A new atlas to study embryonic cell types in Xenopus.

This paper introduces a single-cell atlas for pivotal developmental stages in Xenopus, encompassing gastrulation, neurulation, and early tailbud. Notably surpassing its predecessors, the new atlas enhances gene mapping, read counts, and gene/cell type nomenclature. Leveraging the latest Xenopus tropicalis genome version, alongside advanced alignment pipelines and machine learning for cell type assignment, this release maintains consistency with previous cell type annotations while rectifying nomenclature issues. Employing an unbiased approach for cell type assignment proves especially apt for embryonic contexts, given the considerable number of non-terminally differentiated cell types. An alternative cell type attribution here adopts a fuzzy, non-deterministic stance, capturing the transient nature of early embryo progenitor cells by presenting an ensemble of types in superposition. The value of the new resource is emphasized through numerous examples, with a focus on previously unexplored germ cell populations where we uncover novel transcription onset features. Offering interactive exploration via a user-friendly web portal and facilitating complete data downloads, this atlas serves as a comprehensive and accessible reference.

Two induced pluripotent stem cell (iPSC) lines derived from patients affected by Waardenburg syndrome type 1 retain potential to activate neural crest markers.

Waardenburg syndrome type 1 (WS1), a rare genetic disease characterized by pigmentation defects and mild craniofacial anomalies often associated with congenital deafness is caused by heterozygous mutations in the PAX3 gene (2q36.1). We have generated two induced pluripotent stem cell lines (PCli029-A and PCli031-A) from two patients from the same family both carrying the same heterozygous deletion in PAX3 exon 1 (c.-70_85 + 366del). These cells are pluripotent as they can differentiate into ectoderm, mesoderm and endoderm. They also can activate the early neural crest marker SNAI2. These cells will be useful for studying the human neural crest-derived pigment cells.

PFKFB4 interacts with ICMT and activates RAS/AKT signaling-dependent cell migration in melanoma.

Cell migration is a complex process, tightly regulated during embryonic development and abnormally activated during cancer metastasis. RAS-dependent signaling is a major nexus controlling essential cell parameters including proliferation, survival, and migration, utilizing downstream effectors such as the PI3K/AKT signaling pathway. In melanoma, oncogenic mutations frequently enhance RAS, PI3K/AKT, or MAP kinase signaling and trigger other cancer hallmarks among which the activation of metabolism regulators. PFKFB4 is one of these critical regulators of glycolysis and of the Warburg effect. Here, however, we explore a novel function of PFKFB4 in melanoma cell migration. We find that PFKFB4 interacts with ICMT, a posttranslational modifier of RAS. PFKFB4 promotes ICMT/RAS interaction, controls RAS localization at the plasma membrane, activates AKT signaling and enhances cell migration. We thus provide evidence of a novel and glycolysis-independent function of PFKFB4 in human cancer cells. This unconventional activity links the metabolic regulator PFKFB4 to RAS-AKT signaling and impacts melanoma cell migration.

Xenopus, an emerging model for studying pathologies of the neural crest.

Neural crest cells are a multipotent embryonic stem cell population that emerges from the lateral border of the neural plate after an epithelium-to-mesenchyme transition. These cells then migrate extensively in the embryo and generate a large variety of differentiated cell types and tissues. Alterations in almost any of the processes involved in neural crest development can cause severe congenital defects in humans. Moreover, the malignant transformation of one of the many neural crest derivatives, during childhood or in adults, can cause the development of aggressive tumors prone to metastasis such as melanoma and neuroblastoma. Collectively these diseases are called neurocristopathies. Here we review how a variety of approaches implemented using the amphibian Xenopus as an experimental model have shed light on the molecular basis of numerous neurocristopathies, and how this versatile yet underused vertebrate animal model could help accelerate discoveries in the field. Using the current framework of the neural crest gene regulatory network, we review the pathologies linked to defects at each step of neural crest formation and highlight studies that have used the Xenopus model to decipher the cellular and molecular aspects of neurocristopathies.

BMP signaling is enhanced intracellularly by FHL3 controlling WNT-dependent spatiotemporal emergence of the neural crest.

The spatiotemporal coordination of multiple morphogens is essential for embryonic patterning yet poorly understood. During neural crest (NC) formation, dynamic bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and WNT signals cooperate by acting on mesoderm and ectoderm. Here, we show that Fhl3, a scaffold LIM domain protein, modulates BMP gradient interpretation during NC induction. During gastrulation, low BMP signaling neuralizes the neural border (NB) ectoderm, while Fhl3 enhances Smad1 intracellular response in underlying paraxial mesoderm, triggering the high WNT8 signals needed to pattern the NB. During neurulation, fhl3 activation in NC ectoderm promotes simultaneous high BMP and BMP-dependent WNT activity required for specification. Mechanistically, Fhl3 interacts with Smad1 and promotes Smad1 binding to wnt8 promoter in a BMP-dependent manner. Consequently, differential Fhl3 expression in adjacent cells ensures a finely tuned coordination of BMP and WNT signaling at several stages of NC development, starting by positioning the NC-inducing mesoderm center under competent NB ectoderm.

Neural crest multipotency and specification: power and limits of single cell transcriptomic approaches.

The neural crest is a unique population of multipotent cells forming in vertebrate embryos. Their vast cell fate potential enables the generation of a diverse array of differentiated cell types in vivo. These include, among others, connective tissue, cartilage and bone of the face and skull, neurons and glia of the peripheral nervous system (including enteric nervous system), and melanocytes. Following migration, these derivatives extensively populate multiple germ layers. Within the competent neural border ectoderm, an area located at the junction between the neural and non-neural ectoderm during embryonic development, neural crest cells form in response to a series of inductive secreted cues including BMP, Wnt, and FGF signals. As cells become progressively specified, they express transcriptional modules conducive with their stage of fate determination or cell state. Those sequential states include the neural border state, the premigratory neural crest state, the epithelium-to-mesenchyme transitional state, and the migratory state to end with post-migratory and differentiation states. However, despite the extensive knowledge accumulated over 150 years of neural crest biology, many key questions remain open, in particular the timing of neural crest lineage determination, the control of potency during early developmental stages, and the lineage relationships between different subpopulations of neural crest cells. In this review, we discuss the recent advances in understanding early neural crest formation using cutting-edge high-throughput single cell sequencing approaches. We will discuss how this new transcriptomic data, from 2017 to 2021, has advanced our knowledge of the steps in neural crest cell lineage commitment and specification, the mechanisms driving multipotency, and diversification. We will then discuss the questions that remain to be resolved and how these approaches may continue to unveil the biology of these fascinating cells.

Insights Into the Early Gene Regulatory Network Controlling Neural Crest and Placode Fate Choices at the Neural Border.

The neural crest (NC) cells and cranial placodes are two ectoderm-derived innovations in vertebrates that led to the acquisition of a complex head structure required for a predatory lifestyle. They both originate from the neural border (NB), a portion of the ectoderm located between the neural plate (NP), and the lateral non-neural ectoderm. The NC gives rise to a vast array of tissues and cell types such as peripheral neurons and glial cells, melanocytes, secretory cells, and cranial skeletal and connective cells. Together with cells derived from the cranial placodes, which contribute to sensory organs in the head, the NC also forms the cranial sensory ganglia. Multiple in vivo studies in different model systems have uncovered the signaling pathways and genetic factors that govern the positioning, development, and differentiation of these tissues. In this literature review, we give an overview of NC and placode development, focusing on the early gene regulatory network that controls the formation of the NB during early embryonic stages, and later dictates the choice between the NC and placode progenitor fates.

The vertebrate-specific VENTX/NANOG gene empowers neural crest with ectomesenchyme potential.

During Cambrian, unipotent progenitors located at the neural (plate) border (NB) of an Olfactoria chordate embryo acquired the competence to form ectomesenchyme, pigment cells and neurons, initiating the rise of the multipotent neural crest cells (NC) specific to vertebrates. Surprisingly, the known vertebrate NB/NC transcriptional circuitry is a constrained feature also found in invertebrates. Therefore, evidence for vertebrate-specific innovations endowing vertebrate NC with multipotency is still missing. Here, we identified VENTX/NANOG and POU5/OCT4 as vertebrate-specific innovations. When VENTX was depleted in vivo and in directly-induced NC, the NC lost its early multipotent state and its skeletogenic potential, but kept sensory neuron and pigment identity, thus reminiscent of invertebrate NB precursors. In vivo, VENTX gain-of-function enabled NB specifiers to reprogram embryonic non-neural ectoderm towards early NC identity. We propose that skeletogenic NC evolved by acquiring VENTX/NANOG activity, promoting a novel multipotent progenitor regulatory state into the pre-existing sensory neuron/pigment NB program.

The neural border: Induction, specification and maturation of the territory that generates neural crest cells.

The neural crest is induced at the edge between the neural plate and the nonneural ectoderm, in an area called the neural (plate) border, during gastrulation and neurulation. In recent years, many studies have explored how this domain is patterned, and how the neural crest is induced within this territory, that also participates to the prospective dorsal neural tube, the dorsalmost nonneural ectoderm, as well as placode derivatives in the anterior area. This review highlights the tissue interactions, the cell-cell signaling and the molecular mechanisms involved in this dynamic spatiotemporal patterning, resulting in the induction of the premigratory neural crest. Collectively, these studies allow building a complex neural border and early neural crest gene regulatory network, mostly composed by transcriptional regulations but also, more recently, including novel signaling interactions.

AKT signaling displays multifaceted functions in neural crest development.

AKT signaling is an essential intracellular pathway controlling cell homeostasis, cell proliferation and survival, as well as cell migration and differentiation in adults. Alterations impacting the AKT pathway are involved in many pathological conditions in human disease. Similarly, during development, multiple transmembrane molecules, such as FGF receptors, PDGF receptors or integrins, activate AKT to control embryonic cell proliferation, migration, differentiation, and also cell fate decisions. While many studies in mouse embryos have clearly implicated AKT signaling in the differentiation of several neural crest derivatives, information on AKT functions during the earliest steps of neural crest development had remained relatively scarce until recently. However, recent studies on known and novel regulators of AKT signaling demonstrate that this pathway plays critical roles throughout the development of neural crest progenitors. Non-mammalian models such as fish and frog embryos have been instrumental to our understanding of AKT functions in neural crest development, both in neural crest progenitors and in the neighboring tissues. This review combines current knowledge acquired from all these different vertebrate animal models to describe the various roles of AKT signaling related to neural crest development in vivo. We first describe the importance of AKT signaling in patterning the tissues involved in neural crest induction, namely the dorsal mesoderm and the ectoderm. We then focus on AKT signaling functions in neural crest migration and differentiation.

Avian models and the study of invariant asymmetry: how the chicken and the egg taught us to tell right from left.

While the external vertebrate body plan appears bilaterally symmetrical with respect to anterior-posterior and dorsal-ventral axes, the internal organs are arranged with a striking and invariant left-right asymmetry. This laterality is important for normal body function, as alterations manifest as numerous human birth defect syndromes. The left-right axis is set up very early during embryogenesis by an initial and still poorly understood break in bilateral symmetry, followed by a cascade of molecular events that was discovered 20 years ago in the chick embryo model. This gene regulatory network leads to activation of the pitx2 gene on the left side of the embryo which ultimately establishes asymmetric organogenesis of the heart, gut, brain, and other organs. In this review, we highlight the crucial contributions of the avian model to the discovery of the differential transcriptional cascades operating on the Left and Right sides, as well as to the physiological events operating upstream of asymmetric gene expression. The chick was not only instrumental in the discovery of mechanisms behind left-right patterning, but stands poised to facilitate inroads into the most fundamental aspects that link asymmetry to the rest of evolutionary developmental biology.

An atlas of Wnt activity during embryogenesis in Xenopus tropicalis.

Wnt proteins form a family of highly conserved secreted molecules that are critical mediators of cell-cell signaling during embryogenesis. Partial data on Wnt activity in different tissues and at different stages have been reported in frog embryos. Our objective here is to provide a coherent and detailed description of Wnt activity throughout embryo development. Using a transgenic Xenopus tropicalis line carrying a Wnt-responsive reporter sequence, we depict the spatial and temporal dynamics of canonical Wnt activity during embryogenesis. We provide a comprehensive series of in situ hybridization in whole-mount embryos and in cross-sections, from gastrula to tadpole stages, with special focus on neural tube, retina and neural crest cell development. This collection of patterns will thus constitute a valuable resource for developmental biologists to picture the dynamics of Wnt activity during development.

PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation.

Neural crest (NC) specification comprises an early phase, initiating immature NC progenitors formation at neural plate stage, and a later phase at neural fold stage, resulting in a functional premigratory NC that is able to delaminate and migrate. We found that the NC gene regulatory network triggers upregulation of pfkfb4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) during this late specification phase. As shown in previous studies, PFKFB4 controls AKT signaling in gastrulas and glycolysis rate in adult cells. Here, we focus on PFKFB4 function in NC during and after neurulation, using time-controlled or hypomorph depletions in vivo We find that PFKFB4 is essential both for specification of functional premigratory NC and for its migration. PFKFB4-depleted embryos fail to activate n-cadherin and late NC specifiers, and exhibit severe migration defects resulting in craniofacial defects. AKT signaling mediates PFKFB4 function in NC late specification, whereas both AKT signaling and glycolysis regulate migration. These findings highlight novel and essential roles of PFKFB4 activity in later stages of NC development that are wired into the NC gene regulatory network.

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates.

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

PAX transcription factors in neural crest development.

The nine vertebrate PAX transcription factors (PAX1-PAX9) play essential roles during early development and organogenesis. Pax genes were identified in vertebrates using their homology with the Drosophila melanogaster paired gene DNA-binding domain. PAX1-9 functions are largely conserved throughout vertebrate evolution, in particular during central nervous system and neural crest development. The neural crest is a vertebrate invention, which gives rise to numerous derivatives during organogenesis, including neurons and glia of the peripheral nervous system, craniofacial skeleton and mesenchyme, the heart outflow tract, endocrine and pigment cells. Human and mouse spontaneous mutations as well as experimental analyses have evidenced the critical and diverse functions of PAX factors during neural crest development. Recent studies have highlighted the role of PAX3 and PAX7 in neural crest induction. Additionally, several PAX proteins - PAX1, 3, 7, 9 - regulate cell proliferation, migration and determination in multiple neural crest-derived lineages, such as cardiac, sensory, and enteric neural crest, pigment cells, glia, craniofacial skeleton and teeth, or in organs developing in close relationship with the neural crest such as the thymus and parathyroids. The diverse PAX molecular functions during neural crest formation rely on fine-tuned modulations of their transcriptional transactivation properties. These modulations are generated by multiple means, such as different roles for the various isoforms (formed by alternative splicing), or posttranslational modifications which alter protein-DNA binding, or carefully orchestrated protein-protein interactions with various co-factors which control PAX proteins activity. Understanding these regulations is the key to decipher the versatile roles of PAX transcription factors in neural crest development, differentiation and disease.

PFKFB4 controls embryonic patterning via Akt signalling independently of glycolysis.

How metabolism regulators play roles during early development remains elusive. Here we show that PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4), a glycolysis regulator, is critical for controlling dorsal ectoderm global patterning in gastrulating frog embryos via a non-glycolytic function. PFKFB4 is required for dorsal ectoderm progenitors to proceed towards more specified fates including neural and non-neural ectoderm, neural crest or placodes. This function is mediated by Akt signalling, a major pathway that integrates cell homeostasis and survival parameters. Restoring Akt signalling rescues the loss of PFKFB4 in vivo. In contrast, glycolysis is not essential for frog development at this stage. Our study reveals the existence of a PFKFB4-Akt checkpoint that links cell homeostasis to the ability of progenitor cells to undergo differentiation, and uncovers glycolysis-independent functions of PFKFB4.

Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers.

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.

Protein tyrosine phosphatase 4A3 (PTP4A3) is required for Xenopus laevis cranial neural crest migration in vivo.

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.

Signaling and transcriptional regulation in neural crest specification and migration: lessons from xenopus embryos.

The neural crest is a population of highly migratory and multipotent cells, which arises from the border of the neural plate in vertebrate embryos. In the last few years, the molecular actors of neural crest early development have been intensively studied, notably by using the frog embryo, as a prime model for the analysis of the earliest embryonic inductions. In addition, tremendous progress has been made in understanding the molecular and cellular basis of Xenopus cranial neural crest migration, by combining in vitro and in vivo analysis. In this review, we examine how the action of previously known neural crest-inducing signals [bone morphogenetic protein (BMP), wingless-int (Wnt), fibroblast growth factor (FGF)] is controlled by newly discovered modulators during early neural plate border patterning and neural crest specification. This regulation controls the induction of key transcription factors that cooperate to pattern the premigratory neural crest progenitors. These data are discussed in the perspective of the gene regulatory network that controls neural and neural crest patterning. We then address recent findings on noncanonical Wnt signaling regulation, cell polarization, and collective cell migration which highlight how cranial neural crest cells populate their target tissue, the branchial arches, in vivo. More than ever, the neural crest stands as a powerful and attractive model to decipher complex vertebrate regulatory circuits in vivo.

Pfkfb (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) isoforms display a tissue-specific and dynamic expression during Xenopus laevis development.

Pfkfb (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) enzymes are bi-functional enzymes encoded by four different genes (pfkfb1, pfkfb2, pfkfb3, pfkfb4) in vertebrates. They are involved in the regulation of glycolysis: they catalyze the synthesis and the degradation of F-2,6-BP (fructose-2,6-bisphosphate), the most potent allosteric activator of phosphofructokinase 1 (Pfk1), a key glycolytic enzyme. By producing F-2,6-BP, Pfkfb enzymes allow glycolysis to proceed, while by degrading F-2,6-BP they block glycolysis. As major regulators of glycolysis, Pfkfb enzymes are involved in cancer: tumor cells have a higher glycolytic rate compared to normal cells, even in the presence of adequate oxygen levels (Warburg effect) and several cancer cell lines express elevated levels of Pfkfb enzymes. Glycolysis is also important for energy and metabolite production in proliferating cells. In embryos, however, the role of glycolysis and the expression of glycolysis regulators remain to be explored. Here, we provide a phylogenetic analysis of Pfkfb enzymes in vertebrates, and we detail the expression pattern of pfk1, pfkfb1, pfkfb2, pfkfb3, and pfkfb4 genes in Xenopus laevis embryos. We show that pfkfb transcripts expression is overlapping at blastula and gastrula stages and that from neurulation to tadpole stages, they display tissue-specific, complementary and dynamic expression patterns.