A CRISPR/Cas9-generated mutation in the zebrafish orthologue of PPP2R3B causes idiopathic scoliosis.

Idiopathic scoliosis (IS) is the deformation and/or abnormal curvature of the spine that develops progressively after birth. It is a very common condition, affecting approximately 4% of the general population, yet the genetic and mechanistic causes of IS are poorly understood. Here, we focus on PPP2R3B, which encodes a protein phosphatase 2A regulatory subunit. We found that PPP2R3B is expressed at sites of chondrogenesis within human foetuses, including the vertebrae. We also demonstrated prominent expression in myotome and muscle fibres in human foetuses, and zebrafish embryos and adolescents. As there is no rodent orthologue of PPP2R3B, we used CRIPSR/Cas9-mediated gene-editing to generate a series of frameshift mutations in zebrafish ppp2r3b. Adolescent zebrafish that were homozygous for this mutation exhibited a fully penetrant kyphoscoliosis phenotype which became progressively worse over time, mirroring IS in humans. These defects were associated with reduced mineralisation of vertebrae, resembling osteoporosis. Electron microscopy demonstrated abnormal mitochondria adjacent to muscle fibres. In summary, we report a novel zebrafish model of IS and reduced bone mineral density. In future, it will be necessary to delineate the aetiology of these defects in relation to bone, muscle, neuronal and ependymal cilia function.

Autosomal recessive cardiomyopathy and sudden cardiac death associated with variants in MYL3.

PURPOSE: Variants in genes encoding sarcomeric proteins are the most common cause of inherited cardiomyopathies. However, the underlying genetic cause remains unknown in many cases. We used exome sequencing to reveal the genetic etiology in patients with recessive familial cardiomyopathy. METHODS: Exome sequencing was carried out in three consanguineous families. Functional assessment of the variants was performed. RESULTS: Affected individuals presented with hypertrophic or dilated cardiomyopathy of variable severity from infantile- to early adulthood-onset and sudden cardiac death. We identified a homozygous missense substitution (c.170C>A, p.[Ala57Asp]), a homozygous translation stop codon variant (c.106G>T, p.[Glu36Ter]), and a presumable homozygous essential splice acceptor variant (c.482-1G>A, predicted to result in skipping of exon 5). Morpholino knockdown of the MYL3 orthologue in zebrafish, cmlc1, resulted in compromised cardiac function, which could not be rescued by reintroduction of MYL3 carrying either the nonsense c.106G>T or the missense c.170C>A variants. Minigene assay of the c.482-1G>A variant indicated a splicing defect likely resulting in disruption of the EF-hand Ca2+ binding domains. CONCLUSIONS: Our data demonstrate that homozygous MYL3 loss-of-function variants can cause of recessive cardiomyopathy and occurrence of sudden cardiac death, most likely due to impaired or loss of myosin essential light chain function.

Fgf-driven Tbx protein activities directly induce myf5 and myod to initiate zebrafish myogenesis.

Skeletal muscle derives from dorsal mesoderm formed during vertebrate gastrulation. Fibroblast growth factor (Fgf) signalling cooperates with Tbx transcription factors to promote dorsal mesoderm formation, but their role in myogenesis has been unclear. Using zebrafish, we show that dorsally derived Fgf signals act through Tbx16 and Tbxta to induce slow and fast trunk muscle precursors at distinct dorsoventral positions. Tbx16 binds to and directly activates the myf5 and myod genes, which are required for commitment to myogenesis. Tbx16 activity depends on Fgf signalling from the organiser. In contrast, Tbxta is not required for myf5 expression, but binds a specific site upstream of myod that is not bound by Tbx16 and drives (dependent on Fgf signals) myod expression in adaxial slow precursors, thereby initiating trunk myogenesis. After gastrulation, when similar muscle cell populations in the post-anal tail are generated from tailbud, declining Fgf signalling is less effective at initiating adaxial myogenesis, which is instead initiated by Hedgehog signalling from the notochord. Our findings suggest a hypothesis for ancestral vertebrate trunk myogenic patterning and how it was co-opted during tail evolution to generate similar muscle by new mechanisms.This article has an associated 'The people behind the papers' interview.

A Technique for Studying Glomerular Filtration Integrity in the Zebrafish Pronephros.

With the advances in next-generation sequencing and rapid filtering of candidate variants in diseased patients, it has been increasingly important to develop translatable in vivo models to study genetic changes. This allows for functional validation of pathogenic mutations and establishes a system to understand the etiology of disease. Due to the ease of genetic manipulation and rapid ex utero development, the zebrafish has become a valuable resource to study important biological processes, including nephrogenesis. The development and function of the zebrafish pronephros are akin to that of mammals. As such, they offer a tractable model to study kidney disease, especially diabetic nephropathy. However, in order to study kidney dysfunction in zebrafish it is imperative that an appropriate readout is available. The appearance of macro-proteins in patient's urine is indicative of defective kidney function. In this technical chapter, we describe the in vivo use of fluorescently tagged dextrans of different molecular weights to reveal the integrity of the zebrafish glomerular filtration barrier.

Bi-allelic variants in RNF170 are associated with hereditary spastic paraplegia.

Alterations of Ca2+ homeostasis have been implicated in a wide range of neurodegenerative diseases. Ca2+ efflux from the endoplasmic reticulum into the cytoplasm is controlled by binding of inositol 1,4,5-trisphosphate to its receptor. Activated inositol 1,4,5-trisphosphate receptors are then rapidly degraded by the endoplasmic reticulum-associated degradation pathway. Mutations in genes encoding the neuronal isoform of the inositol 1,4,5-trisphosphate receptor (ITPR1) and genes involved in inositol 1,4,5-trisphosphate receptor degradation (ERLIN1, ERLIN2) are known to cause hereditary spastic paraplegia (HSP) and cerebellar ataxia. We provide evidence that mutations in the ubiquitin E3 ligase gene RNF170, which targets inositol 1,4,5-trisphosphate receptors for degradation, are the likely cause of autosomal recessive HSP in four unrelated families and functionally evaluate the consequences of mutations in patient fibroblasts, mutant SH-SY5Y cells and by gene knockdown in zebrafish. Our findings highlight inositol 1,4,5-trisphosphate signaling as a candidate key pathway for hereditary spastic paraplegias and cerebellar ataxias and thus prioritize this pathway for therapeutic interventions.

Cardiomyopathy with lethal arrhythmias associated with inactivation of KLHL24.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disorder, yet the genetic cause of up to 50% of cases remains unknown. Here, we show that mutations in KLHL24 cause HCM in humans. Using genome-wide linkage analysis and exome sequencing, we identified homozygous mutations in KLHL24 in two consanguineous families with HCM. Of the 11 young affected adults identified, 3 died suddenly and 1 had a cardiac transplant due to heart failure. KLHL24 is a member of the Kelch-like protein family, which acts as substrate-specific adaptors to Cullin E3 ubiquitin ligases. Endomyocardial and skeletal muscle biopsies from affected individuals of both families demonstrated characteristic alterations, including accumulation of desmin intermediate filaments. Knock-down of the zebrafish homologue klhl24a results in heart defects similar to that described for other HCM-linked genes providing additional support for KLHL24 as a HCM-associated gene. Our findings reveal a crucial role for KLHL24 in cardiac development and function.

Synaptotagmin 5 regulates Ca2+-dependent Weibel-Palade body exocytosis in human endothelial cells.

Elevations of intracellular free Ca2+ concentration ([Ca2+]i) are a potent trigger for Weibel-Palade body (WPB) exocytosis and secretion of von Willebrand factor (VWF) from endothelial cells; however, the identity of WPB-associated Ca2+-sensors involved in transducing acute increases in [Ca2+]i into granule exocytosis remains unknown. Here, we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVECs) and is recruited to WPBs to regulate Ca2+-driven WPB exocytosis. Western blot analysis of HUVECs identified SYT5 protein, and exogenously expressed SYT5-mEGFP localised almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 (shSYT5) reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca2+-binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca2+ sensor regulating Ca2+-dependent secretion of stored mediators from vascular endothelial cells.

Bi-allelic mutations in MYL1 cause a severe congenital myopathy.

Congenital myopathies are typically characterised by early onset hypotonia, weakness and hallmark features on biopsy. Despite the rapid pace of gene discovery, ∼50% of patients with a congenital myopathy remain without a genetic diagnosis following screening of known disease genes. We performed exome sequencing on two consanguineous probands diagnosed with a congenital myopathy and muscle biopsy showing selective atrophy/hypotrophy or absence of type II myofibres. We identified variants in the gene (MYL1) encoding the skeletal muscle fast-twitch specific myosin essential light chain (ELC) in both probands. A homozygous essential splice acceptor variant (c.479-2A > G, predicted to result in skipping of exon 5 was identified in Proband 1, and a homozygous missense substitution (c.488T>G, p.(Met163Arg)) was identified in Proband 2. Protein modelling of the p.(Met163Arg) substitution predicted it might impede intermolecular interactions that facilitate binding to the IQ domain of myosin heavy chain, thus likely impacting on the structure and functioning of the myosin motor. MYL1 was markedly reduced in skeletal muscle from both probands, suggesting that the missense substitution likely results in an unstable protein. Knock down of myl1 in zebrafish resulted in abnormal morphology, disrupted muscle structure and impaired touch-evoked escape responses, thus confirming that skeletal muscle fast-twitch specific myosin ELC is critical for myofibre development and function. Our data implicate MYL1 as a crucial protein for adequate skeletal muscle function and that MYL1 deficiency is associated with severe congenital myopathy.

WDR11-mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome.

WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11's role in development is poorly understood. Here, we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues. Wdr11-null mice also exhibit early-onset obesity. We find that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 regulates the proteolytic processing of GLI3 and cooperates with the transcription factor EMX1 in the induction of downstream Hh pathway gene expression and gonadotrophin-releasing hormone production. The CHH/KS-associated human mutations result in loss of function of WDR11. Treatment with the Hh agonist purmorphamine partially rescues the WDR11 haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum.

Mutations in INPP5K Cause a Form of Congenital Muscular Dystrophy Overlapping Marinesco-Sjögren Syndrome and Dystroglycanopathy.

Congenital muscular dystrophies display a wide phenotypic and genetic heterogeneity. The combination of clinical, biochemical, and molecular genetic findings must be considered to obtain the precise diagnosis and provide appropriate genetic counselling. Here we report five individuals from four families presenting with variable clinical features including muscular dystrophy with a reduction in dystroglycan glycosylation, short stature, intellectual disability, and cataracts, overlapping both the dystroglycanopathies and Marinesco-Sjögren syndrome. Whole-exome sequencing revealed homozygous missense and compound heterozygous mutations in INPP5K in the affected members of each family. INPP5K encodes the inositol polyphosphate-5-phosphatase K, also known as SKIP (skeletal muscle and kidney enriched inositol phosphatase), which is highly expressed in the brain and muscle. INPP5K localizes to both the endoplasmic reticulum and to actin ruffles in the cytoplasm. It has been shown to regulate myoblast differentiation and has also been implicated in protein processing through its interaction with the ER chaperone HSPA5/BiP. We show that morpholino-mediated inpp5k loss of function in the zebrafish results in shortened body axis, microphthalmia with disorganized lens, microcephaly, reduced touch-evoked motility, and highly disorganized myofibers. Altogether these data demonstrate that mutations in INPP5K cause a congenital muscular dystrophy syndrome with short stature, cataracts, and intellectual disability.

Zebrafish: a vertebrate tool for studying basal body biogenesis, structure, and function.

Understanding the role of basal bodies (BBs) during development and disease has been largely overshadowed by research into the function of the cilium. Although these two organelles are closely associated, they have specific roles to complete for successful cellular development. Appropriate development and function of the BB are fundamental for cilia function. Indeed, there are a growing number of human genetic diseases affecting ciliary development, known collectively as the ciliopathies. Accumulating evidence suggests that BBs establish cell polarity, direct ciliogenesis, and provide docking sites for proteins required within the ciliary axoneme. Major contributions to our knowledge of BB structure and function have been provided by studies in flagellated or ciliated unicellular eukaryotic organisms, specifically Tetrahymena and Chlamydomonas. Reproducing these and other findings in vertebrates has required animal in vivo models. Zebrafish have fast become one of the primary organisms of choice for modeling vertebrate functional genetics. Rapid ex-utero development, proficient egg laying, ease of genetic manipulation, and affordability make zebrafish an attractive vertebrate research tool. Furthermore, zebrafish share over 80 % of disease causing genes with humans. In this article, we discuss the merits of using zebrafish to study BB functional genetics, review current knowledge of zebrafish BB ultrastructure and mechanisms of function, and consider the outlook for future zebrafish-based BB studies.

Evaluation of zebrafish kidney function using a fluorescent clearance assay.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria 'dipstick', which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function.

Clinical predictors of abnormal magnetic resonance imaging findings in patients with asymmetric sensorineural hearing loss.

IMPORTANCE: Asymmetric sensorineural hearing loss (ASNHL) is commonly encountered in an otolaryngologic clinical practice. Determining what factors are associated with abnormal magnetic resonance imaging (MRI) findings will help with diagnostic workup. OBJECTIVE: To evaluate the association between clinical and audiometric factors and abnormal MRI findings in patients with ASNHL. DESIGN, SETTING, AND PARTICIPANTS: Retrospective medical record review from an urban, tertiary referral center of 451 patients with ASNHL who underwent MRI testing between January 2005 and December 2011. MAIN OUTCOMES AND MEASURES: Medical records were reviewed for audiometric parameters as well as clinical presentation and compared with MRI results, which were categorized as abnormal, normal, or incidental. Data analysis included χ2 tests, logistic regression analysis, and multivariate analysis. RESULTS: A total of 48 patients (10.6%) had abnormal MRI findings. Only 21 patients (4.7%) had a mass of the cerebellopontine angle/internal auditory canal on MRI, making up 40% of all abnormal MRI findings. The next most common MRI finding was labyrinthitis (n = 13; 25%). Vertigo/dizziness (n = 20; P = .01), tinnitus (n = 18; P = .02), sudden hearing loss (n = 15; P = .054), and 15-dB asymmetry at 3 kHz (n = 39; P = .01) were associated with abnormal MRI findings. Loud noise exposure was associated with normal MRI findings. Logistic regression analysis showed that vertigo/dizziness (odds ratio [OR], 2.14; 95% CI, 1.15-3.96; P = .02), unilateral tinnitus (OR, 2.15; 95% CI, 1.14-4.03; P = .02), and 15-dB asymmetry at 3 kHz (OR, 2.62; 95% CI, 1.24-5.57; P = .01) were significantly associated with abnormal MRI findings. Multivariate analysis showed that only 15-dB asymmetry at 3 kHz (OR, 2.42; 95% CI, 1.07-5.50; P = .03) was significantly associated with an abnormal MRI finding. CONCLUSIONS AND RELEVANCE: This study found that asymmetry of 15 dB at 3 kHz on audiometry was associated with higher positive yield on use of MRI in evaluating patients with ASNHL. We recommend that patients who present with ASNHL with this audiometric characteristic undergo MRI as part of their diagnostic workup.

Loss of FTO antagonises Wnt signaling and leads to developmental defects associated with ciliopathies.

Common intronic variants in the Human fat mass and obesity-associated gene (FTO) are found to be associated with an increased risk of obesity. Overexpression of FTO correlates with increased food intake and obesity, whilst loss-of-function results in lethality and severe developmental defects. Despite intense scientific discussions around the role of FTO in energy metabolism, the function of FTO during development remains undefined. Here, we show that loss of Fto leads to developmental defects such as growth retardation, craniofacial dysmorphism and aberrant neural crest cells migration in Zebrafish. We find that the important developmental pathway, Wnt, is compromised in the absence of FTO, both in vivo (zebrafish) and in vitro (Fto(-/-) MEFs and HEK293T). Canonical Wnt signalling is down regulated by abrogated ß-Catenin translocation to the nucleus whilst non-canonical Wnt/Ca(2+) pathway is activated via its key signal mediators CaMKII and PKCδ. Moreover, we demonstrate that loss of Fto results in short, absent or disorganised cilia leading to situs inversus, renal cystogenesis, neural crest cell defects and microcephaly in Zebrafish. Congruently, Fto knockout mice display aberrant tissue specific cilia. These data identify FTO as a protein-regulator of the balanced activation between canonical and non-canonical branches of the Wnt pathway. Furthermore, we present the first evidence that FTO plays a role in development and cilia formation/function.

The Bardet-Biedl syndrome-related protein CCDC28B modulates mTORC2 function and interacts with SIN1 to control cilia length independently of the mTOR complex.

CCDC28B encodes a coiled coil domain-containing protein involved in ciliogenesis that was originally identified as a second site modifier of the ciliopathy Bardet-Biedl syndrome. We have previously shown that the depletion of CCDC28B leads to shortened cilia; however, the mechanism underlying how this protein controls ciliary length is unknown. Here, we show that CCDC28B interacts with SIN1, a component of the mTOR complex 2 (mTORC2), and that this interaction is important both in the context of mTOR signaling and in a hitherto unknown, mTORC-independent role of SIN1 in cilia biology. We show that CCDC28B is a positive regulator of mTORC2, participating in its assembly/stability and modulating its activity, while not affecting mTORC1 function. Further, we show that Ccdc28b regulates cilia length in vivo, at least in part, through its interaction with Sin1. Importantly, depletion of Rictor, another core component of mTORC2, does not result in shortened cilia. Taken together, our findings implicate CCDC28B in the regulation of mTORC2, and uncover a novel function of SIN1 regulating cilia length that is likely independent of mTOR signaling.

Asymptomatic bowel perforation by abandoned ventriculoperitoneal shunt.

We report a case of an abandoned abdominal ventriculoperitoneal shunt that migrated into the gastric antrum, colonic hepatic flexure, and liver parenchyma, which was discovered incidentally on an abdominal CT obtained for renal stones. In regards to the migrated abandoned VP shunt, the patient was asymptomatic. Upon review of prior CT scans, these findings had progressed over approximately 7 years. We describe the case and discuss the clinical and radiologic findings, complications resulting from ventriculoperitoneal shunts, and possible approaches to their management.

Characterization of CCDC28B reveals its role in ciliogenesis and provides insight to understand its modifier effect on Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that is generally inherited in an autosomal recessive fashion. However, in some families, trans mutant alleles interact with the primary causal locus to modulate the penetrance and/or the expressivity of the phenotype. CCDC28B (MGC1203) was identified as a second site modifier of BBS encoding a protein of unknown function. Here we report the first functional characterization of this protein and show it affects ciliogenesis both in cultured cells and in vivo in zebrafish. Consistent with this biological role, our in silico analysis shows that the presence of CCDC28B homologous sequences is restricted to ciliated metazoa. Depletion of Ccdc28b in zebrafish results in defective ciliogenesis and consequently causes a number of phenotypes that are characteristic of BBS and other ciliopathy mutants including hydrocephalus, left-right axis determination defects and renal function impairment. Thus, this work reports CCDC28B as a novel protein involved in the process of ciliogenesis whilst providing functional insight into the cellular basis of its modifier effect in BBS patients.

Cesarean scar pregnancy: sonographic and magnetic resonance imaging findings, complications, and treatment.

A cesarean scar (ectopic) pregnancy occurs when a pregnancy implants on a cesarean scar. This condition is an uncommon but potentially devastating occurrence. The incidence is increasing as cesarean deliveries become more common. Early recognition of the salient sonographic findings is critical because a delay can lead to increased maternal morbidity and mortality. Magnetic resonance imaging is a valuable troubleshooting tool when sonography is equivocal or inconclusive before therapy or intervention. Early diagnosis by sonography directs therapy and improves outcomes by allowing preservation of the uterus and future fertility. We review the imaging features, differential diagnosis, complications, and treatment of cesarean scar pregnancies in the first trimester.

Heat shock induces rapid resorption of primary cilia.

Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90.

Mutations in lectin complement pathway genes COLEC11 and MASP1 cause 3MC syndrome.

3MC syndrome has been proposed as a unifying term encompassing the overlapping Carnevale, Mingarelli, Malpuech and Michels syndromes. These rare autosomal recessive disorders exhibit a spectrum of developmental features, including characteristic facial dysmorphism, cleft lip and/or palate, craniosynostosis, learning disability and genital, limb and vesicorenal anomalies. Here we studied 11 families with 3MC syndrome and identified two mutated genes, COLEC11 and MASP1, both of which encode proteins in the lectin complement pathway (collectin kidney 1 (CL-K1) and MASP-1 and MASP-3, respectively). CL-K1 is highly expressed in embryonic murine craniofacial cartilage, heart, bronchi, kidney and vertebral bodies. Zebrafish morphants for either gene develop pigmentary defects and severe craniofacial abnormalities. Finally, we show that CL-K1 serves as a guidance cue for neural crest cell migration. Together, these findings demonstrate a role for complement pathway factors in fundamental developmental processes and in the etiology of 3MC syndrome.