Genome-wide analysis of copy-number variation in humans with cleft lip and/or cleft palate identifies COBLL1, RIC1, and ARHGEF38 as clefting genes.

Cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex, heterogeneous etiology. It is well established that common and rare sequence variants contribute to the formation of CL/P, but the contribution of copy-number variants (CNVs) to cleft formation remains relatively understudied. To fill this knowledge gap, we conducted a large-scale comparative analysis of genome-wide CNV profiles of 869 individuals from the Philippines and 233 individuals of European ancestry with CL/P with three primary goals: first, to evaluate whether differences in CNV number, amount of genomic content, or amount of coding genomic content existed within clefting subtypes; second, to assess whether CNVs in our cohort overlapped with known Mendelian clefting loci; and third, to identify unestablished Mendelian clefting genes. Significant differences in CNVs across cleft types or in individuals with non-syndromic versus syndromic clefts were not observed; however, several CNVs in our cohort overlapped with known syndromic and non-syndromic Mendelian clefting loci. Moreover, employing a filtering strategy relying on population genetics data that rare variants are on the whole more deleterious than common variants, we identify several CNV-associated gene losses likely driving non-syndromic clefting phenotypes. By prioritizing genes deleted at a rare frequency across multiple individuals with clefts yet enriched in our cohort of individuals with clefts compared to control subjects, we identify COBLL1, RIC1, and ARHGEF38 as clefting genes. CRISPR-Cas9 mutagenesis of these genes in Xenopus laevis and Danio rerio yielded craniofacial dysmorphologies, including clefts analogous to those seen in human clefting disorders.

Maternal Wnt11b regulates cortical rotation during Xenopus axis formation: analysis of maternal-effect wnt11b mutants.

Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions (e.g. amphibian dorsal morula, mammalian anterior visceral endoderm) require stabilised nuclear ß-catenin, but the role of localised Wnt ligand signalling activity in their establishment remains unclear. In Xenopus, dorsal ß-catenin is initiated by vegetal microtubule-mediated symmetry breaking in the fertilised egg, known as 'cortical rotation'. Localised wnt11b mRNA and ligand-independent activators of ß-catenin have been implicated in dorsal ß-catenin activation, but the extent to which each contributes to axis formation in this paradigm remains unclear. Here, we describe a CRISPR-mediated maternal-effect mutation in Xenopus laevis wnt11b.L. We find that wnt11b is maternally required for robust dorsal axis formation and for timely gastrulation, and zygotically for left-right asymmetry. Importantly, we show that vegetal microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs. In addition, we show that activated Wnt coreceptor Lrp6 and Dishevelled lack behaviour consistent with roles in early ß-catenin stabilisation, and that neither is regulated by Wnt11b. This work thus implicates Wnt11b in the distribution of putative dorsal determinants rather than in comprising the determinants themselves. This article has an associated 'The people behind the papers' interview.

A single KH domain in Bicaudal-C links mRNA binding and translational repression functions to maternal development.

Bicaudal-C (Bicc1) is a conserved RNA-binding protein that represses the translation of selected mRNAs to control development. In Xenopus embryos, Bicc1 binds and represses specific maternal mRNAs to control anterior-posterior cell fates. However, it is not known how Bicc1 binds its RNA targets or how binding affects Bicc1-dependent embryogenesis. Focusing on the KH domains, we analyzed Bicc1 mutants for their ability to bind RNA substrates in vivo and in vitro Analyses of these Bicc1 mutants demonstrated that a single KH domain, KH2, was crucial for RNA binding in vivo and in vitro, while the KH1 and KH3 domains contributed minimally. The Bicc1 mutants were also assayed for their ability to repress translation, and results mirrored the RNA-binding data, with KH2 being the only domain essential for repression. Finally, maternal knockdown and rescue experiments indicated that the KH domains were essential for the regulation of embryogenesis by Bicc1. These data advance our understanding of how Bicc1 selects target mRNAs and provide the first direct evidence that the RNA binding functions of Bicc1 are essential for both Bicc1-dependent translational repression and maternal vertebrate development.

A gradient of maternal Bicaudal-C controls vertebrate embryogenesis via translational repression of mRNAs encoding cell fate regulators.

Vertebrate Bicaudal-C (Bicc1) has important biological roles in the formation and homeostasis of multiple organs, but direct experiments to address the role of maternal Bicc1 in early vertebrate embryogenesis have not been reported. Here, we use antisense phosphorothioate-modified oligonucleotides and the host-transfer technique to eliminate specifically maternal stores of both bicc1 mRNA and Bicc1 protein from Xenopus laevis eggs. Fertilization of these Bicc1-depleted eggs produced embryos with an excess of dorsal-anterior structures and overexpressed organizer-specific genes, indicating that maternal Bicc1 is crucial for normal embryonic patterning of the vertebrate embryo. Bicc1 is an RNA-binding protein with robust translational repression function. Here, we show that the maternal mRNA encoding the cell-fate regulatory protein Wnt11b is a direct target of Bicc1-mediated repression. It is well established that the Wnt signaling pathway is crucial to vertebrate embryogenesis. Thus, the work presented here links the molecular function of Bicc1 in mRNA target-specific translation repression to its biological role in the maternally controlled stages of vertebrate embryogenesis.

Role of maternal Xenopus syntabulin in germ plasm aggregation and primordial germ cell specification.

The localization and organization of mitochondria- and ribonucleoprotein granule-rich germ plasm is essential for many aspects of germ cell development. In Xenopus, germ plasm is maternally inherited and is required for the specification of primordial germ cells (PGCs). Germ plasm is aggregated into larger patches during egg activation and cleavage and is ultimately translocated perinuclearly during gastrulation. Although microtubule dynamics and a kinesin (Kif4a) have been implicated in Xenopus germ plasm localization, little is known about how germ plasm distribution is regulated. Here, we identify a role for maternal Xenopus Syntabulin in the aggregation of germ plasm following fertilization. We show that depletion of sybu mRNA using antisense oligonucleotides injected into oocytes results in defects in the aggregation and perinuclear transport of germ plasm and subsequently in reduced PGC numbers. Using live imaging analysis, we also characterize a novel role for Sybu in the collection of germ plasm in vegetal cleavage furrows by surface contraction waves. Additionally, we show that a localized kinesin-like protein, Kif3b, is also required for germ plasm aggregation and that Sybu functionally interacts with Kif3b and Kif4a in germ plasm aggregation. Overall, these data suggest multiple coordinate roles for kinesins and adaptor proteins in controlling the localization and distribution of a cytoplasmic determinant in early development.

Vertebrate Axial Patterning: From Egg to Asymmetry.

The emergence of the bilateral embryonic body axis from a symmetrical egg has been a long-standing question in developmental biology. Historical and modern experiments point to an initial symmetry-breaking event leading to localized Wnt and Nodal growth factor signaling and subsequent induction and formation of a self-regulating dorsal "organizer." This organizer forms at the site of notochord cell internalization and expresses primarily Bone Morphogenetic Protein (BMP) growth factor antagonists that establish a spatiotemporal gradient of BMP signaling across the embryo, directing initial cell differentiation and morphogenesis. Although the basics of this model have been known for some time, many of the molecular and cellular details have only recently been elucidated and the extent that these events remain conserved throughout vertebrate evolution remains unclear. This chapter summarizes historical perspectives as well as recent molecular and genetic advances regarding: (1) the mechanisms that regulate symmetry-breaking in the vertebrate egg and early embryo, (2) the pathways that are activated by these events, in particular the Wnt pathway, and the role of these pathways in the formation and function of the organizer, and (3) how these pathways also mediate anteroposterior patterning and axial morphogenesis. Emphasis is placed on comparative aspects of the egg-to-embryo transition across vertebrates and their evolution. The future prospects for work regarding self-organization and gene regulatory networks in the context of early axis formation are also discussed.

The dynamics of plus end polarization and microtubule assembly during Xenopus cortical rotation.

The self-organization of dorsally-directed microtubules during cortical rotation in the Xenopus egg is essential for dorsal axis formation. The mechanisms controlling this process have been problematic to analyze, owing to difficulties in visualizing microtubules in living egg. Also, the order of events occurring at the onset of cortical rotation have not been satisfactorily visualized in vivo and have been inferred from staged fixed samples. To address these issues, we have characterized the dynamics of total microtubule and plus end behavior continuously throughout cortical rotation, as well as in oocytes and unfertilized eggs. Here, we show that the nascent microtubule network forms in the cortex but associates with the deep cytoplasm at the start of rotation. Importantly, plus ends remain cortical and become increasingly more numerous and active prior to rotation, with dorsal polarization occurring rapidly after the onset of rotation. Additionally, we show that vegetally localized Trim36 is required to attenuate dynamic plus end growth, suggesting that vegetal factors are needed to locally coordinate growth in the cortex.

Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification.

Vertebrate axis specification is an evolutionarily conserved developmental process that relies on asymmetric activation of Wnt signaling and subsequent organizer formation on the future dorsal side of the embryo. Although roles of Wnt signaling during organizer formation have been studied extensively, it is unclear how the Wnt pathway is asymmetrically activated. In Xenopus and zebrafish, the Wnt pathway is triggered by dorsal determinants, which are translocated from the vegetal pole to the future dorsal side of the embryo shortly after fertilization. The transport of dorsal determinants requires a unique microtubule network formed in the vegetal cortex shortly after fertilization. However, molecular mechanisms governing the formation of vegetal cortical microtubule arrays are not fully understood. Here we report that Dead-End 1 (Dnd1), an RNA-binding protein required for primordial germ cell development during later stages of embryogenesis, is essential for Xenopus axis specification. We show that knockdown of maternal Dnd1 specifically interferes with the formation of vegetal cortical microtubules. This, in turn, impairs translocation of dorsal determinants, the initiation of Wnt signaling, organizer formation, and ultimately results in ventralized embryos. Furthermore, we found that Dnd1 binds to a uridine-rich sequence in the 3'-UTR of trim36, a vegetally localized maternal RNA essential for vegetal cortical microtubule assembly. Dnd1 anchors trim36 to the vegetal cortex in the egg, promoting high concentrations of Trim36 protein there. Our work thus demonstrates a novel and surprising function for Dnd1 during early development and provides an important link between Dnd1, mRNA localization, the microtubule cytoskeleton and axis specification.

Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene.

Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development.

Regulation of neurogenesis by Fgf8a requires Cdc42 signaling and a novel Cdc42 effector protein.

Fibroblast growth factor (FGF) signaling is required for numerous aspects of neural development, including neural induction, CNS patterning and neurogenesis. The ability of FGFs to activate Ras/MAPK signaling is thought to be critical for these functions. However, it is unlikely that MAPK signaling can fully explain the diversity of responses to FGFs. We have characterized a Cdc42-dependent signaling pathway operating downstream of the Fgf8a splice isoform. We show that a Cdc42 effector 4-like protein (Cdc42ep4-l or Cep4l) has robust neuronal-inducing activity in Xenopus embryos. Furthermore, we find that Cep4l and Cdc42 itself are necessary and sufficient for sensory neurogenesis in vivo. Furthermore, both proteins are involved in Fgf8a-induced neuronal induction, and Cdc42/Cep4l association is promoted specifically by the Fgf8a isoform of Fgf8, but not by Fgf8b, which lacks neuronal inducing activity. Overall, these data suggest a novel role for Cdc42 in an Fgf8a-specific signaling pathway essential for vertebrate neuronal development.

Vegetally localized Xenopus trim36 regulates cortical rotation and dorsal axis formation.

Specification of the dorsoventral axis in Xenopus depends on rearrangements of the egg vegetal cortex following fertilization, concomitant with activation of Wnt/beta-catenin signaling. How these processes are tied together is not clear, but RNAs localized to the vegetal cortex during oogenesis are known to be essential. Despite their importance, few vegetally localized RNAs have been examined in detail. In this study, we describe the identification of a novel localized mRNA, trim36, and characterize its function through maternal loss-of-function experiments. We find that trim36 is expressed in the germ plasm and encodes a ubiquitin ligase of the Tripartite motif-containing (Trim) family. Depletion of maternal trim36 using antisense oligonucleotides results in ventralized embryos and reduced organizer gene expression. We show that injection of wnt11 mRNA rescues this effect, suggesting that Trim36 functions upstream of Wnt/beta-catenin activation. We further find that vegetal microtubule polymerization and cortical rotation are disrupted in trim36-depleted embryos, in a manner dependent on Trim36 ubiquitin ligase activity. Additionally, these embryos can be rescued by tipping the eggs 90 degrees relative to the animal-vegetal axis. Taken together, our results suggest a role for Trim36 in controlling the stability of proteins regulating microtubule polymerization during cortical rotation, and subsequently axis formation.

Use of fully modified 2'-O-methyl antisense oligos for loss-of-function studies in vertebrate embryos.

Antisense oligonucleotides are commonly employed to study the roles of genes in development. Although morpholino phosphorodiamidate oligonucleotides (morpholinos) are widely used to block translation or splicing of target gene products' the usefulness of other modifications in mediating RNase-H independent inhibition of gene activity in embryos has not been investigated. In this study, we investigated the extent that fully modified 2'-O-methyl oligonucleotides (2'-OMe oligos) that can function as translation inhibiting reagents in vivo, using Xenopus and zebrafish embryos. We find that oligos against Xenopus ß-catenin, wnt11, and bmp4 and against zebrafish chordin (chd), which can efficiently and specifically generate embryonic loss-of-function phenotypes comparable with morpholino injection and other methods. These results show that fully modified 2'-OMe oligos can function as RNase-H independent antisense reagents in vertebrate embryos and can thus serve as an alternative modification to morpholinos in some cases.

The use of antisense oligonucleotides in Xenopus oocytes.

The ability to manipulate gene expression in Xenopus oocytes and then generate fertilized embryos by transfer into host females has made it possible to rapidly characterize maternal signaling pathways in vertebrate development. Maternal mRNAs in particular can be efficiently depleted using antisense deoxyoligonucleotides (oligos), mediated by endogenous RNase-H activity. Since the microinjection of antisense reagents or mRNAs into eggs after fertilization often fails to affect maternal signaling pathways, mRNA depletion in the Xenopus oocyte is uniquely suited to assessing maternal functions. In this review, we highlight the advantages of using antisense in Xenopus oocytes and describe basic methods for designing and choosing effective oligos. We also summarize the procedures for fertilizing cultured oocytes by host-transfer and interpreting the specificity of antisense effects. Although these methods can be technically demanding, the use of antisense in oocytes can be used to address biological questions that are intractable in other experimental settings.

Identification of germ plasm-associated transcripts by microarray analysis of Xenopus vegetal cortex RNA.

RNA localization is a common mechanism for regulating cell structure and function. Localized RNAs in Xenopus oocytes are critical for early development, including germline specification by the germ plasm. Despite the importance of these localized RNAs, only approximately 25 have been identified and fewer are functionally characterized. Using microarrays, we identified a large set of localized RNAs from the vegetal cortex. Overall, our results indicate a minimum of 275 localized RNAs in oocytes, or 2-3% of maternal transcripts, which are in general agreement with previous findings. We further validated vegetal localization for 24 candidates and further characterized three genes expressed in the germ plasm. We identified novel germ plasm expression for reticulon 3.1, exd2 (a novel exonuclease-domain encoding gene), and a putative noncoding RNA. Further analysis of these and other localized RNAs will likely identify new functions of germ plasm and facilitate the identification of cis-acting RNA localization elements.

Maternal Interferon Regulatory Factor 6 is required for the differentiation of primary superficial epithelia in Danio and Xenopus embryos.

Early in the development of animal embryos, superficial cells of the blastula form a distinct lineage and adopt an epithelial morphology. In different animals, the fate of these primary superficial epithelial (PSE) cells varies, and it is unclear whether pathways governing segregation of blastomeres into the PSE lineage are conserved. Mutations in the gene encoding Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic forms of cleft lip and palate, consistent with a role for Irf6 in development of oral epithelia, and mouse Irf6 targeted null mutant embryos display abnormal differentiation of oral epithelia and skin. In Danio rerio (zebrafish) and Xenopus laevis (African clawed frog) embryos, zygotic irf6 transcripts are present in many epithelial tissues including the presumptive PSE cells and maternal irf6 transcripts are present throughout all cells at the blastula stage. Injection of antisense oligonucleotides with ability to disrupt translation of irf6 transcripts caused little or no effect on development. By contrast, injection of RNA encoding a putative dominant negative Irf6 caused epiboly arrest, loss of gene expression characteristic of the EVL, and rupture of the embryo at late gastrula stage. The dominant negative Irf6 disrupted EVL gene expression in a cell autonomous fashion. These results suggest that Irf6 translated in the oocyte or unfertilized egg suffices for early development. Supporting the importance of maternal Irf6, we show that depletion of maternal irf6 transcripts in X. laevis embryos leads to gastrulation defects and rupture of the superficial epithelium. These experiments reveal a conserved role for maternally-encoded Irf6 in differentiation of a simple epithelium in X. laevis and D. rerio. This epithelium constitutes a novel model tissue in which to explore the Irf6 regulatory pathway.

Culture and Host Transfer of Xenopus Oocytes for Maternal mRNA Depletion and Genome Editing Experiments.

The early development of Xenopus critically depends on maternal components stored in the egg. Because important events such as axis formation are triggered immediately after fertilization, it is often desirable to perturb gene function before this occurs. Oocytes can be experimentally manipulated in vitro, prior to maturation, and subsequently fertilized or otherwise activated to develop, and then observed for any embryological defects. Available methods for fertilizing cultured oocytes include in vitro fertilization following oocyte vitelline envelope removal, nuclear transplantation, intracytoplasmic sperm injection, and transferring oocytes to the body cavity of ovulating host females (host transfer). This chapter outlines this host transfer method, which has been used to elucidate basic mechanisms of axis formation, germ-layer induction, and primordial germ cell specification. Methods for obtaining, culturing, transferring, and fertilizing Xenopus oocytes are described. This method has typically been used to alter maternal gene function by antisense oligonucleotide-mediated mRNA knockdown, but is also useful for mRNA or protein overexpression, including the expression of genome-editing reagents prior to fertilization.

Topologically correct central projections of tetrapod inner ear afferents require Fzd3.

Inner ear sensory afferent connections establish sensory maps between the inner ear hair cells and the vestibular and auditory nuclei to allow vestibular and sound information processing. While molecular guidance of sensory afferents to the periphery has been well studied, molecular guidance of central projections from the ear is only beginning to emerge. Disorganized central projections of spiral ganglion neurons in a Wnt/PCP pathway mutant, Prickle1, suggest the Wnt/PCP pathway plays a role in guiding cochlear afferents to the cochlear nuclei in the hindbrain, consistent with known expression of the Wnt receptor, Frizzled3 (Fzd3) in inner ear neurons. We therefore investigated the role of Wnt signaling in central pathfinding in Fzd3 mutant mice and Fzd3 morpholino treated frogs and found aberrant central projections of vestibular afferents in both cases. Ear transplantations from knockdown to control Xenopus showed that it is the Fzd3 expressed within the ear that mediates this guidance. Also, cochlear afferents of Fzd3 mutant mice lack the orderly topological organization observed in controls. Quantification of Fzd3 expression in spiral ganglion neurons show a gradient of expression with Fzd3 being higher in the apex than in the base. Together, these results suggest that a gradient of Fzd3 in inner ear afferents directs projections to the correct dorsoventral column within the hindbrain.

Image analysis of calcium release dynamics.

Many aspects of animal development are dependent on the dynamic release of calcium (Ca2+) ions. Although Ca2+ release within a cell is tightly controlled, how the release dynamics result in a specific biological output in embryonic development is less clear. The integration of pharmacological and molecular-genetic studies with in vivo imaging in zebrafish and Xenopus has linked endogenous Ca2+ release to the Wnt signaling network. Our data suggests that distinct Ca2+ release dynamics lead to antagonism of the developmentally important Wnt/beta-catenin pathway while sustained Ca2+ release modulates polarized cell movements.

Oocyte Host-Transfer and Maternal mRNA Depletion Experiments in Xenopus.

This protocol details the oocyte host-transfer method in Xenopus, using transplantation by intraperitoneal injection. This approach is suitable for the overexpression of mRNAs and for the use of antisense oligonucleotides to deplete maternal mRNAs, which are not replaced until zygotic genome activation in the mid-blastula transition. Xenopus oocyte host-transfer can also be used for highly efficient mutagenesis in the F0 generation by prefertilization injection of genome editing reagents.