Mowat-Wilson syndrome factor ZEB2 controls early formation of human neural crest through BMP signaling modulation.

Mowat-Wilson syndrome is caused by mutations in ZEB2, with patients exhibiting characteristics indicative of neural crest (NC) defects. We examined the contribution of ZEB2 to human NC formation using a model based on human embryonic stem cells. We found ZEB2 to be one of the earliest factors expressed in prospective human NC, and knockdown revealed a role for ZEB2 in establishing the NC state while repressing pre-placodal and non-neural ectoderm genes. Examination of ZEB2 N-terminal mutant NC cells demonstrates its requirement for the repression of enhancers in the NC gene network and proper NC cell terminal differentiation into osteoblasts and peripheral neurons and neuroglia. This ZEB2 mutation causes early misexpression of BMP signaling ligands, which can be rescued by the attenuation of BMP. Our findings suggest that ZEB2 regulates early human NC specification by modulating proper BMP signaling and further elaborate the molecular defects underlying Mowat-Wilson syndrome.

Uncovering the mesendoderm gene regulatory network through multi-omic data integration.

Mesendodermal specification is one of the earliest events in embryogenesis, where cells first acquire distinct identities. Cell differentiation is a highly regulated process that involves the function of numerous transcription factors (TFs) and signaling molecules, which can be described with gene regulatory networks (GRNs). Cell differentiation GRNs are difficult to build because existing mechanistic methods are low throughput, and high-throughput methods tend to be non-mechanistic. Additionally, integrating highly dimensional data composed of more than two data types is challenging. Here, we use linked self-organizing maps to combine chromatin immunoprecipitation sequencing (ChIP-seq)/ATAC-seq with temporal, spatial, and perturbation RNA sequencing (RNA-seq) data from Xenopus tropicalis mesendoderm development to build a high-resolution genome scale mechanistic GRN. We recover both known and previously unsuspected TF-DNA/TF-TF interactions validated through reporter assays. Our analysis provides insights into transcriptional regulation of early cell fate decisions and provides a general approach to building GRNs using highly dimensional multi-omic datasets.

Distinct molecular profile and restricted stem cell potential defines the prospective human cranial neural crest from embryonic stem cell state.

Neural crest cells are an embryonic multipotent stem cell population. Recent studies in model organisms have suggested that neural crest cells are specified earlier than previously thought, at blastula stages. However, the molecular dynamics of early neural crest specification, and functional changes from pluripotent precursors to early specified NC, remain to be elucidated. In this report, we utilized a robust human model of cranial neural crest formation to address the distinct molecular character of the earliest stages of neural crest specification and assess the functional differences from its embryonic stem cell precursor. Our human neural crest model reveals a rapid change in the epigenetic state of neural crest and pluripotency genes, accompanied by changes in gene expression upon Wnt-based induction from embryonic stem cells. These changes in gene expression are directly regulated by the transcriptional activity of ß-catenin. Furthermore, prospective cranial neural crest cells are characterized by restricted stem cell potential compared to embryonic stem cells. Our results suggest that human neural crest induced by Wnt/ß-catenin signaling from human embryonic stem cells rapidly acquire a prospective neural crest cell state defined by a unique molecular signature and endowed with limited potential compared to pluripotent stem cells.

Foxh1/Nodal Defines Context-Specific Direct Maternal Wnt/ß-Catenin Target Gene Regulation in Early Development.

Although Wnt/ß-catenin signaling is generally conserved and well understood, the regulatory mechanisms controlling context-specific direct Wnt target gene expression in development and disease are still unclear. The onset of zygotic gene transcription in early embryogenesis represents an ideal, accessible experimental system to investigate context-specific direct Wnt target gene regulation. We combine transcriptomics using RNA-seq with genome-wide ß-catenin association using ChIP-seq to identify stage-specific direct Wnt target genes. We propose coherent feedforward regulation involving two distinct classes of direct maternal Wnt target genes, which differ both in expression and persistence of ß-catenin association. We discover that genomic ß-catenin association overlaps with Foxh1-associated regulatory sequences and demonstrate that direct maternal Wnt target gene expression requires Foxh1 function and Nodal/Tgfß signaling. Our results support a new paradigm for direct Wnt target gene co-regulation with context-specific mechanisms that will inform future studies of embryonic development and more widely stem cell-mediated homeostasis and human disease.

Disrupted ER membrane protein complex-mediated topogenesis drives congenital neural crest defects.

Multipass membrane proteins have a myriad of functions, including transduction of cell-cell signals, ion transport, and photoreception. Insertion of these proteins into the membrane depends on the endoplasmic reticulum (ER) membrane protein complex (EMC). Recently, birth defects have been observed in patients with variants in the gene encoding a member of this complex, EMC1. Patient phenotypes include congenital heart disease, craniofacial malformations, and neurodevelopmental disease. However, a molecular connection between EMC1 and these birth defects is lacking. Using Xenopus, we identified defects in neural crest cells (NCCs) upon emc1 depletion. We then used unbiased proteomics and discovered a critical role for emc1 in WNT signaling. Consistent with this, readouts of WNT signaling and Frizzled (Fzd) levels were reduced in emc1-depleted embryos, while NCC defects could be rescued with ß-catenin. Interestingly, other transmembrane proteins were mislocalized upon emc1 depletion, providing insight into additional patient phenotypes. To translate our findings back to humans, we found that EMC1 was necessary for human NCC development in vitro. Finally, we tested patient variants in our Xenopus model and found the majority to be loss-of-function alleles. Our findings define molecular mechanisms whereby EMC1 dysfunction causes disease phenotypes through dysfunctional multipass membrane protein topogenesis.

Blastula stage specification of avian neural crest.

Cell fate specification defines the earliest steps towards a distinct cell lineage. Neural crest, a multipotent stem cell population, is thought to be specified from the ectoderm, but its varied contributions defy canons of segregation potential and challenges its embryonic origin. Aiming to resolve this conflict, we have assayed the earliest specification of neural crest using blastula stage chick embryos. Specification assays on isolated chick epiblast explants identify an intermediate region specified towards the neural crest cell fate. Furthermore, low density culture suggests that the specification of intermediate cells towards the neural crest lineage is independent of contact mediated induction and Wnt-ligand induced signaling, but is, however, dependent on transcriptional activity of ß-catenin. Finally, we have validated the regional identity of the intermediate region towards the neural crest cell fate using fate map studies. Our results suggest a model of neural crest specification within a restricted epiblast region in blastula stage chick embryos.

WNT/ß-catenin modulates the axial identity of embryonic stem cell-derived human neural crest.

WNT/ß-catenin signaling is crucial for neural crest (NC) formation, yet the effects of the magnitude of the WNT signal remain ill-defined. Using a robust model of human NC formation based on human pluripotent stem cells (hPSCs), we expose that the WNT signal modulates the axial identity of NCs in a dose-dependent manner, with low WNT leading to anterior OTX+ HOX- NC and high WNT leading to posterior OTX- HOX+ NC. Differentiation tests of posterior NC confirm expected derivatives, including posterior-specific adrenal derivatives, and display partial capacity to generate anterior ectomesenchymal derivatives. Furthermore, unlike anterior NC, posterior NC exhibits a transient TBXT+/SOX2+ neuromesodermal precursor-like intermediate. Finally, we analyze the contributions of other signaling pathways in posterior NC formation, which suggest a crucial role for FGF in survival/proliferation, and a requirement of BMP for NC maturation. As expected retinoic acid (RA) and FGF are able to modulate HOX expression in the posterior NC. Surprisingly, early RA supplementation prohibits NC formation. This work reveals for the first time that the amplitude of WNT signaling can modulate the axial identity of NC cells in humans.

FGF Modulates the Axial Identity of Trunk hPSC-Derived Neural Crest but Not the Cranial-Trunk Decision.

The neural crest is a transient embryonic tissue that gives rise to a multitude of derivatives in an axially restricted manner. An in vitro counterpart to neural crest can be derived from human pluripotent stem cells (hPSCs) and can be used to study neural crest ontogeny and neurocristopathies, and to generate cells for therapeutic purposes. In order to successfully do this, it is critical to define the specific conditions required to generate neural crest of different axial identities, as regional restriction in differentiation potential is partly cell intrinsic. WNT and FGF signaling have been implicated as inducers of posterior fate, but the exact role that these signals play in trunk neural crest formation remains unclear. Here, we present a fully defined, xeno-free system for generating trunk neural crest from hPSCs and show that FGF signaling directs cells toward different axial identities within the trunk compartment while WNT signaling is the primary determinant of trunk versus cranial identity.

Specification and formation of the neural crest: Perspectives on lineage segregation.

The neural crest is a fascinating embryonic population unique to vertebrates that is endowed with remarkable differentiation capacity. Thought to originate from ectodermal tissue, neural crest cells generate neurons and glia of the peripheral nervous system, and melanocytes throughout the body. However, the neural crest also generates many ectomesenchymal derivatives in the cranial region, including cell types considered to be of mesodermal origin such as cartilage, bone, and adipose tissue. These ectomesenchymal derivatives play a critical role in the formation of the vertebrate head, and are thought to be a key attribute at the center of vertebrate evolution and diversity. Further, aberrant neural crest cell development and differentiation is the root cause of many human pathologies, including cancers, rare syndromes, and birth malformations. In this review, we discuss the current findings of neural crest cell ontogeny, and consider tissue, cell, and molecular contributions toward neural crest formation. We further provide current perspectives into the molecular network involved during the segregation of the neural crest lineage.

Electroporation and in vitro culture of early rabbit embryos.

The functional interrogation of factors underlying early mammalian development is necessary for the understanding and amelioration of human health conditions. The associated article [1] reports on the molecular characterization of markers of neural crest cells in gastrula and neurula stage rabbit embryos. This article presents survival data of rabbit embryos cultured in vitro, as well as immunofluorescence data for molecular markers of neural crest cells following approximately 24-h of culture. Lastly, towards the functional analysis of early neural crest and other developmental genes, this article provides data on the introduction of exogenous DNA into early stage rabbit embryos using electroporation.

Early specification and development of rabbit neural crest cells.

The phenomenal migratory and differentiation capacity of neural crest cells has been well established across model organisms. While the earliest stages of neural crest development have been investigated in non-mammalian model systems such as Xenopus and Aves, the early specification of this cell population has not been evaluated in mammalian embryos, of which the murine model is the most prevalent. Towards a more comprehensive understanding of mammalian neural crest formation and human comparative studies, we have used the rabbit as a mammalian system for the study of early neural crest specification and development. We examine the expression profile of well-characterized neural crest markers in rabbit embryos across developmental time from early gastrula to later neurula stages, and provide a comparison to markers of migratory neural crest in the chick. Importantly, we apply explant specification assays to address the pivotal question of mammalian neural crest ontogeny, and provide the first evidence that a specified population of neural crest cells exists in the rabbit gastrula prior to the overt expression of neural crest markers. Finally, we demonstrate that FGF signaling is necessary for early rabbit neural crest formation, as SU5402 treatment strongly represses neural crest marker expression in explant assays. This study pioneers the rabbit as a model for neural crest development, and provides the first demonstration of mammalian neural crest specification and the requirement of FGF signaling in this process.

A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs.

Germ layer formation is among the earliest differentiation events in metazoan embryos. In triploblasts, three germ layers are formed, among which the endoderm gives rise to the epithelial lining of the gut tube and associated organs including the liver, pancreas and lungs. In frogs (Xenopus), where early germ layer formation has been studied extensively, the process of endoderm specification involves the interplay of dozens of transcription factors. Here, we review the interactions between these factors, summarized in a transcriptional gene regulatory network (GRN). We highlight regulatory connections conserved between frog, fish, mouse, and human endodermal lineages. Especially prominent is the conserved role and regulatory targets of the Nodal signaling pathway and the T-box transcription factors, Vegt and Eomes. Additionally, we highlight network topologies and motifs, and speculate on their possible roles in development.

Foxh1 Occupies cis-Regulatory Modules Prior to Dynamic Transcription Factor Interactions Controlling the Mesendoderm Gene Program.

The interplay between transcription factors and chromatin dictates gene regulatory network activity. Germ layer specification is tightly coupled with zygotic gene activation and, in most metazoans, is dependent upon maternal factors. We explore the dynamic genome-wide interactions of Foxh1, a maternal transcription factor that mediates Nodal/TGF-ß signaling, with cis-regulatory modules (CRMs) during mesendodermal specification. Foxh1 marks CRMs during cleavage stages and recruits the co-repressor Tle/Groucho in the early blastula. We highlight a population of CRMs that are continuously occupied by Foxh1 and show that they are marked by H3K4me1, Ep300, and Fox/Sox/Smad motifs, suggesting interplay between these factors in gene regulation. We also propose a molecular "hand-off" between maternal Foxh1 and zygotic Foxa at these CRMs to maintain enhancer activation. Our findings suggest that Foxh1 functions at the top of a hierarchy of interactions by marking developmental genes for activation, beginning with the onset of zygotic gene expression.