E-liquids and vanillin flavoring disrupts retinoic acid signaling and causes craniofacial defects in Xenopus embryos.

Environmental teratogens such as smoking are known risk factors for developmental disorders such as cleft palate. While smoking rates have declined, a new type of smoking, called vaping is on the rise. Vaping is the use of e-cigarettes to vaporize and inhale an e-liquid containing nicotine and food-like flavors. There is the potential that, like smoking, vaping could also pose a danger to the developing human. Rather than waiting for epidemiological and mammalian studies, we have turned to an aquatic developmental model, Xenopus laevis, to more quickly assess whether e-liquids contain teratogens that could lead to craniofacial malformations. Xenopus, like zebrafish, has the benefit of being a well-established developmental model and has also been effective in predicting whether a chemical could be a teratogen. We have determined that embryonic exposure to dessert flavored e-liquids can cause craniofacial abnormalities, including an orofacial cleft in Xenopus. To better understand the underlying mechanisms contributing to these defects, transcriptomic analysis of the facial tissues of embryos exposed to a representative dessert flavored e-liquid vapor extract was performed. Analysis of differentially expressed genes in these embryos revealed several genes associated with retinoic acid metabolism or the signaling pathway. Consistently, retinoic acid receptor inhibition phenocopied the craniofacial defects as those embryos exposed to the vapor extract of the e-liquid. Such malformations also correlated with a group of common differentially expressed genes, two of which are associated with midface birth defects in humans. Further, e-liquid exposure sensitized embryos to forming craniofacial malformations when they already had depressed retinoic acid signaling. Moreover, 13-cis-retinoic acid treatment could significantly reduce the e-liquid induced malformation in the midface. Such results suggest the possibility of an interaction between retinoic acid signaling and e-liquid exposure. One of the most popular and concentrated flavoring chemicals in dessert flavored e-liquids is vanillin. Xenopus embryos exposed to this chemical closely resembled embryos exposed to dessert-like e-liquids and a retinoic acid receptor antagonist. In summary, we determined that e-liquid chemicals, in particular vanillin, can cause craniofacial defects potentially by dysregulating retinoic acid signaling. This work warrants the evaluation of vanillin and other such flavoring additives in e-liquids on mammalian development.

Deep learning is widely applicable to phenotyping embryonic development and disease.

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.

Cilia, ciliopathies and hedgehog-related forebrain developmental disorders.

Development of the forebrain critically depends on the Sonic Hedgehog (Shh) signaling pathway, as illustrated in humans by the frequent perturbation of this pathway in holoprosencephaly, a condition defined as a defect in the formation of midline structures of the forebrain and face. The Shh pathway requires functional primary cilia, microtubule-based organelles present on virtually every cell and acting as cellular antennae to receive and transduce diverse chemical, mechanical or light signals. The dysfunction of cilia in humans leads to inherited diseases called ciliopathies, which often affect many organs and show diverse manifestations including forebrain malformations for the most severe forms. The purpose of this review is to provide the reader with a framework to understand the developmental origin of the forebrain defects observed in severe ciliopathies with respect to perturbations of the Shh pathway. We propose that many of these defects can be interpreted as an imbalance in the ratio of activator to repressor forms of the Gli transcription factors, which are effectors of the Shh pathway. We also discuss the complexity of ciliopathies and their relationships with forebrain disorders such as holoprosencephaly or malformations of cortical development, and emphasize the need for a closer examination of forebrain defects in ciliopathies, not only through the lens of animal models but also taking advantage of the increasing potential of the research on human tissues and organoids.

Grainyhead-like Transcription Factors in Craniofacial Development.

Craniofacial development in vertebrates involves the coordinated growth, migration, and fusion of several facial prominences during embryogenesis, processes governed by strict genetic and molecular controls. A failure in any of the precise spatiotemporal sequences of events leading to prominence fusion often leads to anomalous facial, skull, and jaw formation-conditions termed craniofacial defects (CFDs). Affecting approximately 0.1% to 0.3% of live births, CFDs are a highly heterogeneous class of developmental anomalies, which are often underpinned by genetic mutations. Therefore, identifying novel disease-causing mutations in genes that regulate craniofacial development is a critical prerequisite to develop new preventive or therapeutic measures. The Grainyhead-like ( GRHL) transcription factors are one such gene family, performing evolutionarily conserved roles in craniofacial patterning. The antecedent member of this family, Drosophila grainyhead ( grh), is required for head skeleton development in fruit flies, loss or mutation of Grhl family members in mouse and zebrafish models leads to defects of both maxilla and mandible, and recently, mutations in human GRHL3 have been shown to cause or contribute to both syndromic (Van Der Woude syndrome) and nonsyndromic palatal clefts. In this review, we summarize the current knowledge regarding the craniofacial-specific function of the Grainyhead-like family in multiple model species, identify some of the major target genes regulated by the Grhl transcription factors in craniofacial patterning, and, by examining animal models, draw inferences as to how these data will inform the likely roles of GRHL factors in human CFDs comprising palatal clefting. By understanding the molecular networks regulated by Grhl2 and Grhl3 target genes in other systems, we can propose likely pathways that mediate the effects of these transcription factors in human palatogenesis.

Matrix Gla protein deficiency impairs nasal septum growth, causing midface hypoplasia.

Genetic and environmental factors may lead to abnormal growth of the orofacial skeleton, affecting the overall structure of the face. In this study, we investigated the craniofacial abnormalities in a mouse model for Keutel syndrome, a rare genetic disease caused by loss-of-function mutations in the matrix Gla protein (MGP) gene. Keutel syndrome patients show diffuse ectopic calcification of cartilaginous tissues and impaired midface development. Our comparative cephalometric analyses of micro-computed tomography images revealed a severe midface hypoplasia in Mgp-/- mice. In vivo reporter studies demonstrated that the Mgp promoter is highly active at the cranial sutures, cranial base synchondroses, and nasal septum. Interestingly, the cranial sutures of the mutant mice showed normal anatomical features. Although we observed a mild increase in mineralization of the spheno-occipital synchondrosis, it did not reduce the relative length of the cranial base in comparison with total skull length. Contrary to this, we found the nasal septum to be abnormally mineralized and shortened in Mgp-/- mice. Transgenic restoration of Mgp expression in chondrocytes fully corrected the craniofacial anomalies caused by MGP deficiency, suggesting a local role for MGP in the developing nasal septum. Although there was no up-regulation of markers for hypertrophic chondrocytes, a TUNEL assay showed a marked increase in apoptotic chondrocytes in the calcified nasal septum. Transmission electron microscopy confirmed unusual mineral deposits in the septal extracellular matrix of the mutant mice. Of note, the systemic reduction of the inorganic phosphate level was sufficient to prevent abnormal mineralization of the nasal septum in Mgp-/-;Hyp compound mutants. Our work provides evidence that modulation of local and systemic factors regulating extracellular matrix mineralization can be possible therapeutic strategies to prevent ectopic cartilage calcification and some forms of congenital craniofacial anomalies in humans.

Fraser syndrome: features suggestive of prenatal diagnosis in a review of 38 cases.

OBJECTIVE: Fraser syndrome (FS) is a rare malformation recessive disorder. Major criteria are cryptophtalmos, syndactyly, respiratory, genital and urinary tract anomalies. Few prenatal presentations have been reported. METHOD: We analyzed the prenatal and postnatal fetal phenotype in 38 cases of FS, including 25 pregnancy termination cases, 8 intra-uterine death cases and 4 cases that died after birth. RESULTS: Including both prenatal and postnatal fetal phenotypic evaluation, all cases presented dysmorphic features with nose and ear dysplasia. Renal anomalies and syndactyly were present in 37/38 cases, cryptophtalmos in 36/38, airways anomalies in 30/37 and genital anomalies in 30/35 cases. Anomalies of the abdominal wall such as low set umbilicus and omphalocele were found in 31 cases. Among the 26 cases for which ultrasound data were available, detectable anomalies included oligohydramnios (22), ascites/hydrops (9), renal anomalies (20), evidence for high airways obstruction (11), ophthalmologic anomalies (4), ear dysplasia (2) and syndactyly (2). CONCLUSION: This study shows that the postnatal phenotype of FS is very specific, whereas oligohydramnios hampers the prenatal recognition of the cardinal FS diagnosis criteria. Association of oligohydramnios, kidney agenesis and CHAOS should lead to consider this diagnosis. © 2016 John Wiley & Sons, Ltd.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

A novel ciliopathic skull defect arising from excess neural crest.

The skull is essential for protecting the brain from damage, and birth defects involving disorganization of skull bones are common. However, the developmental trajectories and molecular etiologies by which many craniofacial phenotypes arise remain poorly understood. Here, we report a novel skull defect in ciliopathic Fuz mutant mice in which only a single bone pair encases the forebrain, instead of the usual paired frontal and parietal bones. Through genetic lineage analysis, we show that this defect stems from a massive expansion of the neural crest-derived frontal bone. This expansion occurs at the expense of the mesodermally-derived parietal bones, which are either severely reduced or absent. A similar, though less severe, phenotype was observed in Gli3 mutant mice, consistent with a role for Gli3 in cilia-mediated signaling. Excess crest has also been shown to drive defective palate morphogenesis in ciliopathic mice, and that defect is ameliorated by reduction of Fgf8 gene dosage. Strikingly, skull defects in Fuz mutant mice are also rescued by loss of one allele of fgf8, suggesting a potential route to therapy. In sum, this work is significant for revealing a novel skull defect with a previously un-described developmental etiology and for suggesting a common developmental origin for skull and palate defects in ciliopathies.

[Prenatal genetic diagnosis for a fetus with atypical neurofibromatosis type 1 microdeletion].

OBJECTIVE: To analyze the correlation between atypical neurofibromatosis type 1(NF1) microdeletion and fetal phenotype. METHODS: Fetal blood sampling was carried out for a woman bearing a fetus with talipes equinovarus. G-banded karyotyping and single nucleotide polymorphism array (SNP-array) were performed on the fetal blood sample. Fluorescence in situ hybridization (FISH) was used to confirm the result of SNP array analysis. FISH assay was also carried out on peripheral blood specimens from the parents to ascertain the origin of mutation. RESULTS: The karyotype of fetus was found to be 46, XY by G-banding analysis. However, a 3.132 Mb microdeletion was detected in chromosome region 17q11.2 by SNP array, which overlaped with the region of NF1 microdeletion syndrome. Analyzing of the specimens from the fetus and its parents with FISH has confirmed it to be a de novo deletion. CONCLUSION: Talipes equinovarus may be an abnormal sonographic feature of fetus with atypical NF1 microdeletion which can be accurately diagnosed with SNP array.

Utilizing the chicken as an animal model for human craniofacial ciliopathies.

The chicken has been a particularly useful model for the study of craniofacial development and disease for over a century due to their relatively large size, accessibility, and amenability for classical bead implantation and transplant experiments. Several naturally occurring mutant lines with craniofacial anomalies also exist and have been heavily utilized by developmental biologist for several decades. Two of the most well known lines, talpid(2) (ta(2)) and talpid(3) (ta(3)), represent the first spontaneous mutants to have the causative genes identified. Despite having distinct genetic causes, both mutants have recently been identified as ciliopathic. Excitingly, both of these mutants have been classified as models for human craniofacial ciliopathies: Oral-facial-digital syndrome (ta(2)) and Joubert syndrome (ta(3)). Herein, we review and compare these two models of craniofacial disease and highlight what they have revealed about the molecular and cellular etiology of ciliopathies. Furthermore, we outline how applying classical avian experiments and new technological advances (transgenics and genome editing) with naturally occurring avian mutants can add a tremendous amount to what we currently know about craniofacial ciliopathies.

Augmented Indian hedgehog signaling in cranial neural crest cells leads to craniofacial abnormalities and dysplastic temporomandibular joint in mice.

Extensive studies have pinpointed the crucial role of Indian hedgehog (Ihh) signaling in the development of the appendicular skeleton and the essential function of Ihh in the formation of the temporomandibular joint (TMJ). In this study, we have investigated the effect of augmented Ihh signaling in TMJ development. We took a transgenic gain-of-function approach by overexpressing Ihh in the cranial neural crest (CNC) cells using a conditional Ihh transgenic allele and the Wnt1-Cre allele. We found that Wnt1-Cre-mediated tissue-specific overexpression of Ihh in the CNC lineage caused severe craniofacial abnormalities, including cleft lip/palate, encephalocele, anophthalmos, micrognathia, and defective TMJ development. In the mutant TMJ, the glenoid fossa was completely absent, whereas the condyle and the articular disc appeared relatively normal with slightly delayed chondrocyte differentiation. Our findings thus demonstrate that augmented Ihh signaling is detrimental to craniofacial development, and that finely tuned Ihh signaling is critical for TMJ formation. Our results also provide additional evidence that the development of the condyle and articular disc is independent of the glenoid fossa.

Combining Foxc2 and Connexin37 deletions in mice leads to severe defects in lymphatic vascular growth and remodeling.

Connexins (Cxs), proteins that are vital for intercellular communication in vertebrates, have recently been shown to play a critical role in lymphatic development. However, our knowledge is currently limited regarding the functional relationships of Cxs with other proteins and signaling pathways. Cell culture studies have shown that Cx37 is necessary for coordinated activation of the transcription factor NFATc1, which cooperates with Foxc2 (another transcription factor) during lymphatic endothelial development. These data suggest that Cxs, Foxc2, and NFATc1 are part of a common developmental pathway. Here, we present a detailed characterization of Foxc2(+/-)Cx37(-/-) mice, demonstrating that lymphatic network architecture and valve formation rely on the concurrent embryonic expression and function of Foxc2 and Cx37. Foxc2(+/-)Cx37(-/-) mice have lymphedema in utero, exhibit craniofacial abnormalities, show severe dilation of intestinal lymphatics, display abnormal lacteal development, lack lymphatic valves, and typically die perinatally (outcomes not seen in Foxc2(+/-) or Cx37(-/-) mice separately). We provide a rigorous, quantitative documentation of lymphatic vascular network changes that highlight the specific structural alterations that occur in Foxc2(+/-)Cx37(-/-) mice. These data provide further evidence suggesting that Foxc2 and Cx37 are elements in a common molecular pathway directing lymphangiogenesis.

Midline craniofacial malformations with a lipomatous cephalocele are associated with insufficient closure of the neural tube in the tuft mouse.

BACKGROUND: Genetic variations affecting neural tube closure along the head result in malformations to the face and brain, posing a significant impact on health care costs and the quality of life. METHODS: We have established a mouse line from a mutation that arose spontaneously in our wild-type colony that we called tuft. Tuft mice have heritable midline craniofacial defects featuring an anterior lipomatous cephalocele. RESULTS: Whole-mount skeletal stains indicated that affected newborns had a broader interfrontal suture where the cephalocele emerged between the frontal bones. Mice with a cephalocele positioned near the rostrum also presented craniofacial malformations such as ocular hypertelorism and midfacial cleft of the nose. Gross and histological examination revealed that the lipomatous cephalocele originated as a fluid filled cyst no earlier than E14.5 while embryos with a midfacial cleft was evident during craniofacial development at E11.5. Histological sections of embryos with a midfacial cleft revealed the cephalic neuroectoderm remained proximal or fused to the frontonasal ectoderm about the closure site of the anterior neuropore, indicating a defect to neural tube closure. We found the neural folds along the rostrum of E9 to E10.5 embryos curled inward and failed to close as well as embryos with exencephaly and anencephaly at later stages. Whole-mount in situ hybridization of anterior markers Fgf8 and Sonic hedgehog indicated closure of the rostral site was compromised in severe cases. CONCLUSION: We present a model demonstrating how anterior cranial cephaloceles are generated following a defect to neural tube closure and relevance to subsequent craniofacial morphogenesis in the tuft mouse.

Metabolic characterization of all-trans-retinoic acid (ATRA)-induced craniofacial development of murine embryos using in vivo proton magnetic resonance spectroscopy.

AIM: To characterize the abnormal metabolic profile of all-trans-retinoic acid (ATRA)-induced craniofacial development in mouse embryos using proton magnetic resonance spectroscopy (1H-MRS). METHODS: Timed-pregnant mice were treated by oral gavage on the morning of embryonic gestation day 11 (E11) with all-trans-retinoic acid (ATRA). Dosing solutions were adjusted by maternal body weight to provide 30, 70, or 100 mg/kg RA. The control group was given an equivalent volume of the carrier alone. Using an Agilent 7.0 T MR system and a combination of surface coil coils, a 3 mm×3 mm×3 mm 1H-MRS voxel was selected along the embryonic craniofacial tissue. 1H-MRS was performed with a single-voxel method using PRESS sequence and analyzed using LCModel software. Hematoxylin and eosin was used to detect and confirm cleft palate. RESULT: 1H-MRS revealed elevated choline levels in embryonic craniofacial tissue in the RA70 and RA100 groups compared to controls (P<0.05). Increased choline levels were also found in the RA70 and RA100 groups compared with the RA30 group (P<0.01). High intra-myocellular lipids at 1.30 ppm (IMCL13) in the RA100 group compared to the RA30 group were found (P<0.01). There were no significant changes in taurine, intra-myocellular lipids at 2.10 ppm (IMCL21), and extra-myocellular lipids at 2.30 ppm (EMCL23). Cleft palate formation was observed in all fetuses carried by mice administered 70 and 100 mg/kg RA. CONCLUSIONS: This novel study suggests that the elevated choline and lipid levels found by 1H-MRS may represent early biomarkers of craniofacial defects. Further studies will determine performance of this test and pathogenetic mechanisms of craniofacial malformation.

Craniofacial and dental development in Costello syndrome.

Costello syndrome (CS) is a RASopathy characterized by a wide range of cardiac, musculoskeletal, dermatological, and developmental abnormalities. The RASopathies are defined as a group of syndromes caused by activated Ras/mitogen-activated protein kinase (MAPK) signaling. Specifically, CS is caused by activating mutations in HRAS. Although receptor tyrosine kinase (RTK) signaling, which is upstream of Ras/MAPK, is known to play a critical role in craniofacial and dental development, the craniofacial and dental features of CS have not been systematically defined in a large group of individuals. In order to address this gap in our understanding and fully characterize the CS phenotype, we evaluated the craniofacial and dental phenotype in a large cohort (n = 41) of CS individuals. We confirmed that the craniofacial features common in CS include macrocephaly, bitemporal narrowing, convex facial profile, full cheeks, and large mouth. Additionally, CS patients have a characteristic dental phenotype that includes malocclusion with anterior open bite and posterior crossbite, enamel hypo-mineralization, delayed tooth development and eruption, gingival hyperplasia, thickening of the alveolar ridge, and high palate. Comparison of the craniofacial and dental phenotype in CS with other RASopathies, such as cardio-facio-cutaneous syndrome (CFC), provides insight into the complexities of Ras/MAPK signaling in human craniofacial and dental development.

Craniofacial divergence by distinct prenatal growth patterns in Fgfr2 mutant mice.

BACKGROUND: Differences in cranial morphology arise due to changes in fundamental cell processes like migration, proliferation, differentiation and cell death driven by genetic programs. Signaling between fibroblast growth factors (FGFs) and their receptors (FGFRs) affect these processes during head development and mutations in FGFRs result in congenital diseases including FGFR-related craniosynostosis syndromes. Current research in model organisms focuses primarily on how these mutations change cell function local to sutures under the hypothesis that prematurely closing cranial sutures contribute to skull dysmorphogenesis. Though these studies have provided fundamentally important information contributing to the understanding of craniosynostosis conditions, knowledge of changes in cell function local to the sutures leave change in overall three-dimensional cranial morphology largely unexplained. Here we investigate growth of the skull in two inbred mouse models each carrying one of two gain-of-function mutations in FGFR2 on neighboring amino acids (S252W and P253R) that in humans cause Apert syndrome, one of the most severe FGFR-related craniosynostosis syndromes. We examine late embryonic skull development and suture patency in Fgfr2 Apert syndrome mice between embryonic day 17.5 and birth and quantify the effects of these mutations on 3D skull morphology, suture patency and growth. RESULTS: We show in mice what studies in humans can only infer: specific cranial growth deviations occur prenatally and worsen with time in organisms carrying these FGFR2 mutations. We demonstrate that: 1) distinct skull morphologies of each mutation group are established by E17.5; 2) cranial suture patency patterns differ between mice carrying these mutations and their unaffected littermates; 3) the prenatal skull grows differently in each mutation group; and 4) unique Fgfr2-related cranial morphologies are exacerbated by late embryonic growth patterns. CONCLUSIONS: Our analysis of mutation-driven changes in cranial growth provides a previously missing piece of knowledge necessary for explaining variation in emergent cranial morphologies and may ultimately be helpful in managing human cases carrying these same mutations. This information is critical to the understanding of craniofacial development, disease and evolution and may contribute to the evaluation of incipient therapeutic strategies.

Facial clefts and facial dysplasia: revisiting the classification.

Most craniofacial malformations are identified by their appearance. The majority of the classification systems are mainly clinical or anatomical, not related to the different levels of development of the malformation, and underlying pathology is usually not taken into consideration. In 1976, Tessier first emphasized the relationship between soft tissues and the underlying bone stating that "a fissure of the soft tissue corresponds, as a general rule, with a cleft of the bony structure". He introduced a cleft numbering system around the orbit from 0 to 14 depending on its relationship to the zero line (ie, the vertical midline cleft of the face). The classification, easy to understand, became widely accepted because the recording of the malformations was simple and communication between observers facilitated. It represented a great breakthrough in identifying craniofacial malformations, named clefts by him. In the present paper, the embryological-based classification of craniofacial malformations, proposed in 1983 and in 1990 by us, has been revisited. Its aim was to clarify some unanswered questions regarding apparently atypical or bizarre anomalies and to establish as much as possible the moment when this event occurred. In our opinion, this classification system may well integrate the one proposed by Tessier and tries at the same time to find a correlation between clinical observation and morphogenesis.Terminology is important. The overused term cleft should be reserved to true clefts only, developed from disturbances in the union of the embryonic facial processes, between the lateronasal and maxillary process (or oro-naso-ocular cleft); between the medionasal and maxillary process (or cleft of the lip); between the maxillary processes (or cleft of the palate); and between the maxillary and mandibular process (or macrostomia).For the other types of defects, derived from alteration of bone production centers, the word dysplasia should be used instead. Facial dysplasias have been ranged in a helix form and named after the site of the developmental arrest. Thus, an internasal, nasal, nasomaxillary, maxillary and malar dysplasia, depending on the involved area, have been identified.The classification may provide a useful guide in better understanding the morphogenesis of rare craniofacial malformations.

Construction and characterization of a sox9b transgenic reporter line.

The transcription factor SOX9 is a member of the SRY-related high-mobility-group box (SOX) superfamily of genes. In mammals, Sox9 plays important roles in many developmental processes including craniofacial, skeletal and heart morphogenesis, retinal and brain development, and gonad differentiation. Human mutations in SOX9 or the SOX9 promoter result in campomelic dysplasia, a severe genetic disorder, which disrupts skeletal, craniofacial, cardiac, neural and reproductive development. Due to the duplication of the teleost fish genome, zebrafish (Danio rerio) have two Sox9 genes: sox9a and sox9b. Loss of sox9b in zebrafish results in loss of function phenotypes that are similar to those observed in humans and mice. In order to generate a transgenic sox9b:EGFP reporter line, we cloned a 2450 bp fragment of the sox9b promoter and fused it to an EGFP reporter. Consistent with reported sox9b expression and function, we observed sox9b:EGFP in the developing heart, skeletal and craniofacial structures, brain, retina, and ovaries. Our resulting transgenic line is a useful tool for identifying and studying sox9b function in development and visualizing a number of zebrafish organs and tissues in which sox9b is normally expressed.

Transitions in craniofacial biology: a tribute to Bernard G. Sarnat.

The surge of advances in understanding ontological, phylogenetic, pathological, paleoanthropological and genetic insights in development and evolution over the past century has resulted in a profound revolution of our cognizance of biological sciences. The past decade of the human genome project has provided opportunities for diagnosis, prognosis, therapeutic interventions and prevention of dysgenesis, dysmorphology, and disease. The new technologies of imaging and experimental inquiries into developmental phenomena provide insights into evolutionary advances that have taken place to result in the present human structure.