Pluripotency of a founding field: rebranding developmental biology.

The field of developmental biology has declined in prominence in recent decades, with off-shoots from the field becoming more fashionable and highly funded. This has created inequity in discovery and opportunity, partly due to the perception that the field is antiquated or not cutting edge. A 'think tank' of scientists from multiple developmental biology-related disciplines came together to define specific challenges in the field that may have inhibited innovation, and to provide tangible solutions to some of the issues facing developmental biology. The community suggestions include a call to the community to help 'rebrand' the field, alongside proposals for additional funding apparatuses, frameworks for interdisciplinary innovative collaborations, pedagogical access, improved science communication, increased diversity and inclusion, and equity of resources to provide maximal impact to the community.

Small molecule-mediated reprogramming of Xenopus blastula stem cells to a neural crest state.

Neural crest cells are a stem cell population unique to vertebrates that give rise to a diverse array of derivatives, including much of the peripheral nervous system, pigment cells, cartilage, mesenchyme, and bone. Acquisition of these cells drove the evolution of vertebrates and defects in their development underlies a broad set of neurocristopathies. Moreover, studies of neural crest can inform differentiation protocols for pluripotent stem cells and regenerative medicine applications. Xenopus embryos are an important system for studies of the neural crest and have provided numerous insights into the signals and transcription factors that control the formation and later lineage diversification of these stem cells. Pluripotent animal pole explants are a particularly powerful tool in this system as they can be cultured in simple salt solution and instructed to give rise to any cell type including the neural crest. Here we report a protocol for small molecule-mediated induction of the neural crest state from blastula stem cells and validate it using transcriptome analysis and grafting experiments. This is an powerful new tool for generating this important cell type that will facilitate future studies of neural crest development and mutations and variants linked to neurocristopathies.

SoxB1 transcription factors are essential for initiating and maintaining the neural plate border gene expression.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis , we set out to determine if SoxB1 transcription factors have a regulatory function in NPB formation. Herein, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and, surprisingly, in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as a potential SoxB1 partners in regulating the formation of the NPB and show their combined activity is needed to maintain NPB gene expression. Together, these data identify a novel role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

Production and characterization of monoclonal antibodies to Xenopus proteins.

Monoclonal antibodies are powerful and versatile tools that enable the study of proteins in diverse contexts. They are often utilized to assist with identification of subcellular localization and characterization of the function of target proteins of interest. However, because there can be considerable sequence diversity between orthologous proteins in Xenopus and mammals, antibodies produced against mouse or human proteins often do not recognize Xenopus counterparts. To address this issue, we refined existing mouse monoclonal antibody production protocols to generate antibodies against Xenopus proteins of interest. Here, we describe several approaches for the generation of useful mouse anti-Xenopus antibodies to multiple Xenopus proteins and their validation in various experimental approaches. These novel antibodies are now available to the research community through the Developmental Study Hybridoma Bank (DSHB).

The developmental and evolutionary origins of cellular pluripotency in the vertebrate neural crest.

Neural crest cells are central to vertebrate development and evolution, endowing vertebrates with a "new head" that resulted in morphological, physiological, and behavioral features that allowed vertebrates to become active predators. One remarkable feature of neural crest cells is their multi-germ layer potential that allows for the formation of both ectodermal (pigmentation, peripheral glia, sensory neurons) and mesenchymal (connective tissue, cartilage/bone, dermis) cell types. Understanding the cellular and evolutionary origins of this broad cellular potential in the neural crest has been a long-standing focus for developmental biologists. Here, we review recent work that has demonstrated that neural crest cells share key features with pluripotent blastula stem cells, including expression of the Yamanaka stem cell factors (Oct3/4, Klf4, Sox2, c-Myc). These shared features suggest that pluripotency is either retained in the neural crest from blastula stages or subsequently reactivated as the neural crest forms. We highlight the cellular and molecular parallels between blastula stem cells and neural crest cells and discuss the work that has led to current models for the cellular origins of broad potential in the crest. Finally, we explore how these themes can provide new insights into how and when neural crest cells and pluripotency evolved in vertebrates and the evolutionary relationship between these populations.

Shared features of blastula and neural crest stem cells evolved at the base of vertebrates.

The neural crest is vertebrate-specific stem cell population that helped drive the origin and evolution of the vertebrate clade. A distinguishing feature of these stem cells is their multi-germ layer potential, which has drawn developmental and evolutionary parallels to another stem cell population-pluripotent embryonic stem cells (animal pole cells or ES cells) of the vertebrate blastula. Here, we investigate the evolutionary origins of neural crest potential by comparing neural crest and pluripotency gene regulatory networks (GRNs) in both jawed ( Xenopus ) and jawless (lamprey) vertebrates. Through comparative gene expression analysis and transcriptomics, we reveal an ancient evolutionary origin of shared regulatory factors between neural crest and pluripotency GRNs that dates back to the last common ancestor of extant vertebrates. Focusing on the key pluripotency factor pou5 (formerly oct4), we show that the lamprey genome encodes a pou5 ortholog that is expressed in animal pole cells, as in jawed vertebrates, but is absent from the neural crest. However, gain-of-function experiments show that both lamprey and Xenopus pou5 enhance neural crest formation, suggesting that pou5 was lost from the neural crest of jawless vertebrates. Finally, we show that pou5 is required for neural crest specification in jawed vertebrates and that it acquired novel neural crest-enhancing activity after evolving from an ancestral pou3 -like clade that lacks this functionality. We propose that a pluripotency-neural crest GRN was assembled in stem vertebrates and that the multi-germ layer potential of the neural crest evolved by deploying this regulatory program.

Quantitative analysis of transcriptome dynamics provides novel insights into developmental state transitions.

BACKGROUND: During embryogenesis, the developmental potential of initially pluripotent cells becomes progressively restricted as they transit to lineage restricted states. The pluripotent cells of Xenopus blastula-stage embryos are an ideal system in which to study cell state transitions during developmental decision-making, as gene expression dynamics can be followed at high temporal resolution. RESULTS: Here we use transcriptomics to interrogate the process by which pluripotent cells transit to four different lineage-restricted states: neural progenitors, epidermis, endoderm and ventral mesoderm, providing quantitative insights into the dynamics of Waddington's landscape. Our findings provide novel insights into why the neural progenitor state is the default lineage state for pluripotent cells and uncover novel components of lineage-specific gene regulation. These data reveal an unexpected overlap in the transcriptional responses to BMP4/7 and Activin signaling and provide mechanistic insight into how the timing of signaling inputs such as BMP are temporally controlled to ensure correct lineage decisions. CONCLUSIONS: Together these analyses provide quantitative insights into the logic and dynamics of developmental decision making in early embryos. They also provide valuable lineage-specific time series data following the acquisition of specific lineage states during development.

Metal ion fluxes controlling amphibian fertilization.

Mammalian oocytes undergo major changes in zinc content and localization to be fertilized, the most striking being the rapid exocytosis of over 10 billion zinc ions in what are known as zinc sparks. Here, we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese. Synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through electron-nuclear double-resonance studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs, but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy.

Sorting Sox: Diverse Roles for Sox Transcription Factors During Neural Crest and Craniofacial Development.

Sox transcription factors play many diverse roles during development, including regulating stem cell states, directing differentiation, and influencing the local chromatin landscape. Of the twenty vertebrate Sox factors, several play critical roles in the development the neural crest, a key vertebrate innovation, and the subsequent formation of neural crest-derived structures, including the craniofacial complex. Herein, we review the specific roles for individual Sox factors during neural crest cell formation and discuss how some factors may have been essential for the evolution of the neural crest. Additionally, we describe how Sox factors direct neural crest cell differentiation into diverse lineages such as melanocytes, glia, and cartilage and detail their involvement in the development of specific craniofacial structures. Finally, we highlight several SOXopathies associated with craniofacial phenotypes.

A transition from SoxB1 to SoxE transcription factors is essential for progression from pluripotent blastula cells to neural crest cells.

The neural crest is a stem cell population unique to vertebrate embryos that gives rise to derivatives from multiple embryonic germ layers. The molecular underpinnings of potency that govern neural crest potential are highly conserved with that of pluripotent blastula stem cells, suggesting that neural crest cells may have evolved through retention of aspects of the pluripotency gene regulatory network (GRN). A striking difference in the regulatory factors utilized in pluripotent blastula cells and neural crest cells is the deployment of different sub-families of Sox transcription factors; SoxB1 factors play central roles in the pluripotency of naïve blastula and ES cells, whereas neural crest cells require SoxE function. Here we explore the shared and distinct activities of these factors to shed light on the role that this molecular hand-off of Sox factor activity plays in the genesis of neural crest and the lineages derived from it. Our findings provide evidence that SoxB1 and SoxE factors have both overlapping and distinct activities in regulating pluripotency and lineage restriction in the embryo. We hypothesize that SoxE factors may transiently replace SoxB1 factors to control pluripotency in neural crest cells, and then poise these cells to contribute to glial, chondrogenic and melanocyte lineages at stages when SoxB1 factors promote neuronal progenitor formation.

Histone deacetylase activity has an essential role in establishing and maintaining the vertebrate neural crest.

The neural crest, a progenitor population that drove vertebrate evolution, retains the broad developmental potential of the blastula cells it is derived from, even as neighboring cells undergo lineage restriction. The mechanisms that enable these cells to preserve their developmental potential remain poorly understood. Here, we explore the role of histone deacetylase (HDAC) activity in this process in Xenopus We show that HDAC activity is essential for the formation of neural crest, as well as for proper patterning of the early ectoderm. The requirement for HDAC activity initiates in naïve blastula cells; HDAC inhibition causes loss of pluripotency gene expression and blocks the ability of blastula stem cells to contribute to lineages of the three embryonic germ layers. We find that pluripotent naïve blastula cells and neural crest cells are both characterized by low levels of histone acetylation, and show that increasing HDAC1 levels enhance the ability of blastula cells to be reprogrammed to a neural crest state. Together, these findings elucidate a previously uncharacterized role for HDAC activity in establishing the neural crest stem cell state.

FGF mediated MAPK and PI3K/Akt Signals make distinct contributions to pluripotency and the establishment of Neural Crest.

Early vertebrate embryos possess cells with the potential to generate all embryonic cell types. While this pluripotency is progressively lost as cells become lineage restricted, Neural Crest cells retain broad developmental potential. Here, we provide novel insights into signals essential for both pluripotency and neural crest formation in Xenopus. We show that FGF signaling controls a subset of genes expressed by pluripotent blastula cells, and find a striking switch in the signaling cascades activated by FGF signaling as cells lose pluripotency and commence lineage restriction. Pluripotent cells display and require Map Kinase signaling, whereas PI3 Kinase/Akt signals increase as developmental potential is restricted, and are required for transit to certain lineage restricted states. Importantly, retaining a high Map Kinase/low Akt signaling profile is essential for establishing Neural Crest stem cells. These findings shed important light on the signal-mediated control of pluripotency and the molecular mechanisms governing genesis of Neural Crest.

NEURODEVELOPMENT. Shared regulatory programs suggest retention of blastula-stage potential in neural crest cells.

Neural crest cells, which are specific to vertebrates, arise in the ectoderm but can generate cell types that are typically categorized as mesodermal. This broad developmental potential persists past the time when most ectoderm-derived cells become lineage-restricted. The ability of neural crest to contribute mesodermal derivatives to the bauplan has raised questions about how this apparent gain in potential is achieved. Here, we describe shared molecular underpinnings of potency in neural crest and blastula cells. We show that in Xenopus, key neural crest regulatory factors are also expressed in blastula animal pole cells and promote pluripotency in both cell types. We suggest that neural crest cells may have evolved as a consequence of a subset of blastula cells retaining activity of the regulatory network underlying pluripotency.

Sox5 Is a DNA-binding cofactor for BMP R-Smads that directs target specificity during patterning of the early ectoderm.

The SoxD factor, Sox5, is expressed in ectodermal cells at times and places where BMP signaling is active, including the cells of the animal hemisphere at blastula stages and the neural plate border and neural crest at neurula stages. Sox5 is required for proper ectoderm development, and deficient embryos display patterning defects characteristic of perturbations of BMP signaling, including loss of neural crest and epidermis and expansion of the neural plate. We show that Sox5 is essential for activation of BMP target genes in embryos and explants, that it physically interacts with BMP R-Smads, and that it is essential for recruitment of Smad1/4 to BMP regulatory elements. Our findings identify Sox5 as the long-sought DNA-binding partner for BMP R-Smads essential to plasticity and pattern in the early ectoderm.

Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis.

X-ray phase-contrast microtomography (XPCµT) is a label-free, high-resolution imaging modality for analyzing early development of vertebrate embryos in vivo by using time-lapse sequences of 3D volumes. Here we provide a detailed protocol for applying this technique to study gastrulation in Xenopus laevis (African clawed frog) embryos. In contrast to µMRI, XPCµT images optically opaque embryos with subminute temporal and micrometer-range spatial resolution. We describe sample preparation, culture and suspension of embryos, tomographic imaging with a typical duration of 2 h (gastrulation and neurulation stages), intricacies of image pre-processing, phase retrieval, tomographic reconstruction, segmentation and motion analysis. Moreover, we briefly discuss our present understanding of X-ray dose effects (heat load and radiolysis), and we outline how to optimize the experimental configuration with respect to X-ray energy, photon flux density, sample-detector distance, exposure time per tomographic projection, numbers of projections and time-lapse intervals. The protocol requires an interdisciplinary effort of developmental biologists for sample preparation and data interpretation, X-ray physicists for planning and performing the experiment and applied mathematicians/computer scientists/physicists for data processing and analysis. Sample preparation requires 9-48 h, depending on the stage of development to be studied. Data acquisition takes 2-3 h per tomographic time-lapse sequence. Data processing and analysis requires a further 2 weeks, depending on the availability of computing power and the amount of detail required to address a given scientific problem.

Setting appropriate boundaries: fate, patterning and competence at the neural plate border.

The neural crest and craniofacial placodes are two distinct progenitor populations that arise at the border of the vertebrate neural plate. This border region develops through a series of inductive interactions that begins before gastrulation and progressively divide embryonic ectoderm into neural and non-neural regions, followed by the emergence of neural crest and placodal progenitors. In this review, we describe how a limited repertoire of inductive signals-principally FGFs, Wnts and BMPs-set up domains of transcription factors in the border region which establish these progenitor territories by both cross-inhibitory and cross-autoregulatory interactions. The gradual assembly of different cohorts of transcription factors that results from these interactions is one mechanism to provide the competence to respond to inductive signals in different ways, ultimately generating the neural crest and cranial placodes.

X-ray phase-contrast in vivo microtomography probes new aspects of Xenopus gastrulation.

An ambitious goal in biology is to understand the behaviour of cells during development by imaging-in vivo and with subcellular resolution-changes of the embryonic structure. Important morphogenetic movements occur throughout embryogenesis, but in particular during gastrulation when a series of dramatic, coordinated cell movements drives the reorganization of a simple ball or sheet of cells into a complex multi-layered organism. In Xenopus laevis, the South African clawed frog and also in zebrafish, cell and tissue movements have been studied in explants, in fixed embryos, in vivo using fluorescence microscopy or microscopic magnetic resonance imaging. None of these methods allows cell behaviours to be observed with micrometre-scale resolution throughout the optically opaque, living embryo over developmental time. Here we use non-invasive in vivo, time-lapse X-ray microtomography, based on single-distance phase contrast and combined with motion analysis, to examine the course of embryonic development. We demonstrate that this powerful four-dimensional imaging technique provides high-resolution views of gastrulation processes in wild-type X. laevis embryos, including vegetal endoderm rotation, archenteron formation, changes in the volumes of cavities within the porous interstitial tissue between archenteron and blastocoel, migration/confrontation of mesendoderm and closure of the blastopore. Differential flow analysis separates collective from relative cell motion to assign propulsion mechanisms. Moreover, digitally determined volume balances confirm that early archenteron inflation occurs through the uptake of external water. A transient ectodermal ridge, formed in association with the confrontation of ventral and head mesendoderm on the blastocoel roof, is identified. When combined with perturbation experiments to investigate molecular and biomechanical underpinnings of morphogenesis, our technique should help to advance our understanding of the fundamentals of development.

Interactions between Twist and other core epithelial-mesenchymal transition factors are controlled by GSK3-mediated phosphorylation.

A subset of transcription factors classified as neural crest 'specifiers' are also core epithelial-mesenchymal transition regulatory factors, both in the neural crest and in tumour progression. The bHLH factor Twist is among the least well studied of these factors. Here we demonstrate that Twist is required for cranial neural crest formation and fate determination in Xenopus. We further show that Twist function in the neural crest is dependent upon its carboxy-terminal WR domain. The WR domain mediates physical interactions between Twist and other core epithelial-mesenchymal transition factors, including Snail1 and Snail2, which are essential for proper function. Interaction with Snail1/2, and Twist function more generally, is regulated by GSK-3-ß-mediated phosphorylation of conserved sites in the WR domain. Together, these findings elucidate a mechanism for coordinated control of a group of structurally diverse factors that function as a regulatory unit in both developmental and pathological epithelial-mesenchymal transitions.