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.

Human stem cell model of neural crest cell differentiation reveals a requirement of SF3B4 in survival, maintenance, and differentiation.

In vitro modeling is a powerful approach to investigate the pathomechanisms driving human congenital conditions. Here we use human embryonic stem cells (hESCs) to model Nager and Rodriguez syndromes, two craniofacial conditions characterized by hypoplastic neural crest-derived craniofacial bones, caused by pathogenic variants of SF3B4, a core component of the spliceosome. We observed that siRNA-mediated knockdown of SF3B4 interferes with the production of hESC-derived neural crest cells, as seen by a marked reduction in neural crest gene expression. This phenotype is associated with an increase in neural crest cell apoptosis and their premature neuronal differentiation. Together, these results point at a role of SF3B4 in neural crest cell survival, maintenance, and differentiation as the primary cause of Nager/Rodriguez syndrome associated craniofacial defects, and illustrate the benefit of in vitro human stem cell models to understand congenital diseases.

Sf3b4 mutation in Xenopus tropicalis causes RNA splicing defects followed by massive gene dysregulation that disrupt cranial neural crest development.

Nager syndrome is a rare craniofacial and limb disorder characterized by midface retrusion, micrognathia, absent thumbs, and radial hypoplasia. This disorder results from haploinsufficiency of SF3B4 (splicing factor 3b, subunit 4) a component of the pre-mRNA spliceosomal machinery. The spliceosome is a complex of RNA and proteins that function together to remove introns and join exons from transcribed pre-mRNA. While the spliceosome is present and functions in all cells of the body, most spliceosomopathies - including Nager syndrome - are cell/tissue-specific in their pathology. In Nager syndrome patients, it is the neural crest (NC)-derived craniofacial skeletal structures that are primarily affected. To understand the pathomechanism underlying this condition, we generated a Xenopus tropicalis sf3b4 mutant line using the CRISPR/Cas9 gene editing technology. Here we describe the sf3b4 mutant phenotype at neurula, tail bud, and tadpole stages, and performed temporal RNA-sequencing analysis to characterize the splicing events and transcriptional changes underlying this phenotype. Our data show that while loss of one copy of sf3b4 is largely inconsequential in Xenopus tropicalis, homozygous deletion of sf3b4 causes major splicing defects and massive gene dysregulation, which disrupt cranial NC cell migration and survival, thereby pointing at an essential role of Sf3b4 in craniofacial development.

In vitro modeling of cranial placode differentiation: Recent advances, challenges, and perspectives.

Cranial placodes are transient ectodermal thickenings that contribute to a diverse array of organs in the vertebrate head. They develop from a common territory, the pre-placodal region that over time segregates along the antero-posterior axis into individual placodal domains: the adenohypophyseal, olfactory, lens, trigeminal, otic, and epibranchial placodes. These placodes terminally differentiate into the anterior pituitary, the lens, and contribute to sensory organs including the olfactory epithelium, and inner ear, as well as several cranial ganglia. To study cranial placodes and their derivatives and generate cells for therapeutic purposes, several groups have turned to in vitro derivation of placodal cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs). In this review, we summarize the signaling cues and mechanisms involved in cranial placode induction, specification, and differentiation in vivo, and discuss how this knowledge has informed protocols to derive cranial placodes in vitro. We also discuss the benefits and limitations of these protocols, and the potential of in vitro cranial placode modeling in regenerative medicine to treat cranial placode-related pathologies.

Gene expression in notochord and nuclei pulposi: a study of gene families across the chordate phylum.

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Developmental roles of natriuretic peptides and their receptors.

Natriuretic peptides and their receptors are implicated in the physiological control of blood pressure, bone growth, and cardiovascular and renal homeostasis. They mediate their action through the modulation of intracellular levels of cGMP and cAMP, two second-messengers that have broad biological roles. In this review, we briefly describe the major players of this signaling pathway and their physiological roles in the adult, and discuss several reports describing their activity in the control of various aspects of embryonic development in several species. While the core components of this signaling pathway are well conserved, their functions have diverged in the embryo and the adult to control a diverse array of biological processes.

Paracrine regulation of neural crest EMT by placodal MMP28.

Epithelial-mesenchymal transition (EMT) is an early event in cell dissemination from epithelial tissues. EMT endows cells with migratory, and sometimes invasive, capabilities and is thus a key process in embryo morphogenesis and cancer progression. So far, matrix metalloproteinases (MMPs) have not been considered as key players in EMT but rather studied for their role in matrix remodelling in later events such as cell migration per se. Here, we used Xenopus neural crest cells to assess the role of MMP28 in EMT and migration in vivo. We show that a catalytically active MMP28, expressed by neighbouring placodal cells, is required for neural crest EMT and cell migration. We provide strong evidence indicating that MMP28 is imported in the nucleus of neural crest cells where it is required for normal Twist expression. Our data demonstrate that MMP28 can act as an upstream regulator of EMT in vivo raising the possibility that other MMPs might have similar early roles in various EMT-related contexts such as cancer, fibrosis, and wound healing.

Npr3 regulates neural crest and cranial placode progenitors formation through its dual function as clearance and signaling receptor.

Natriuretic peptide signaling has been implicated in a broad range of physiological processes, regulating blood volume and pressure, ventricular hypertrophy, fat metabolism, and long bone growth. Here, we describe a completely novel role for natriuretic peptide signaling in the control of neural crest (NC) and cranial placode (CP) progenitors formation. Among the components of this signaling pathway, we show that natriuretic peptide receptor 3 (Npr3) plays a pivotal role by differentially regulating two developmental programs through its dual function as clearance and signaling receptor. Using a combination of MO-based knockdowns, pharmacological inhibitors and rescue assays we demonstrate that Npr3 cooperate with guanylate cyclase natriuretic peptide receptor 1 (Npr1) and natriuretic peptides (Nppa/Nppc) to regulate NC and CP formation, pointing at a broad requirement of this signaling pathway in early embryogenesis. We propose that Npr3 acts as a clearance receptor to regulate local concentrations of natriuretic peptides for optimal cGMP production through Npr1 activation, and as a signaling receptor to control cAMP levels through inhibition of adenylyl cyclase. The intracellular modulation of these second messengers therefore participates in the segregation of NC and CP cell populations.

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.

The Core Splicing Factors EFTUD2, SNRPB and TXNL4A Are Essential for Neural Crest and Craniofacial Development.

Mandibulofacial dysostosis (MFD) is a human congenital disorder characterized by hypoplastic neural-crest-derived craniofacial bones often associated with outer and middle ear defects. There is growing evidence that mutations in components of the spliceosome are a major cause for MFD. Genetic variants affecting the function of several core splicing factors, namely SF3B4, SF3B2, EFTUD2, SNRPB and TXNL4A, are responsible for MFD in five related but distinct syndromes known as Nager and Rodriguez syndromes (NRS), craniofacial microsomia (CFM), mandibulofacial dysostosis with microcephaly (MFDM), cerebro-costo-mandibular syndrome (CCMS) and Burn-McKeown syndrome (BMKS), respectively. Animal models of NRS and MFDM indicate that MFD results from an early depletion of neural crest progenitors through a mechanism that involves apoptosis. Here we characterize the knockdown phenotype of Eftud2, Snrpb and Txnl4a in Xenopus embryos at different stages of neural crest and craniofacial development. Our results point to defects in cranial neural crest cell formation as the likely culprit for MFD associated with EFTUD2, SNRPB and TXNL4A haploinsufficiency, and suggest a commonality in the etiology of these craniofacial spliceosomopathies.

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.

Xbp1 and Brachyury establish an evolutionarily conserved subcircuit of the notochord gene regulatory network.

Gene regulatory networks coordinate the formation of organs and structures that compose the evolving body plans of different organisms. We are using a simple chordate model, the Ciona embryo, to investigate the essential gene regulatory network that orchestrates morphogenesis of the notochord, a structure necessary for the proper development of all chordate embryos. Although numerous transcription factors expressed in the notochord have been identified in different chordates, several of them remain to be positioned within a regulatory framework. Here, we focus on Xbp1, a transcription factor expressed during notochord formation in Ciona and other chordates. Through the identification of Xbp1-downstream notochord genes in Ciona, we found evidence of the early co-option of genes involved in the unfolded protein response to the notochord developmental program. We report the regulatory interplay between Xbp1 and Brachyury, and by extending these results to Xenopus, we show that Brachyury and Xbp1 form a cross-regulatory subcircuit of the notochord gene regulatory network that has been consolidated during chordate evolution.

Anterior patterning genes induced by Zic1 are sensitive to retinoic acid and its metabolite, 4-oxo-RA.

BACKGROUND: Development of paired sensory organs is a highly complex and coordinated process. These organs arise from ectodermal thickenings in the cephalic region known as cranial placodes. We have previously shown that Zic1 is a critical regulator for the formation of the pre-placodal region (PPR), the common territory for the development of all cranial placodes in Xenopus laevis. RESULTS: In this study, we have analyzed a number of Zic1 targets for their expression during PPR patterning, as well as their regulation by retinoic acid (RA) and one of its major metabolites, 4-oxo-RA. Our findings show that anteriorly Zic1 regulates several transcription factors, Crx, Fezf2, Nkx3-1, and Xanf1 as well as a serine/threonine/tyrosine kinase, Pkdcc.2. These factors are all expressed in the vicinity of the PPR and as such are candidate regulators of placode formation downstream of Zic1. In addition to their differential regulation by RA, we find that 4-oxo-RA is also capable of modulating the expression of these genes, as well as a broad array of RA-regulated genes. CONCLUSION: Our data highlight the complexity of retinoid-mediated regulation required for Zic1-activated anterior structure specification in Xenopus, and the potential physiological role of 4-oxo-RA in cranial placode development.

Function of chromatin modifier Hmgn1 during neural crest and craniofacial development.

The neural crest is a dynamic embryonic structure that plays a major role in the formation of the vertebrate craniofacial skeleton. Neural crest formation is regulated by a complex sequence of events directed by a network of transcription factors working in concert with chromatin modifiers. The high mobility group nucleosome binding protein 1 (Hmgn1) is a nonhistone chromatin architectural protein, associated with transcriptionally active chromatin. Here we report the expression and function of Hmgn1 during Xenopus neural crest and craniofacial development. Hmgn1 is broadly expressed at the gastrula and neurula stages, and is enriched in the head region at the tailbud stage, especially in the eyes and the pharyngeal arches. Hmgn1 knockdown affected the expression of several neural crest specifiers, including sox8, sox10, foxd3, and twist1, while other genes (sox9 and snai2) were only marginally affected. The specificity of this phenotype was confirmed by rescue, where injection of Hmgn1 mRNA was able to restore sox10 expression in morphant embryos. The reduction in neural crest gene expression at the neurula stage in Hmgn1 morphant embryos correlated with a decreased number of sox10- and twist1-positive cells in the pharyngeal arches at the tailbud stage, and hypoplastic craniofacial cartilages at the tadpole stage. These results point to a novel role for Hmgn1 in the control of gene expression essential for neural crest and craniofacial development. Future work will investigate the precise mode of action of Hmgn1 in this context.

Haploinsufficiency of SF3B2 causes craniofacial microsomia.

Craniofacial microsomia (CFM) is the second most common congenital facial anomaly, yet its genetic etiology remains unknown. We perform whole-exome or genome sequencing of 146 kindreds with sporadic (n = 138) or familial (n = 8) CFM, identifying a highly significant burden of loss of function variants in SF3B2 (P = 3.8 × 10-10), a component of the U2 small nuclear ribonucleoprotein complex, in probands. We describe twenty individuals from seven kindreds harboring de novo or transmitted haploinsufficient variants in SF3B2. Probands display mandibular hypoplasia, microtia, facial and preauricular tags, epibulbar dermoids, lateral oral clefts in addition to skeletal and cardiac abnormalities. Targeted morpholino knockdown of SF3B2 in Xenopus results in disruption of cranial neural crest precursor formation and subsequent craniofacial cartilage defects, supporting a link between spliceosome mutations and impaired neural crest development in congenital craniofacial disease. The results establish haploinsufficient variants in SF3B2 as the most prevalent genetic cause of CFM, explaining ~3% of sporadic and ~25% of familial cases.

Molecular mechanisms of hearing loss in Nager syndrome.

Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome.

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.

Retinoic acid production, regulation and containment through Zic1, Pitx2c and Cyp26c1 control cranial placode specification.

All paired sensory organs arise from a common precursor domain called the pre-placodal region (PPR). In Xenopus, Zic1 non-cell autonomously regulates PPR formation by activating retinoic acid (RA) production. Here, we have identified two Zic1 targets, the RA catabolizing enzyme Cyp26c1 and the transcription factor Pitx2c, expressed in the vicinity of the PPR as being crucially required for maintaining low RA levels in a spatially restricted, PPR-adjacent domain. Morpholino- or CRISPR/Cas9-mediated Cyp26c1 knockdown abrogated PPR gene expression, yielding defective cranial placodes. Direct measurement of RA levels revealed that this is mediated by a mechanism involving excess RA accumulation. Furthermore, we show that pitx2c is activated by RA and required for Cyp26c1 expression in a domain-specific manner through induction of FGF8. We propose that Zic1 anteriorly establishes a program of RA containment and regulation through activation of Cyp26c1 and Pitx2c that cooperates to promote PPR specification in a spatially restricted domain.