Neural Crest Research Articles

7206 Perioperative Management of Pheochromocytoma: The Mayo Clinic Florida Experience

Abstract Disclosure: A. Verma: None. F. Elli: None. S. Rao: None. M. Smith: None. A. Shapiro: None. C. Moshe: None. R. Chadha: None. Introduction: Pheochromocytomas are rare, neuroendocrine tumors derived from chromaffin cells of the neural crest, typically within the adrenal medulla. The pathophysiologic effect of primary concern is the uncontrolled release of catecholamines into the bloodstream, which carry risk for episodic hypertension, palpitations, and life-threatening crises. Preoperative preparation with drugs that block adrenergic receptors is utilized to improve hemodynamic oscillation force and surgical outcomes. Widely used premedication include alpha-blockers, beta-blockers, and calcium channel blockers. Despite adequate pretreatment, hemodynamic instability continues to evoke massive risk. The objective of this study was to assess the incidence of intraoperative hypertensive episodes in patients undergoing robotic assisted laparoscopic adrenalectomy. Methods: This study is an observational retrospective analysis of patients who underwent elective laparoscopic robotic-assisted adrenalectomies from February 2019 to April 2023. 17 patients total patients were included. Preoperative information included age, BMI, ASA physical classification, preoperative blockade regiment, and urine metanephrines. Intraoperative information included operation duration, tumor size, intraoperative anti-hypertensives, and hypertensive spikes defined as a SBP greater than 175 mmHg for 5 consecutive minutes. Data analysis was performed to obtain frequency counts (%), means (standard deviations), and median (range) as appropriate. Results: The mean age at the time of surgery was 57.12 ± 17.2 years. The majority of patients (76%) were ASA status class III. 7 of the 17 patients did not have active pheochromocytoma based on symptomatology, the incidental diagnoses being made by imaging and workup. The average tumor diameter was 3.90 ± 1.8, with a range of 2.0 - 8.0. Mean operative time was 153.76 ± 53.9 minutes. Preoperative urinary metanephrines were observed to have a mean of 1,248.44 ± 2,866.9 pg/mL. In terms of preoperative preparation, 65% of patients received Doxazosin, and 29% of patients received an alternative anti-hypertensive. Yet, 64.7% of patients were found to have at least 1 intraoperative hypertensive spike Conclusion: Despite premedication with anti-hypertensives, a significant percentage of patients experienced intraoperative hemodynamic instability. This emphasizes the importance of anesthesiologist preparation for intraoperative hypertension management to aggressively prevent hypertensive crises. In addition, it questions the routine use of alpha blockade for all cases preoperatively as has been suggested in certain studies, and whether or not it should be driven by other patient characteristics. Presentation: 6/1/2024

8715 Cholinergic Signaling in the Mouse Embryonic Pancreas Targets Early Endocrine Cells

Abstract Disclosure: C. Duarte: None. J.K. Panzer: None. N. Borowsky: None. A. Caicedo: None. The parasympathetic vagus nerve affects pancreas function by activating local intrapancreatic neurons that release acetylcholine (ACh). These neurons are derived from progenitors of the vagal neural crest and colonize the pancreas during embryonic development. The specific mechanisms by which ACh is released from these neurons influence embryonic development of the islet and, specifically, beta cells remain largely unknown. The purpose of this study was to identify cholinergic signaling components and assess early physiological responses to ACh during murine development of endocrine lineage cells. We extracted pancreatic rudiments from mice at embryonic stages E15 to E18 and stained them for neuronal and endocrine markers, along with components associated with cholinergic signaling. We further performed Ca2+ imaging of the pancreatic rudiments from NGN3CRE-GCaMP3 mice to record responses to ACh in cells of the endocrine lineage. At E15, we observed a significantly higher concentration of glucagon+ cells compared to insulin+ cells, consistent with the earlier differentiation of alpha cells in the developing pancreas. Neuronal body clusters were identified in close proximity to endocrine lineage cells from E15 to E18, with the neuronal network becoming more dispersed and interconnected. At E15, wide distribution of the presynaptic vesicular acetylcholine transporter (VAChT) was observed later becoming localized around insulin+ and glucagon+ cells at E18. Postsynaptic acetylcholinesterase (AChE) colocalized with glucagon+ cells from E15 to E18 and with insulin+ cells by E18. ACh elicited Ca2+ responses in individual endocrine cells as early as E15. Our results show that cholinergic nerve terminals target early endocrine cells and elicit responses to ACh, indicating that local cholinergic neurons innervate and activate endocrine progenitors in the embryonic pancreas. Our current studies are aimed at determining how cholinergic signaling contributes to beta cell differentiation and function. We expect this knowledge to guide future protocols for deriving beta cells from stem cells. Presentation: 6/2/2024

Comparison of human pluripotent stem cell differentiation protocols to generate neuroblastoma tumors

Neuroblastoma is the most common pediatric extracranial solid tumor and is derived from trunk neural crest cells (tNCC) and its progenitor sympathoadrenal (SA) cells. While human pluripotent stem cell (PSC) models of neuroblastoma have been described, the PSC were differentiated using protocols that made neural crest cells, but not specifically the trunk subtype. Here, we compared four recent protocols to differentiate pluripotent stem cells (PSC) toward SA cells and examined their efficiency at generating SA cells along with earlier cell states (neuromesodermal progenitors [NMP], tNCC), as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two PSC lines of different genders. This protocol also generated tumors with the highest level of PHOX2B, a marker of neuroblastoma. Transcriptionally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma.

Conditional heterozygous loss of kit receptor tyrosine kinase in neural crest cell lineage is associated with midline cleft lip and bifid nose deformity

ObjectivesThe receptor tyrosine kinase Kit is expressed in cells derived from the trunk neural crest (NC), such as melanocytes; however, its role in cranial NC cell development is not fully understood. MethodsWe investigated the effects of the heterozygous loss of Kit in NC cells during embryonic development by mating Kit2lox/+ mice with Wnt1-Cre mice to produce Wnt1-Cre; Kit2lox/+ embryos. In addition, Wnt1-Cre mice were mated with Rosa26R-yellow fluorescent protein (YFP) mice to visualize the tissue regions expressing Cre recombinase. Histological studies of the craniofacial regions of these mice were performed using samples from embryonic day (E) 12.5 and postnatal day (P) 1. Cellular apoptosis and proliferation were both analyzed through the immunostaining of tissue sections collected on E13.5 and E14.5 using anti-cleaved caspase 3 (CC3) to detect apoptosis and anti-Ki67 to detect proliferation. Cells from YFP-positive tissue regions of the facial areas of Wnt1-Cre; Kit+/+; Rosa26R–YFP embryos and Wnt1-Cre; Kit2lox/+; Rosa26R–YFP embryos collected on E12.5 and E15.5 were cultured and evaluated for cell proliferation. ResultsCompared with control littermates, Wnt1-Cre; Kit2lox/+ embryos exhibited midline cleft lip and bifid nose deformities. Substantial early (P1) postnatal lethality was observed in Wnt1-Cre; Kit2lox/+ mice, with none surviving to 3 weeks of age. YFP-positive cells from the maxillary regions of Wnt1-Cre; Kit2lox/+; Rosa26R–YFP embryos exhibited defective cell growth and self-renewal in vitro. ConclusionConditional heterozygous loss of Kit in Wnt1-Cre; Kit2lox/+ embryos is associated with craniofacial dysplasia and exhibit defective NC development in vitro and in vivo.

Neural crest precursors from the skin are the primary source of directly reprogrammed neurons

Direct reprogramming involves the conversion of differentiated cell types without returning to an earlier developmental state. Here, we explore how heterogeneity in developmental lineage and maturity of the starting cell population contributes to direct reprogramming using the conversion of murine fibroblasts into neurons. Our hypothesis is that a single lineage of cells contributes to most reprogramming and that a rare elite precursor with intrinsic bias is the source of reprogrammed neurons. We find that nearly all reprogrammed neurons are derived from the neural crest (NC) lineage. Moreover, when rare proliferating NC precursors are selectively ablated, there is a large reduction in the number of reprogrammed neurons. Previous interpretations of this paradigm are that it demonstrates a cell fate conversion across embryonic germ layers (mesoderm to ectoderm). Our interpretation is that this is actually directed differentiation of a neural lineage stem cell in the skin that has intrinsic bias to produce neuronal progeny.

Validation of non-destructive morphology-based selection of cerebral cortical organoids by paired morphological and single-cell RNA-seq analyses

Organoids, self-organized cell aggregates, contribute significantly to developing disease models and cell-based therapies. Organoid-to-organoid variations, however, are inevitable despite the use of the latest differentiation protocols. Here, we focused on the morphology of organoids formed in a cerebral organoid differentiation culture and assessed their cellular compositions by single-cell RNA sequencing analysis. The data revealed that organoids primarily composed of non-neuronal cells, such as those from the neural crest and choroid plexus, showed unique morphological features. Moreover, we demonstrate that non-destructive morphological analysis can accurately distinguish organoids composed of cerebral cortical tissues from other cerebral tissues, thus enhancing experimental accuracy and reliability to ensure the safety of cell-based therapies.

Variations in non-local interaction range lead to emergent chase-and-run in heterogeneous populations.

In a chase-and-run dynamic, the interaction between two individuals is such that one moves towards the other (the chaser), while the other moves away (the runner). Examples can be found in both interacting cells and animals. Here, we investigate the behaviours that can emerge at a population level, for a heterogeneous group that contains subpopulations of chasers and runners. We show that a wide variety of patterns can form, from stationary patterns to oscillatory and population-level chase-and-run, where the latter describes a synchronized collective movement of the two populations. We investigate the conditions under which different behaviours arise, specifically focusing on the interaction ranges: the distances over which cells or organisms can sense one another's presence. We find that when the interaction range of the chaser is sufficiently larger than that of the runner-or when the interaction range of the chase is sufficiently larger than that of the run-population-level chase-and-run emerges in a robust manner. We discuss the results in the context of phenomena observed in cellular and ecological systems, with particular attention to the dynamics observed experimentally within populations of neural crest and placode cells.

Cleft Palate Induced by Augmented Fibroblast Growth Factor-9 Signaling in Cranial Neural Crest Cells in Mice.

Although enhanced fibroblast growth factor (FGF) signaling has been demonstrated to be crucial in many cases of syndromic cleft palate caused by tongue malposition in humans, animal models that recapitulate this phenotype are limited, and the precise mechanisms remain elusive. Mutations in FGF9 with the effect of either loss- or gain-of-function effects have been identified to be associated with cleft palate in humans. Here, we generated a mouse model with a transgenic Fgf9 allele specifically activated in cranial neural crest cells, aiming to elucidate the gain-of-function effects of Fgf9 in palatogenesis. We observed cleft palate with 100% penetrance in mutant mice. Further analysis demonstrated that no inherent defects in the morphogenic competence of palatal shelves could be found, but a passively lifted tongue prevented the elevation of palatal shelves, leading to the cleft palate. This tongue malposition was induced by posterior spatial confinement that was exerted by temporomandibular joint (TMJ) dysplasia characterized by a reduction in Sox9+ progenitors within the condyle and a structural decrease in the posterior dimension of the lower jaw. Our findings highlight the critical role of excessive FGF signaling in disrupting spatial coordination during palate development and suggest a potential association between palatal shelf elevation and early TMJ development.

Single-cell transcriptomic resolution of osteogenesis during craniofacial morphogenesis

Craniofacial morphogenesis depends on complex cell fate decisions during the differentiation of post-migratory cranial neural crest cells. Molecular mechanisms of cell differentiation of mesenchymal cells to developing bones, cartilage, teeth, tongue, and other craniofacial tissues are still poorly understood. We performed single-cell transcriptomic analysis of craniofacial mesenchymal cells derived from cranial NCCs in mouse embryo. Using FACS sorting of Wnt1-Cre2 progeny, we carefully mapped the cell heterogeneity in the craniofacial region during the initial stages of cartilage and bone formation. Transcriptomic data and in vivo validations identified molecular determinants of major cell populations involved in the development of lower and upper jaw, teeth, tongue, dermis, or periocular mesenchyme. Single-cell transcriptomic analysis of Meis2-deficient mice revealed critical gene expression differences, including increased osteogenic and cell adhesion markers. This leads to affected mesenchymal cell differentiation and increased ossification, resulting in impaired bone, cartilage, and tongue formation.

The primary cilia: Orchestrating cranial neural crest cell development

Primary cilia (hereafter “cilia”) are microtubule-based antenna-like organelles projecting from the surface of vertebrate cells. Cilia can serve as cellular antennae controlling cell growth and differentiation. Absent or dysfunctional cilia frequently lead to craniofacial anomalies known as craniofacial ciliopathies. However, the detailed pathological mechanisms of craniofacial ciliopathies remain unclear. This perspective discusses our current understanding of the role of cilia in cranial neural crest cells. We also describe potential mechanisms of ciliogenesis in cranial neural crest cells, which may contribute to unraveling the complex pathogenesis of craniofacial ciliopathies.

The important role of the Wnt/β-catenin signaling pathway in small molecules mediated gingival mesenchymal stem cells transdifferentiate into neuron-like cells

ObjectiveGiven their neural crest origin, gingival mesenchymal stem cells (GMSCs) possess high neurogenic potential, which makes them suitable for cell replacement therapy against neurodegenerative diseases. This study investigated whether GMSCs can be transdifferentiated into neurons in vitro using a protocol involving small molecules VCRFY (VPA, CHIR99021, Repsox, Forskolin, and Y-27632). The regulatory mechanisms of key signaling pathways were also investigated. MethodsNeuronal induction of GMSCs was conducted using a small molecules-based protocol over 7 days, which included the evaluation of cell morphology, proliferation, expressions of neurogenic markers, and intracellular calcium oscillation. The activation of canonical the Wnt signaling pathway was assessed by examining the protein content and subcellular localization of β-catenin. ResultsSmall molecules-treated GMSCs displayed neuronal morphology and increased expression of neurogenic markers, including class III beta-tubulin (TUJ1), neuron-specific enolase (NSE), microtube-associated protein 2 (MAP2), and neurofilament medium (NFM), verified through RT-qPCR, western blotting, and immunocytochemistry. Based on the results of Fluo-4 AM calcium flux assay, small molecules-treated GMSCs exhibited enhanced electrophysiological activity. GMSC proliferation halted after 2 days of treatment. Among the small molecules, CHIR99021 exhibited the highest neuronal induction efficiency. Furthermore, activation of the Wnt/β-catenin signaling pathway augmented neuronal differentiation. ConclusionsSmall molecule-based cellular reprogramming can efficiently generate neurons from GMSCs, with Wnt/β-catenin signaling to play a critical role in neuronal induction.

Alternative Balance between Transcriptional and Epigenetic Regulation during Developmental Proliferation of Human Cranial Neural Crest Cells.

Cranial neural crest cells are implicated in multiple transcriptional events at the different stages of differentiation during development. The alteration of some transcription factors expressed during neural crest development, like PAX7, could be implicated in the etiology of face malformation in murine models. Epigenetic regulation has been shown to be an important mechanistic actor in the control of timing and the level of gene expression at different stages of neural crest development. During this work, we investigated the interconnection between epigenetics and transcription factors across a diversity of human development cranial neural crest cells. Across a diversity of neural cells from human developing cranial tissues, in accordance with their proliferation stage, an alternative balance of regulation between transcription factors and epigenetic factors was identified.

Retinoic acid, an essential component of the roof plate organizer, promotes the spatiotemporal segregation of dorsal neural fates.

Dorsal neural tube-derived retinoic acid promotes the end of neural crest production and transition into a definitive roof plate. Here, we analyze how this impacts the segregation of central and peripheral lineages, a process essential for tissue patterning and function. Localized in ovo inhibition in quail embryos of retinoic acid activity followed by single-cell transcriptomics unraveled a comprehensive list of differentially expressed genes relevant to these processes. Importantly, progenitors co-expressed neural crest, roof plate and dI1 interneuron markers, indicating a failure in proper lineage segregation. Furthermore, separation between roof plate and dI1 interneurons is mediated by Notch activity downstream of retinoic acid, highlighting their crucial role in establishing the roof plate-dI1 boundary. Within the peripheral branch, where absence of retinoic acid resulted in neural crest production and emigration extending into the roof plate stage, sensory progenitors failed to separate from melanocytes, leading to formation of a common glia-melanocyte cell with aberrant migratory patterns. In summary, the implementation of single-cell RNA sequencing facilitated the discovery and characterization of a molecular mechanism responsible for the segregation of dorsal neural fates during development.

Pharmacologically Targeting Ferroptosis and Cuproptosis in Neuroblastoma.

Neuroblastoma is a deadly pediatric cancer that originates from the neural crest and frequently develops in the abdomen or adrenal gland. Although multiple approaches, including chemotherapy, radiotherapy, targeted therapy, and immunotherapy, are recommended for treating neuroblastoma, the tumor will eventually develop resistance, leading to treatment failure and cancer relapse. Therefore, a firm understanding of the molecular mechanisms underlying therapeutic resistance is vital for the development of new effective therapies. Recent research suggests that cancer-specific modifications to multiple subtypes of nonapoptotic regulated cell death (RCD), such as ferroptosis and cuproptosis, contribute to therapeutic resistance in neuroblastoma. Targeting these specific types of RCD may be viable novel targets for future drug discovery in the treatment of neuroblastoma. In this review, we summarize the core mechanisms by which the inability to properly execute ferroptosis and cuproptosis can enhance the pathogenesis of neuroblastoma. Therefore, we focus on emerging therapeutic compounds that can induce ferroptosis or cuproptosis, delineating their beneficial pharmacodynamic effects in neuroblastoma treatment. Cumulatively, we suggest that the pharmacological stimulation of ferroptosis and ferroptosis may be a novel and therapeutically viable strategy to target neuroblastoma.

Genomic mosaicism reveals developmental organization of trunk neural crest-derived ganglia.

The neural crest generates numerous cell types, but conflicting results leave developmental origins unresolved. Here using somatic mosaic variants as cellular barcodes, we infer embryonic clonal dynamics of trunk neural crest, focusing on the sensory and sympathetic ganglia. From three independent adult neurotypical human donors, we identified 1,278 mosaic variants using deep whole-genome sequencing, then profiled allelic fractions in 187 anatomically dissected ganglia. We found a massive rostrocaudal spread of progenitor clones specific to sensory or sympathetic ganglia, which unlike in the brain, showed robust bilateral distributions. Computational modeling suggested neural crest progenitor fate specification preceded delamination from neural tube. Single-cell multiomic analysis suggested both neurons and glia contributed to the rostrocaudal clonal organization. CRISPR barcoding in mice and live imaging in quail embryos confirmed these clonal dynamics across multiple somite levels. Our findings reveal an evolutionarily conserved clonal spread of cells populating peripheral neural ganglia.

Microgravity Effect on Pancreatic Islets.

We previously demonstrated that boundary cap neural crest stem cells (BCs) induce the proliferation of beta-cells in vitro, increase survival of pancreatic islets (PIs) in vivo after transplantation, and themselves strongly increase their proliferation capacity after exposure to space conditions. Therefore, we asked if space conditions can induce the proliferation of beta-cells when PIs are alone or together with BCs in free-floating or 3D-printed form. During the MASER 15 sounding rocket experiment, half of the cells were exposed to 6 min of microgravity (µg), whereas another group of cells were kept in 1 g conditions in a centrifuge onboard. The proliferation marker EdU was added to the cells just before the rocket reached µg conditions. The morphological assessment revealed that PIs successfully survived and strongly proliferated, particularly in the free-floating condition, though the fusion of PIs hampered statistical analysis. Proliferation of beta-cells was displayed in 3D-printed islets two weeks after µg exposure, suggesting that the effects of µg may be delayed. Thus, PIs in 3D-printed scaffolds did not fuse, and this preparation is more suitable than free-floating specimens for morphological analysis in µg studies. PIs maintained their increased proliferation capacity for weeks after µg exposure, an effect that may not appear directly, but can emerge after a delay.

Immunohistochemical evaluation of CD34, CD117, and calretinin for diagnosis of hirschsprung’s disease

IntroductionHirschsprung’s disease (HD) is a neurogenic intestinal disorder attributed to incomplete neural crest cell migration during fetal intestinal development, leading to an aganglionic segment of the colon and functional obstruction. Associated malformations like intestinal atresia, hydronephrosis, and imperforate anus can accompany Hirschsprung’s disease. this study aims to evaluate the efficacy of Calretinin and Cajal cells (CD34 and CD117) immunohistochemical staining in improving HD diagnosis.MethodsThe study involved 70 pediatric patients suspected of Hirschsprung’s disease. Clinical, histopathological, and immunohistochemical analyses were conducted, focusing on calretinin, CD34, and CD117 markers to identify ganglion cells and Cajal cells. Data were statistically analyzed using SPSS software.ResultsIn the examination of the samples, the calretinin marker exhibited a consistent accuracy of 100% in diagnosing Hirschsprung’s disease (with sensitivity and specificity both at 100%). Regarding the markers for Cajal cells in cases of Hirschsprung’s disease, an irregularity in the arrangement of Cajal cells was observed, which was absent in normal cases. These markers also demonstrated a specificity and sensitivity of 100% in diagnosing the disease.ConclusionHirschsprung’s disease remains a complex condition with multifaceted pathophysiological mechanisms. Calretinin immunohistochemical staining offers enhanced diagnostic accuracy, while the debate surrounding ICC distribution underscores the need for advanced diagnostic techniques. Further research is warranted to unravel the intricacies of Hirschsprung’s disease and its associated complications.

BMP signaling pathway member expression is enriched in enteric neural progenitors and required for zebrafish enteric nervous system development

AbstractBackgroundThe vertebrate enteric nervous system (ENS) consists of a series of interconnected ganglia within the gastrointestinal (GI) tract, formed during development following migration of enteric neural crest cells (ENCCs) into the primitive gut tube. Much work has been done to unravel the complex nature of extrinsic and intrinsic factors that regulate processes that direct migration, proliferation, and differentiation of ENCCs. However, ENS development is a complex process, and we still have much to learn regarding the signaling factors that regulate ENCC development.ResultsHere in zebrafish, through transcriptomic, in situ transcript expression, immunohistochemical analysis, and chemical attenuation, we identified a time‐dependent role for bone morphogenetic protein (BMP) in the maintenance of Phox2bb+ enteric progenitor numbers and/or time of differentiation of the progenitor pool. In support of our in silico transcriptomic analysis, we identified expression of a novel ENS ligand‐encoding transcript, bmp5, within developmental regions of ENCCs. Through generation of a novel mutant bmp5wmr2 and bmp5 crispants, we identified a functional role for BMP5 in proper GI tract colonization, whereby phox2bb+ enteric progenitor numbers were reduced.ConclusionAltogether, this work identified time‐dependent roles for BMP signaling and a novel extrinsic factor, BMP5, that is necessary for vertebrate ENS formation.

Expansion of a neural crest gene signature following ectopic MYCN expression in sympathoadrenal lineage cells in vivo.

Neural crest cells (NCC) are multipotent migratory stem cells that originate from the neural tube during early vertebrate embryogenesis. NCCs give rise to a variety of cell types within the developing organism, including neurons and glia of the sympathetic nervous system. It has been suggested that failure in correct NCC differentiation leads to several diseases, including neuroblastoma (NB). During normal NCC development, MYCN is transiently expressed to promote NCC migration, and its downregulation precedes neuronal differentiation. Overexpression of MYCN has been linked to high-risk and aggressive NB progression. For this reason, understanding the effect overexpression of this oncogene has on the development of NCC-derived sympathoadrenal progenitors (SAP), which later give rise to sympathetic nerves, will help elucidate the developmental mechanisms that may prime the onset of NB. Here, we found that overexpressing human EGFP-MYCN within SAP lineage cells in zebrafish led to the transient formation of an abnormal SAP population, which displayed expanded and elevated expression of NCC markers while paradoxically also co-expressing SAP and neuronal differentiation markers. The aberrant NCC signature was corroborated with in vivo time-lapse confocal imaging in zebrafish larvae, which revealed transient expansion of sox10 reporter expression in MYCN overexpressing SAPs during the early stages of SAP development. In these aberrant MYCN overexpressing SAP cells, we also found evidence of dampened BMP signaling activity, indicating that BMP signaling disruption occurs following elevated MYCN expression. Furthermore, we discovered that pharmacological inhibition of BMP signaling was sufficient to create an aberrant NCC gene signature in SAP cells, phenocopying MYCN overexpression. Together, our results suggest that MYCN overexpression in SAPs disrupts their differentiation by eliciting abnormal NCC gene expression programs, and dampening BMP signaling response, having developmental implications for the priming of NB in vivo.

Etiology of craniofacial and cardiac malformations in a mouse model of SF3B4-related syndromes

Pathogenic variants in SF3B4, a component of the U2 snRNP complex important for branchpoint sequence recognition and splicing, are responsible for the acrofacial disorders Nager and Rodriguez Syndrome, also known as SF3B4-related syndromes. Patients exhibit malformations in the head, face, limbs, vertebrae as well as the heart. To uncover the etiology of craniofacial malformations found in SF3B4-related syndromes, mutant mouse lines with homozygous deletion of Sf3b4 in neural crest cells (NCC) were generated. Like in human patients, these embryos had craniofacial and cardiac malformations with variable expressivity and penetrance. The severity and survival of Sf3b4 NCC mutants was modified by the level of Sf3b4 in neighboring non-NCC. RNA sequencing analysis of heads of embryos prior to morphological abnormalities revealed significant changes in expression of genes forming the NCC regulatory network, as well as an increase in exon skipping. Additionally, several key histone modifiers involved in craniofacial and cardiac development showed increased exon skipping. Increased exon skipping was also associated with use of a more proximal branch point, as well as an enrichment in thymidine bases in the 50 bp around the branch points. We propose that decrease in Sf3b4 causes changes in the expression and splicing of transcripts required for proper craniofacial and cardiac development, leading to abnormalities.