Endothelin signaling in development.

Since the discovery of endothelin 1 (EDN1) in 1988, the role of endothelin ligands and their receptors in the regulation of blood pressure in normal and disease states has been extensively studied. However, endothelin signaling also plays crucial roles in the development of neural crest cell-derived tissues. Mechanisms of endothelin action during neural crest cell maturation have been deciphered using a variety of in vivo and in vitro approaches, with these studies elucidating the basis of human syndromes involving developmental differences resulting from altered endothelin signaling. In this Review, we describe the endothelin pathway and its functions during the development of neural crest-derived tissues. We also summarize how dysregulated endothelin signaling causes developmental differences and how this knowledge may lead to potential treatments for individuals with gene variants in the endothelin pathway.

GATA3 is essential for separating patterning domains during facial morphogenesis.

Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

The society for craniofacial genetics and developmental biology 46th annual meeting.

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 46th Annual Meeting at Cincinnati Children's Hospital Medical Center in Cincinnati, Ohio on October 10th-12th, 2023. On the first day of the meeting, Drs. Sally Moody and Justin Cotney were each honored with the SCGDB Distinguished Scientist Awards for their exceptional contributions to the field of craniofacial biology. The following two days of the meeting featured five sessions that highlighted new discoveries in signaling and genomic mechanisms regulating craniofacial development, human genetics, translational and regenerative approaches, and clinical management of craniofacial differences. Interactive workshops on spatial transcriptomics and scientific communication, as well as a poster session facilitated meaningful interactions among the 122 attendees representing diverse career stages and research backgrounds in developmental biology and genetics, strengthened the SCGDB community.

Transcriptomic analysis reveals the role of SIX1 in mouse cranial neural crest patterning and bone development.

BACKGROUND: Genetic variants of the transcription factor SIX1 and its co-factor EYA1 underlie 50% of Branchio-oto-renal syndrome (BOR) cases. BOR is characterized by craniofacial defects, including malformed middle ear ossicles leading to conductive hearing loss. In this work, we expand our knowledge of the Six1 gene regulatory network by using a Six1-null mouse line to assess gene expression profiles of E10.5 mandibular arches, which give rise to the neural crest (NC)-derived middle ear ossicles and lower jaw, via bulk RNA sequencing. RESULTS: Our transcriptomic analysis led to the identification of 808 differentially expressed genes that are related to translation, NC cell differentiation, osteogenesis, and chondrogenesis including components of the WNT signaling pathway. As WNT signaling is a known contributor to bone development, we demonstrated that SIX1 is required for expression of the WNT antagonist Frzb in the mandibular arch, and determined that SIX1 expression results in repression of WNT signaling. CONCLUSION: Our results clarify the mechanisms by which SIX1 regulates the development of NC-derived craniofacial elements that are altered in SIX1-associated disorders. In addition, this work identifies novel genes that could be causative to this birth defect and establishes a link between SIX1 and WNT signaling during patterning of NC cells.

The Society for Craniofacial Genetics and Developmental Biology 45th Annual Meeting.

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 45th Annual Meeting at the Sanford Consortium for Regenerative Medicine at the University of California, San Diego on October 20th-21st, 2022. The meeting included presentation of the SCGDB Distinguished Scientists in Craniofacial Research Awards to Drs. Ralph Marcucio and Loydie Jerome-Majewska and four scientific sessions that highlighted new discoveries in signaling in craniofacial development, genomics of craniofacial development, human genetics of craniofacial development and translational and regenerative approaches in craniofacial biology. The meeting also included workshops on analysis of single cell RNA sequencing datasets and using human sequencing data from the Gabriella Miller Kids First Pediatric Research Program. There were 110 faculty and trainees in attendance that represent a diverse group of researchers from all career stages in the fields of developmental biology and genetics. The meeting, which also included outdoor poster presentations, provided opportunities for participant interactions and discussions, thus strengthening the SCGDB community.

Piloting a multidisciplinary approach to improve outcomes of fetal whole exome sequencing: An overview of workflow and case example.

INTRODUCTION: Whole exome sequencing (WES) has increasingly become integrated into prenatal care and genetic testing pathways. Current studies of prenatal WES have focused on diagnostic yield. The possibility of obtaining a variant of uncertain significance and lack of provider expertise are frequently described as common barriers to clinical integration of prenatal WES. We describe the implementation and workflow for a multidisciplinary approach to effectively integrate prenatal WES into maternal-fetal care to overcome these barriers. METHODS: A multidisciplinary team reviews and approves potential cases for WES. This team reviews WES results, reclassifying variants as appropriate and provides recommendations for postnatal care. A detailed description of this workflow is provided, and a case example is included to demonstrate effectiveness of this approach. Our team has approved 62 cases for WES with 45 patients ultimately pursuing WES. We have achieved a diagnostic yield of 40% and the multidisciplinary team has played a role in variant interpretation in 50% of the reported variants of uncertain significance. CONCLUSIONS: This approach facilitates communication between prenatal and postnatal care teams and provides accurate interpretation and recommendations for identified fetal variants. This model can be replicated to ensure appropriate patient care and effective integration of novel genomic technologies into prenatal settings.

The Society for Craniofacial Genetics and Developmental Biology 44th Annual Meeting.

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 44th Annual Meeting in a virtual format on October 18-19, 2021. The SCGDB meeting included presentation of the SCGDB Distinguished Scientists in Craniofacial Research Awards to Drs. Paul Trainor and Jeff Bush and four scientific sessions on the genomics of craniofacial development, craniofacial morphogenesis and regeneration, translational craniofacial biology and signaling during craniofacial development. The meeting also included workshops on professional development for faculty and trainees, National Institutes of Health (NIH)/National Institute of Craniofacial and Dental Research funding and usage of Genomics Software, as well as two poster sessions. An exhibitor booth run by FaceBase was also present to facilitate the upload and download of datasets relevant to the craniofacial community. Over 200 attendees from 12 countries and 23 states, representing over 80 different scientific institutions, participated. This diverse group of scientists included cell biologists, developmental biologists, and clinical geneticists. Although the continuing COVID-19 pandemic forced a virtual meeting format for a second year in a row, the meeting platform provided ample opportunities for participant interactions and discussions, thus strengthening the community.

The society for craniofacial genetics and developmental biology 43rd annual meeting.

The Society for Craniofacial Genetics and Developmental Biology (SCGDB) held its 43rd annual meeting in a virtual format on October 19-20, 2020. The SCGDB meeting included the presentation of the SCGDB Distinguished Scientists in Craniofacial Research Awards to Marilyn Jones and Kerstin Ludwig and four scientific sessions on the molecular regulation of craniofacial development, craniofacial morphogenesis, translational craniofacial biology, and signaling during craniofacial development. The meeting also included workshops on career development, NIH/NIDCR funding, and the utility of the FaceBase database, as well as two poster sessions. Over 190 attendees from 21 states, representing over 50 different scientific institutions, participated. This diverse group of scientists included cell biologists, developmental biologists, and clinical geneticists. While in-person interactions were missed due to the virtual meeting format imposed by the COVID-19 pandemic, the meeting platform provided ample opportunities for participant interactions and discussions, thus strengthening the community.

Negative regulation of endothelin signaling by SIX1 is required for proper maxillary development.

Jaw morphogenesis is a complex event mediated by inductive signals that establish and maintain the distinct developmental domains required for formation of hinged jaws, the defining feature of gnathostomes. The mandibular portion of pharyngeal arch 1 is patterned dorsally by Jagged-Notch signaling and ventrally by endothelin receptor A (EDNRA) signaling. Loss of EDNRA signaling disrupts normal ventral gene expression, the result of which is homeotic transformation of the mandible into a maxilla-like structure. However, loss of Jagged-Notch signaling does not result in significant changes in maxillary development. Here we show in mouse that the transcription factor SIX1 regulates dorsal arch development not only by inducing dorsal Jag1 expression but also by inhibiting endothelin 1 (Edn1) expression in the pharyngeal endoderm of the dorsal arch, thus preventing dorsal EDNRA signaling. In the absence of SIX1, but not JAG1, aberrant EDNRA signaling in the dorsal domain results in partial duplication of the mandible. Together, our results illustrate that SIX1 is the central mediator of dorsal mandibular arch identity, thus ensuring separation of bone development between the upper and lower jaws.

Loss-of-function of Endothelin receptor type A results in Oro-Oto-Cardiac syndrome.

Craniofacial morphogenesis is regulated in part by signaling from the Endothelin receptor type A (EDNRA). Pathogenic variants in EDNRA signaling pathway components EDNRA, GNAI3, PCLB4, and EDN1 cause Mandibulofacial Dysostosis with Alopecia (MFDA), Auriculocondylar syndrome (ARCND) 1, 2, and 3, respectively. However, cardiovascular development is normal in MFDA and ARCND individuals, unlike Ednra knockout mice. One explanation may be that partial EDNRA signaling remains in MFDA and ARCND, as mice with reduced, but not absent, EDNRA signaling also lack a cardiovascular phenotype. Here we report an individual with craniofacial and cardiovascular malformations mimicking the Ednra -/- mouse phenotype, including a distinctive micrognathia with microstomia and a hypoplastic aortic arch. Exome sequencing found a novel homozygous missense variant in EDNRA (c.1142A>C; p.Q381P). Bioluminescence resonance energy transfer assays revealed that this amino acid substitution in helix 8 of EDNRA prevents recruitment of G proteins to the receptor, abrogating subsequent receptor activation by its ligand, Endothelin-1. This homozygous variant is thus the first reported loss-of-function EDNRA allele, resulting in a syndrome we have named Oro-Oto-Cardiac Syndrome. Further, our results illustrate that EDNRA signaling is required for both normal human craniofacial and cardiovascular development, and that limited EDNRA signaling is likely retained in ARCND and MFDA individuals. This work illustrates a straightforward approach to identifying the functional consequence of novel genetic variants in signaling molecules associated with malformation syndromes.

Transcriptomics reveals complex kinetics of dorsal-ventral patterning gene expression in the mandibular arch.

The mandibular or first pharyngeal arch forms the upper and lower jaws in all gnathostomes. A gene regulatory network that defines ventral, intermediate, and dorsal domains along the dorsal-ventral (D-V) axis of the arch has emerged from studies in zebrafish and mice, but the temporal dynamics of this process remain unclear. To define cell fate trajectories in the arches we have performed quantitative gene expression analyses of D-V patterning genes in pharyngeal arch primordia in zebrafish and mice. Using NanoString technology to measure transcript numbers per cell directly we show that, in many cases, genes expressed in similar D-V domains and induced by similar signals vary dramatically in their temporal profiles. This suggests that cellular responses to D-V patterning signals are likely shaped by the baseline kinetics of target gene expression. Furthermore, similarities in the temporal dynamics of genes that occupy distinct pathways suggest novel shared modes of regulation. Incorporating these gene expression kinetics into our computational models for the mandibular arch improves the accuracy of patterning, and facilitates temporal comparisons between species. These data suggest that the magnitude and timing of target gene expression help diversify responses to patterning signals during craniofacial development.

Exclusion of Dlx5/6 expression from the distal-most mandibular arches enables BMP-mediated specification of the distal cap.

Cranial neural crest cells (crNCCs) migrate from the neural tube to the pharyngeal arches (PAs) of the developing embryo and, subsequently, differentiate into bone and connective tissue to form the mandible. Within the PAs, crNCCs respond to local signaling cues to partition into the proximo-distally oriented subdomains that convey positional information to these developing tissues. Here, we show that the distal-most of these subdomains, the distal cap, is marked by expression of the transcription factor Hand1 (H1) and gives rise to the ectomesenchymal derivatives of the lower incisors. We uncover a H1 enhancer sufficient to drive reporter gene expression within the crNCCs of the distal cap. We show that bone morphogenic protein (BMP) signaling and the transcription factor HAND2 (H2) synergistically regulate H1 distal cap expression. Furthermore, the homeodomain proteins distal-less homeobox 5 (DLX5) and DLX6 reciprocally inhibit BMP/H2-mediated H1 enhancer regulation. These findings provide insights into how multiple signaling pathways direct transcriptional outcomes that pattern the developing jaw.

Mutations in the endothelin receptor type A cause mandibulofacial dysostosis with alopecia.

The endothelin receptor type A (EDNRA) signaling pathway is essential for the establishment of mandibular identity during development of the first pharyngeal arch. We report four unrelated individuals with the syndrome mandibulofacial dysostosis with alopecia (MFDA) who have de novo missense variants in EDNRA. Three of the four individuals have the same substitution, p.Tyr129Phe. Tyr129 is known to determine the selective affinity of EDNRA for endothelin 1 (EDN1), its major physiological ligand, and the p.Tyr129Phe variant increases the affinity of the receptor for EDN3, its non-preferred ligand, by two orders of magnitude. The fourth individual has a somatic mosaic substitution, p.Glu303Lys, and was previously described as having Johnson-McMillin syndrome. The zygomatic arch of individuals with MFDA resembles that of mice in which EDNRA is ectopically activated in the maxillary prominence, resulting in a maxillary to mandibular transformation, suggesting that the p.Tyr129Phe variant causes an EDNRA gain of function in the developing upper jaw. Our in vitro and in vivo assays suggested complex, context-dependent effects of the EDNRA variants on downstream signaling. Our findings highlight the importance of finely tuned regulation of EDNRA signaling during human craniofacial development and suggest that modification of endothelin receptor-ligand specificity was a key step in the evolution of vertebrate jaws.

Nkx2.5 regulates endothelin converting enzyme-1 during pharyngeal arch patterning.

In gnathostomes, dorsoventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches is crucial for the development of hinged jaws. One of the key signals that mediate this process is Endothelin-1 (EDN1). Loss of EDN1 binding to the Endothelin-A receptor (EDNRA) results in loss of EDNRA signaling and subsequent facial birth defects in humans, mice and zebrafish. A rate-limiting step in this crucial signaling pathway is the conversion of immature EDN1 into a mature active form by Endothelin converting enzyme-1 (ECE1). However, surprisingly little is known about how Ece1 transcription is induced or regulated. We show here that Nkx2.5 is required for proper craniofacial development in zebrafish and acts in part by upregulating ece1 expression. Disruption of nkx2.5 in zebrafish embryos results in defects in both ventral and dorsal pharyngeal arch-derived elements, with changes in ventral arch gene expression consistent with a disruption in Ednra signaling. ece1 mRNA rescues the nkx2.5 morphant phenotype, indicating that Nkx2.5 functions through modulating Ece1 expression or function. These studies illustrate a new function for Nkx2.5 in embryonic development and provide new avenues with which to pursue potential mechanisms underlying human facial disorders.

MEMO1 drives cranial endochondral ossification and palatogenesis.

The cranial base is a component of the neurocranium and has a central role in the structural integration of the face, brain and vertebral column. Consequently, alteration in the shape of the human cranial base has been intimately linked with primate evolution and defective development is associated with numerous human facial abnormalities. Here we describe a novel recessive mutant mouse strain that presented with a domed head and fully penetrant cleft secondary palate coupled with defects in the formation of the underlying cranial base. Mapping and non-complementation studies revealed a specific mutation in Memo1 - a gene originally associated with cell migration. Expression analysis of Memo1 identified robust expression in the perichondrium and periosteum of the developing cranial base, but only modest expression in the palatal shelves. Fittingly, although the palatal shelves failed to elevate in Memo1 mutants, expression changes were modest within the shelves themselves. In contrast, the cranial base, which forms via endochondral ossification had major reductions in the expression of genes responsible for bone formation, notably matrix metalloproteinases and markers of the osteoblast lineage, mirrored by an increase in markers of cartilage and extracellular matrix development. Concomitant with these changes, mutant cranial bases showed an increased zone of hypertrophic chondrocytes accompanied by a reduction in both vascular invasion and mineralization. Finally, neural crest cell-specific deletion of Memo1 caused a failure of anterior cranial base ossification indicating a cell autonomous role for MEMO1 in the development of these neural crest cell derived structures. However, palate formation was largely normal in these conditional mutants, suggesting a non-autonomous role for MEMO1 in palatal closure. Overall, these findings assign a new function to MEMO1 in driving endochondral ossification in the cranium, and also link abnormal development of the cranial base with more widespread effects on craniofacial shape relevant to human craniofacial dysmorphology.

Kabuki syndrome genes KMT2D and KDM6A: functional analyses demonstrate critical roles in craniofacial, heart and brain development.

Kabuki syndrome (KS) is a rare multiple congenital anomaly syndrome characterized by distinctive facial features, global developmental delay, intellectual disability and cardiovascular and musculoskeletal abnormalities. While mutations in KMT2D have been identified in a majority of KS patients, a few patients have mutations in KDM6A. We analyzed 40 individuals clinically diagnosed with KS for mutations in KMT2D and KDM6A. Mutations were detected in KMT2D in 12 and KDM6A in 4 cases, respectively. Observed mutations included single-nucleotide variations and indels leading to frame shifts, nonsense, missense or splice-site alterations. In two cases, we discovered overlapping chromosome X microdeletions containing KDM6A. To further elucidate the functional roles of KMT2D and KDM6A, we knocked down the expression of their orthologs in zebrafish. Following knockdown of kmt2d and the two zebrafish paralogs kdm6a and kdm6al, we analyzed morphants for developmental abnormalities in tissues that are affected in individuals with KS, including craniofacial structures, heart and brain. The kmt2d morphants exhibited severe abnormalities in all tissues examined. Although the kdm6a and kdm6al morphants had similar brain abnormalities, kdm6a morphants exhibited craniofacial phenotypes, whereas kdm6al morphants had prominent defects in heart development. Our results provide further support for the similar roles of KMT2D and KDM6A in the etiology of KS by using a vertebrate model organism to provide direct evidence of their roles in the development of organs and tissues affected in KS patients.

Hand1 phosphoregulation within the distal arch neural crest is essential for craniofacial morphogenesis.

In this study we examine the consequences of altering Hand1 phosphoregulation in the developing neural crest cells (NCCs) of mice. Whereas Hand1 deletion in NCCs reveals a nonessential role for Hand1 in craniofacial development and embryonic survival, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, in NCCs results in severe mid-facial clefting and neonatal death. Hand1 phosphorylation mutants exhibit a non-cell-autonomous increase in pharyngeal arch cell death accompanied by alterations in Fgf8 and Shh pathway expression. Together, our data indicate that the extreme distal pharyngeal arch expression domain of Hand1 defines a novel bHLH-dependent activity, and that disruption of established Hand1 dimer phosphoregulation within this domain disrupts normal craniofacial patterning.

Cre recombinase-regulated Endothelin1 transgenic mouse lines: novel tools for analysis of embryonic and adult disorders.

Endothelin-1 (EDN1) influences both craniofacial and cardiovascular development and a number of adult physiological conditions by binding to one or both of the known endothelin receptors, thus initiating multiple signaling cascades. Animal models containing both conventional and conditional loss of the Edn1 gene have been used to dissect EDN1 function in both embryos and adults. However, while transgenic Edn1 over-expression or targeted genomic insertion of Edn1 has been performed to understand how elevated levels of Edn1 result in or exacerbate disease states, an animal model in which Edn1 over-expression can be achieved in a spatiotemporal-specific manner has not been reported. Here we describe the creation of Edn1 conditional over-expression transgenic mouse lines in which the chicken ß-actin promoter and an Edn1 cDNA are separated by a strong stop sequence flanked by loxP sites. In the presence of Cre, the stop cassette is removed, leading to Edn1 expression. Using the Wnt1-Cre strain, in which Cre expression is targeted to the Wnt1-expressing domain of the central nervous system (CNS) from which neural crest cells (NCCs) arise, we show that stable chicken ß-actin-Edn1 (CBA-Edn1) transgenic lines with varying EDN1 protein levels develop defects in NCC-derived tissues of the face, though the severity differs between lines. We also show that Edn1 expression can be achieved in other embryonic tissues utilizing other Cre strains, with this expression also resulting in developmental defects. CBA-Edn1 transgenic mice will be useful in investigating diverse aspects of EDN1-mediated-development and disease, including understanding how NCCs achieve and maintain a positional and functional identity and how aberrant EDN1 levels can lead to multiple physiological changes and diseases.

Downregulation of Dlx5 and Dlx6 expression by Hand2 is essential for initiation of tongue morphogenesis.

Lower jaw development is a complex process in which multiple signaling cascades establish a proximal-distal organization. These cascades are regulated both spatially and temporally and are constantly refined through both induction of normal signals and inhibition of inappropriate signals. The connective tissue of the tongue arises from cranial neural crest cell-derived ectomesenchyme within the mandibular portion of the first pharyngeal arch and is likely to be impacted by this signaling. Although the developmental mechanisms behind later aspects of tongue development, including innervation and taste acquisition, have been elucidated, the early patterning signals driving ectomesenchyme into a tongue lineage are largely unknown. We show here that the basic helix-loop-helix transcription factor Hand2 plays key roles in establishing the proximal-distal patterning of the mouse lower jaw, in part through establishing a negative-feedback loop in which Hand2 represses Dlx5 and Dlx6 expression in the distal arch ectomesenchyme following Dlx5- and Dlx6-mediated induction of Hand2 expression in the same region. Failure to repress distal Dlx5 and Dlx6 expression results in upregulation of Runx2 expression in the mandibular arch and the subsequent formation of aberrant bone in the lower jaw along with proximal-distal duplications. In addition, there is an absence of lateral lingual swelling expansion, from which the tongue arises, resulting in aglossia. Hand2 thus appears to establish a distal mandibular arch domain that is conducive for lower jaw development, including the initiation of tongue mesenchyme morphogenesis.

Redundant roles of PRDM family members in zebrafish craniofacial development.

BACKGROUND: PRDM proteins are evolutionary conserved Zn-Finger transcription factors that share a characteristic protein domain organization. Previous studies have shown that prdm1a is required for the specification and differentiation of neural crest cells in the zebrafish. RESULTS: Here we examine other members of this family, specifically prdm3, 5, and 16, in the differentiation of the zebrafish craniofacial skeleton. prdm3 and prdm16 are strongly expressed in the pharyngeal arches, while prdm5 is expressed specifically in the area of the forming neurocranium. Knockdown of prdm3 and prdm16 results in a reduction in the neural crest markers dlx2a and barx1 and defects in both the viscerocranium and the neurocranium. The knockdown of prdm3 and prdm16 in combination is additive in the neurocranium, but not in the viscerocranium. Injection of sub-optimal doses of prdm1a with prdm3 or prdm16 Morpholinos together leads to more severe phenotypes in the viscerocranium and neurocranium. prdm5 mutants have defects in the neurocranium and prdm1a and prdm5 double mutants also show more severe phenotypes. CONCLUSIONS: Overall, our data reveal that prdm3, 5, and 16 are involved in the zebrafish craniofacial development and that prdm1a may interact with prdm3, 5, and 16 in the formation of the craniofacial skeleton in zebrafish.