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.

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.

RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells.

Current approaches for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated-9 (Cas9)-mediated genome editing in human pluripotent stem (PS) cells mainly employ plasmids or ribonucleoprotein complexes. Here, we devise an improved transfection protocol of in vitro transcribed Cas9 mRNA and crRNA:tracrRNA duplex that can effectively generate indels in four genetic loci (two active and two inactive) and demonstrate utility in four human PS cell lines (one embryonic and three induced PS cell lines). Our improved protocol incorporating a Cas9-linked selection marker and a staggered transfection strategy promotes targeting efficiency up to 85% and biallelic targeting efficiency up to 76.5% of total mutant clones. The superior targeting efficiency and the non-integrative nature of our approach underscore broader applications in high-throughput arrayed CRISPR screening and in generating custom-made or off-the-shelf cell products for human therapy.

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.

Human neural crest induction by temporal modulation of WNT activation.

The developmental biology of neural crest cells in humans remains unexplored due to technical and ethical challenges. The use of pluripotent stem cells to model human neural crest development has gained momentum. We recently introduced a rapid chemically defined approach to induce robust neural crest by WNT/ß-CATENIN activation. Here we investigate the temporal requirements of ectopic WNT activation needed to induce neural crest cells. By altering the temporal activation of canonical WNT/ß-CATENIN with a GSK3 inhibitor we find that a 2 Day pulse of WNT/ß-CATENIN activation via GSK3 inhibition is optimal to generate bona fide neural crest cells, as shown by their capacity to differentiate to neural crest specific fates including peripheral neurons, glia, melanoblasts and ectomesenchymal osteocytes, chondrocytes and adipocytes. Although a 2 Day pulse can impart neural crest character when GSK3 is inhibited days after seeding, optimal results are obtained when WNT is activated from the beginning, and we find that the window of competence to induce NCs from non-neural ectodermal/placodal precursors closes by day 3 of culture. The reduced requirement for exogenous WNT activation offers an approach that is cost-effective, and we show that this adherent 2-dimensional approach is efficient in a broad range of culture platforms ranging from 96-well vessels to 10 cm dishes.

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.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

Defects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

WNT/ß-catenin signaling mediates human neural crest induction via a pre-neural border intermediate.

Neural crest (NC) cells arise early in vertebrate development, migrate extensively and contribute to a diverse array of ectodermal and mesenchymal derivatives. Previous models of NC formation suggested derivation from neuralized ectoderm, via meso-ectodermal, or neural-non-neural ectoderm interactions. Recent studies using bird and amphibian embryos suggest an earlier origin of NC, independent of neural and mesodermal tissues. Here, we set out to generate a model in which to decipher signaling and tissue interactions involved in human NC induction. Our novel human embryonic stem cell (ESC)-based model yields high proportions of multipotent NC cells (expressing SOX10, PAX7 and TFAP2A) in 5 days. We demonstrate a crucial role for WNT/ß-catenin signaling in launching NC development, while blocking placodal and surface ectoderm fates. We provide evidence of the delicate temporal effects of BMP and FGF signaling, and find that NC development is separable from neural and/or mesodermal contributions. We further substantiate the notion of a neural-independent origin of NC through PAX6 expression and knockdown studies. Finally, we identify a novel pre-neural border state characterized by early WNT/ß-catenin signaling targets that displays distinct responses to BMP and FGF signaling from the traditional neural border genes. In summary, our work provides a fast and efficient protocol for human NC differentiation under signaling constraints similar to those identified in vivo in model organisms, and strengthens a framework for neural crest ontogeny that is separable from neural and mesodermal fates.

Pax7 is regulated by cMyb during early neural crest development through a novel enhancer.

The neural crest (NC) is a migratory population of cells unique to vertebrates that generates many diverse derivatives. NC cells arise during gastrulation at the neural plate border (NPB), which is later elevated as the neural folds (NFs) form and fuse in the dorsal region of the closed neural tube, from where NC cells emigrate. In chick embryos, Pax7 is an early marker, and necessary component of NC development. Unlike other early NPB markers, which are co-expressed in lateral ectoderm, medial neural plate or posterior-lateral mesoderm, Pax7 early expression seems more restricted to the NPB. However, the molecular mechanisms controlling early Pax7 expression remain poorly understood. Here, we identify a novel enhancer of Pax7 in avian embryos that replicates the expression of Pax7 associated with early NC development. Expression from this enhancer is found in early NPB, NFs and early emigrating NC, but unlike Pax7, which is also expressed in mesodermal derivatives, this enhancer is not active in somites. Further analysis demonstrates that cMyb is able to interact with this enhancer and modulates reporter and endogenous early Pax7 expression; thus, cMyb is identified as a novel regulator of Pax7 in early NC development.

SUMOylation of Pax7 is essential for neural crest and muscle development.

Regulatory transcription factors of the Pax family play fundamental roles in the function of multipotent cells during vertebrate development, post-natal regeneration, and cancer. Pax7 and its homologue Pax3 are important players in neural crest and muscle development. Both genes are coexpressed in various tissues and are thought to provide similar, but not identical, functions. The mechanisms that allow specific regulation of Pax7 remain largely unknown. Here, we report for the first time that Pax7 is regulated by SUMOylation. We identify the interaction of Pax7 with Ubc9, the SUMO conjugating enzyme, and reveal that SUMOylation machinery is enriched in neural crest precursors and plays a critical role in NC development. We demonstrate that Pax7 becomes SUMOylated and identify an essential role for lysine 85 (K85) in Pax7-SUMOylation. Despite high conservation surrounding K85 amongst Pax genes, we were unable to identify SUMOylation of other Pax proteins tested, including Pax3. Using a non-SUMOylatable Pax7 variant (K85 X R), we demonstrate that SUMOylation is essential for the function of Pax7 in neural crest development, C2C12 myogenic differentiation, and transcriptional transactivation. Our study provides new mechanistic insight into the molecular regulation of Pax7's function by SUMOylation in neural crest and muscle development.

FGF signaling transforms non-neural ectoderm into neural crest.

The neural crest arises at the border between the neural plate and the adjacent non-neural ectoderm. It has been suggested that both neural and non-neural ectoderm can contribute to the neural crest. Several studies have examined the molecular mechanisms that regulate neural crest induction in neuralized tissues or the neural plate border. Here, using the chick as a model system, we address the molecular mechanisms by which non-neural ectoderm generates neural crest. We report that in response to FGF the non-neural ectoderm can ectopically express several early neural crest markers (Pax7, Msx1, Dlx5, Sox9, FoxD3, Snail2, and Sox10). Importantly this response to FGF signaling can occur without inducing ectopic mesodermal tissues. Furthermore, the non-neural ectoderm responds to FGF by expressing the prospective neural marker Sox3, but it does not express definitive markers of neural or anterior neural (Sox2 and Otx2) tissues. These results suggest that the non-neural ectoderm can launch the neural crest program in the absence of mesoderm, without acquiring definitive neural character. Finally, we report that prior to the upregulation of these neural crest markers, the non-neural ectoderm upregulates both BMP and Wnt molecules in response to FGF. Our results provide the first effort to understand the molecular events leading to neural crest development via the non-neural ectoderm in amniotes and present a distinct response to FGF signaling.

Pax7 lineage contributions to the mammalian neural crest.

BACKGROUND: Neural crest cells are vertebrate-specific multipotent cells that contribute to a variety of tissues including the peripheral nervous system, melanocytes, and craniofacial bones and cartilage. Abnormal development of the neural crest is associated with several human maladies including cleft/lip palate, aggressive cancers such as melanoma and neuroblastoma, and rare syndromes, like Waardenburg syndrome, a complex disorder involving hearing loss and pigment defects. We previously identified the transcription factor Pax7 as an early marker, and required component for neural crest development in chick embryos. In mammals, Pax7 is also thought to play a role in neural crest development, yet the precise contribution of Pax7 progenitors to the neural crest lineage has not been determined. METHODOLOGY/PRINCIPAL FINDINGS: Here we use Cre/loxP technology in double transgenic mice to fate map the Pax7 lineage in neural crest derivates. We find that Pax7 descendants contribute to multiple tissues including the cranial, cardiac and trunk neural crest, which in the cranial cartilage form a distinct regional pattern. The Pax7 lineage, like the Pax3 lineage, is additionally detected in some non-neural crest tissues, including a subset of the epithelial cells in specific organs. CONCLUSIONS/SIGNIFICANCE: These results demonstrate a previously unappreciated widespread distribution of Pax7 descendants within and beyond the neural crest. They shed light regarding the regionally distinct phenotypes observed in Pax3 and Pax7 mutants, and provide a unique perspective into the potential roles of Pax7 during disease and development.

Current perspectives of the signaling pathways directing neural crest induction.

The neural crest is a migratory population of embryonic cells with a tremendous potential to differentiate and contribute to nearly every organ system in the adult body. Over the past two decades, an incredible amount of research has given us a reasonable understanding of how these cells are generated. Neural crest induction involves the combinatorial input of multiple signaling pathways and transcription factors, and is thought to occur in two phases from gastrulation to neurulation. In the first phase, FGF and Wnt signaling induce NC progenitors at the border of the neural plate, activating the expression of members of the Msx, Pax, and Zic families, among others. In the second phase, BMP, Wnt, and Notch signaling maintain these progenitors and bring about the expression of definitive NC markers including Snail2, FoxD3, and Sox9/10. In recent years, additional signaling molecules and modulators of these pathways have been uncovered, creating an increasingly complex regulatory network. In this work, we provide a comprehensive review of the major signaling pathways that participate in neural crest induction, with a focus on recent developments and current perspectives. We provide a simplified model of early neural crest development and stress similarities and differences between four major model organisms: Xenopus, chick, zebrafish, and mouse.

FGF/MAPK signaling is required in the gastrula epiblast for avian neural crest induction.

Neural crest induction involves the combinatorial inputs of the FGF, BMP and Wnt signaling pathways. Recently, a two-step model has emerged where BMP attenuation and Wnt activation induces the neural crest during gastrulation, whereas activation of both pathways maintains the population during neurulation. FGF is proposed to act indirectly during the inductive phase by activating Wnt ligand expression in the mesoderm. Here, we use the chick model to investigate the role of FGF signaling in the amniote neural crest for the first time and uncover a novel requirement for FGF/MAPK signaling. Contrary to current models, we demonstrate that FGF is required within the prospective neural crest epiblast during gastrulation and is unlikely to operate through mesodermal tissues. Additionally, we show that FGF/MAPK activity in the prospective neural plate prevents the ectopic expression of lateral ectoderm markers, independently of its role in neural specification. We then investigate the temporal participation of BMP/Smad signaling and suggest a later involvement in neural plate border development, likely due to widespread FGF/MAPK activity in the gastrula epiblast. Our results identify an early requirement for FGF/MAPK signaling in amniote neural crest induction and suggest an intriguing role for FGF-mediated Smad inhibition in ectodermal development.

Embryonic Pax7-expressing progenitors contribute multiple cell types to the postnatal olfactory epithelium.

Prolonged neurogenesis driven by stem/progenitor cells is a hallmark of the olfactory epithelium (OE), beginning at the placodal stages in the embryo and continuing throughout adult life. Despite the progress made to identify and study the regulation of adult OE progenitors, our knowledge of embryonic OE precursors and their cellular contributions to the adult OE has been stalled by the lack of markers able to distinguish individual candidate progenitors. Here we identify embryonic OE Pax7+ progenitors, detected at embryonic day 10.5 (E10.5) in the olfactory pit with an antigen profile and location previously assigned to presumptive OE stem cells. Using Cre-loxP technology (Pax7-cre/ROSA YFP mice), we expose a wide range of derivatives, including CNS and olfactory neurons, non-neuronal cells, and olfactory ensheathing glia, all made from embryonic Pax7+ cells. Importantly, the expression of Pax7 in the embryonic OE is downregulated from E15.5, such that after birth, no Pax7+ cells are found in the OE, and thus the progenitor population here identified is restricted to embryonic stages. Our results provide the first evidence for a population of Pax7-expressing embryonic progenitors that contribute to multiple OE lineages and demonstrate novel insights into the unique spatiotemporal patterning of the postnatal OE.