no signs of excessive cell death or loss of proliferation in the epithelium of the second arch, suggesting that the neural crest cells are not the source of any indispensable mitogenic or survival signals. These results illustrate that Hox genes are not only necessary for proper axial specification of the neural crest but that they also play a vital role in the generation of this population itself. Furthermore, they demonstrate that early patterning of the separate components of the pharyngeal arches can proceed independently of neural crest cell migration.

Conservation and elaboration of Hox gene regulation during evolution of the vertebrate head.

The comparison of Hox genes between vertebrates and their closest invertebrate relatives (amphioxus and ascidia) highlights two derived features of Hox genes in vertebrates: duplication of the Hox gene cluster, and an elaboration of Hox expression patterns and roles compared with non-vertebrate chordates. We have investigated how new expression domains and their associated developmental functions evolved, by testing the cis-regulatory activity of genomic DNA fragments from the cephalochordate amphioxus Hox cluster in transgenic mouse and chick embryos. Here we present evidence for the conservation of cis-regulatory mechanisms controlling gene expression in the neural tube for half a billion years of evolution, including a dependence on retinoic acid signalling. We also identify amphioxus Hox gene regulatory elements that drive spatially localized expression in vertebrate neural crest cells, in derivatives of neurogenic placodes and in branchial arches, despite the fact that cephalochordates lack both neural crest and neurogenic placodes. This implies an elaboration of cis-regulatory elements in the Hox gene cluster of vertebrate ancestors during the evolution of craniofacial patterning.

Dorsal patterning defects in the hindbrain, roof plate and skeleton in the dreher (dr(J)) mouse mutant.

dreher is a spontaneous mouse mutation in which adult animals display a complex phenotype associated with hearing loss, neurological, pigmentation and skeletal abnormalities. During early embryogenesis, the neural tube of dreher mutants is abnormally shaped in the region of the rhomboencephalon, due to problems in the formation of a proper roof plate over the otic hindbrain. We have studied the expression of Hox/lacZ transgenic mouse strains in the dreher background and shown that primary segmentation of the neural tube is not altered in these mutants, although correct morphogenesis is affected resulting in misshapen rhombomeres. Neural crest derivatives from rhombomere 6, such as the glossopharyngeal ganglion, are defective, and the dorsal neural tube marker Wnt1 is absent from this segment. Selected trunk neural crest populations are also altered, as there is a lack of pigmentation in the thoracic region of mutant mice. Skeletal defects include abnormal cranial bones of neural crest origin, and improper fusion of the dorsal aspects of cervical and thoracic vertebrae. Taken together, the gene affected in the dreher mutant is responsible for correct patterning of the dorsal-most cell types of the neural tube, that is, the neural crest and the roof plate, in the hindbrain region. Axial skeletal defects could reflect inductive influence of the dorsal neural tube on proper fusion of the neural arches. It is possible that a common precursor population for both neural crest and roof plate is the cellular target of the dreher mutation.

Plasticity in mouse neural crest cells reveals a new patterning role for cranial mesoderm.

The anteroposterior identity of cranial neural crest cells is thought to be preprogrammed before these cells emigrate from the neural tube. Here we test this assumption by developing techniques for transposing cells in the hindbrain of mouse embryos, using small numbers of cells in combination with genetic and lineage markers. This technique has uncovered a surprising degree of plasticity with respect to the expression of Hox genes, which can be used as markers of different hindbrain segments and cells, in both hindbrain tissue and cranial neural crest cells. Our analysis shows that the patterning of cranial neural crest cells relies on a balance between permissive and instructive signals, and underscores the importance of cell-community effects. These results reveal a new role for the cranial mesoderm in patterning facial tissues. Furthermore, our findings argue against a permanently fixed prepatterning of the cranial neural crest that is maintained by passive transfer of positional information from the hindbrain to the periphery.

Defects in pathfinding by cranial neural crest cells in mice lacking the neuregulin receptor ErbB4.

Mouse embryos with a loss-of-function mutation in the gene encoding the receptor tyrosine kinase ErbB4 exhibit misprojections of cranial sensory ganglion afferent axons. Here we analyse ErbB4-deficient mice, and find that morphological differences between wild-type and mutant cranial ganglia correlate with aberrant migration of a subpopulation of hindbrain-derived cranial neural crest cells within the paraxial mesenchyme environment. In transplantation experiments using new grafting techniques in cultured mouse embryos, we determine that this phenotype is non-cell-autonomous: wild-type and mutant neural crest cells both migrate in a pattern consistent with the host environment, deviating from their normal pathway only when transplanted into mutant embryos. ErbB4 signalling events within the hindbrain therefore provide patterning information to cranial paraxial mesenchyme that is essential for the proper migration of neural crest cells.

Patterning the cranial neural crest: hindbrain segmentation and Hox gene plasticity.

Understanding the patterning mechanisms that control head development--particularly the neural crest and its contribution to bones, nerves and connective tissue--is an important problem, as craniofacial anomalies account for one-third of all human congenital defects. Classical models for craniofacial patterning argue that the morphogenic program and Hox gene identity of the neural crest is pre-patterned, carrying positional information acquired in the hindbrain to the peripheral nervous system and the branchial arches. Recently, however, plasticity of Hox gene expression has been observed in the hindbrain and cranial neural crest of chick, mouse and zebrafish embryos. Hence, craniofacial development is not dependent on neural crest prepatterning, but is regulated by a more complex integration of cell and tissue interactions.

The role of kreisler in segmentation during hindbrain development.

The mouse kreisler gene is expressed in rhombomeres (r) 5 and 6 during neural development and kreisler mutants have patterning defects in the hindbrain that are not fully understood. Here we analyzed this phenotype with a combination of genetic, molecular, and cellular marking techniques. Using Hox/lacZ transgenic mice as reporter lines and by analyzing Eph/ephrin expression, we have found that while r5 fails to form in these mice, r6 is present. This shows that kreisler has an early role in the formation of r5. We also observed patterning defects in r3 and r4 that are outside the normal domain of kreisler expression. In both heterozygous and homozygous kreisler embryos some r5 markers are induced in r3, suggesting that there is a partial change in r3 identity that is not dependent upon the loss of r5. To investigate the cellular character of r6 in kreisler embryos we performed heterotopic grafting experiments in the mouse hindbrain to monitor its mixing properties. Control experiments revealed that cells from even- or odd-numbered segments only mixed freely with themselves, but not with cells of opposite character. Transposition of cells from the r6 territory of kreisler mutants reveals that they adopt mature r6 characteristics, as they freely mix only with cells from even-numbered rhombomeres. Analysis of Phox2b expression shows that some aspects of later neurogenesis in r6 are altered, which may be associated with the additional roles of kreisler in regulating segmental identity. Together these results suggest that the formation of r6 has not been affected in kreisler mutants. This analysis has revealed phenotypic and mechanistic differences between kreisler and its zebrafish equivalent valentino. While valentino is believed to subdivide preexisting segmental units, in the mouse kreisler specifies a particular segment. The formation of r6 independent of r5 argues against a role of kreisler in prorhombomeric segmentation of the mouse hindbrain. We conclude that the mouse kreisler gene regulates multiple steps in segmental patterning involving both the formation of segments and their A-P identity.

The (in)stability of adolescent fears.

This study examined change in different types of fear in a longitudinal study of a birth cohort from age 13-15 yr. When viewed cross-sectionally there were few differences in the frequency and content of fears reported at these ages with the exception of an increase in social fears at age 15. When viewed longitudinally, however, few adolescents who reported fears at age 13 continued to report fears 2 yr later. Implications for developmental theories of fear and phobia are discussed.

Assessing the accuracy of femoral component placement in custom cemented hip replacement.

Leg length disparity is a frequent complication of hip arthroplasty. The most common problem is lengthening of the operative leg, which usually occurs on the femoral side. The Belfast Hip Group has designed a femoral caliper that offers an effective solution to the problem. This instrument is accurate and easy to use and could be applied by the orthopaedic surgeon to control leg length. Patients' perception of the success of their total hip replacement is influenced by their ability to walk without appliances or pain. With minimal additional effort and planning, this caliper will ensure greater control of leg length measurement in total hip replacement patients.

Cranial paraxial mesoderm and neural crest cells of the mouse embryo: co-distribution in the craniofacial mesenchyme but distinct segregation in branchial arches.

The spatial distribution of the cranial paraxial mesoderm and the neural crest cells during craniofacial morphogenesis of the mouse embryo was studied by micromanipulative cell grafting and cell labelling. Results of this study show that the paraxial mesoderm and neural crest cells arising at the same segmental position share common destinations. Mesodermal cells from somitomeres I, III, IV and VI were distributed to the same craniofacial tissues as neural crest cells of the forebrain, the caudal midbrain, and the rostral, middle and caudal hindbrains found respectively next to these mesodermal segments. This finding suggests that a basic meristic pattern is established globally in the neural plate ectoderm and paraxial mesoderm during early mouse development. Cells from these two sources mixed extensively in the peri-ocular, facial, periotic and cervical mesenchyme. However, within the branchial arches a distinct segregation of these two cell populations was discovered. Neural crest cells colonised the periphery of the branchial arches and enveloped the somitomere-derived core tissues on the rostral, lateral and caudal sides of the arch. Such segregation of cell populations in the first three branchial arches is apparent at least until the 10.5-day hindlimb bud stage and could be important for the patterning of the skeletal and myogenic derivatives of the arches.