Biology subjects

LaBonne, C.

Publications and source records attributed to LaBonne, C..

8 recordsLinked to original sources

bioRxiv2025

Dynamic and non-uniform expression of key transcription factors provides novel insights into the emergence of neural crest cells at the neural plate border

The neural crest is a vertebrate stem cell population with broad developmental potential whose emergence requires precise regulation of gene expression at the neural plate border (NPB). While a hierarchical gene regulatory network (GRN) describing signaling pathways and transcription factors underpinning the establishment of definitive neural crest cells has been generated by integrating the work of numerous groups, much remains to be learned about the relationships of distinct GRN components to each other. Here, we use high-resolution fluorescent in situ Hybridization Chain Reaction (HCR-FISH) to quantify the spatiotemporal dynamics of neural crest gene expression in Xenopus laevis embryos. We find that the onset of snai2, sox8, and foxd3 expression during late gastrulation is broad, heterogeneous, and partially overlapping, with distinct anterior-posterior and medio-lateral biases. By neurulation, these markers converge on a shared neural crest domain but retain relative expression differences along axial levels that persist into migratory stages, producing stream-specific gene expression patterns. Computational surface mapping revealed that these differences correlate with dynamic, layered expression of NPB factors, particularly pax3 and zic1. Correlating relative intensities of pax3 and zic1 with the presence or absence of nascent neural crest transcripts predicts that these NPB factors can differentially regulate snai2 and sox8, which we confirm with functional experiments. Strikingly, later stages show an inverse correlation between neural crest and NPB gene expression, suggesting a handoff mechanism in which pax3 and zic1 initially promote neural crest gene activation but are downregulated as neural crest identity emerges and contribute to combinatorial signatures of gene expression along the A-P axis. These finding provide important new insights into the genesis of a centrally important cell type.

developmental biology↗

bioRxiv2025

WDR5 and Myc Cooperate to Regulate Formation of Neural Crest Stem Cells

WDR5 is a multifunctional scaffolding protein with established roles in chromatin regulation and pluripotency, but its functions in early vertebrate development remain poorly understood. Here, we show that Xenopus wdr5 is expressed in blastula stem cells and enriched in neural crest cells during neurulation. Morpholino-mediated depletion of wdr5 abolished neural crest marker expression both in embryos and in reprogrammed explants while unexpectedly expanding neural plate border and neural plate domains. Gain-of-function experiments revealed a striking dose-dependent effect: low levels of wdr5 enhanced neural crest formation, whereas high levels suppressed it, suggesting a requirement for precise stoichiometry with interacting partners. We identify myc as a critical cofactor for wdr5 in neural crest specification--wdr5 and myc physically interact in early embryos, and co-expression at defined ratios rescues neural crest formation while either, individually, is inhibitory. Domain-specific mutagenesis showed that the WBM site of wdr5 is required for myc-dependent activation of neural crest genes, while the WIN site regulates myc expression itself; both domains are necessary to rescue wdr5 loss-of-function phenotypes. Moreover, modulation of myc levels phenocopied wdr5s dose-sensitive effects, reinforcing the importance of balanced wdr5-myc activity. These findings reveal that wdr5 orchestrates neural crest development through multiple, domain-specific mechanisms--integrating stoichiometric control with partner-specific transcriptional regulation--and highlight parallels with Wdr5-Myc cooperation in cancer, underscoring the broader relevance of precise cofactor ratios in cell fate decisions.

developmental biology↗

bioRxiv2025

Evolutionary dynamics of FoxQ2 transcription factors across metazoans: A tale of three ancient paralogs

FoxQ2 is a highly conserved class of Forkhead-box transcription factors expressed on the anterior side of the body in cnidarians and bilaterians. Despite this conserved expression pattern, recent phylogenetic analyses have revealed a complex and rapid evolution of this class, with several taxon-specific duplications and losses. Until recently, FoxQ2 was thought to be lost in most vertebrate lineages, and its presence and localization in different vertebrate groups remains unclear. To reconcile these conflicting reports of conservation and divergence, here we present a comprehensive analysis of the phylogenetic relationships and expression patterns of FoxQ2 genes across metazoans. By combining phylogenetics and synteny analyses of FoxQ2 sequences from 21 animal phyla, we uncover the presence of three ancient FoxQ2 paralogs in bilaterians, which we name FoxQ2a, FoxQ2b and FoxQ2c. All three FoxQ2 paralogs are present in the chordate lineage and two are conserved in vertebrates, indicating a richer repertoire of vertebrate Fox genes than previously estimated. To investigate the expression of FoxQ2 genes across bilaterians, we mined expression data from existing single cell transcriptomic datasets of mollusk, acoel, amphioxus and zebrafish development, and expanded it using fluorescent in situ hybridization in amphioxus, lamprey, skate, zebrafish and chicken. Our analysis demonstrates the conserved anterior expression of FoxQ2a and FoxQ2b paralogs while also revealing a novel domain of FoxQ2c expression within the chordate endoderm, including in amphioxus, lamprey and skate. Finally, we devise a method to predict conserved transcription factor binding sites across the three extant amphioxus genera with specificity to developmental stage and cell-type identity. This suggests conserved regulatory interactions for the expression of FoxQ2a across deuterostomes. Overall, this work clarifies the complex evolutionary history of FoxQ2 genes and identifies a newly discovered endodermally-expressed Fox gene, FoxQ2c. We further propose that the early duplication of FoxQ2a and FoxQ2b, along with their redundant functions, provided the ideal background for subfunctionalization, contributing to the fast evolutionary rate of FoxQ2 sequences observed in bilaterians.

evolutionary biology↗

bioRxiv2025

Kruppel-like Factors Play Essential Roles in Regulating Pluripotency and the Formation of Neural Crest Stem Cells

The evolutionary transition from simple chordate body plans to complex vertebrate body plans was driven by the acquisition of the neural crest, a stem cell population that retains broad, multi-germ layer developmental potential long after most embryonic cells have become lineage restricted. We have previously shown that neural crest cells share significant gene regulatory architecture with pluripotent blastula stem cells. Here we examine the roles that Kruppel-like Family (Klf) transcription factors play in these stem cell populations. Although Klf4 has established roles in regulating pluripotency in mammalian stem cells cultures, we find that in Xenopus it is klf2 that is highly expressed in pluripotent blastula stem cells. klf2 expression is down-regulated as cells transition to a neural crest state while a related klf factor, klf17, is significantly up regulated in response to neural crest induction. We used gain and loss of function studies to compare the activities of these closely related factors and found that they have both shared and distinct activities. Inhibition of either klf2 or klf17 activity led to significantly expanded expression of pluripotency, neural plate border and neural crest factors in neurula stage embryos, leading us to hypothesize that klf factors regulate the exit from pluripotency and proper establishment of the boundary of the neural crest domain. To gain further insights into the role of klf factors in the evolution of the neural crest, we examined their expression in the jawless vertebrate, Petromyzon marinus (sea lamprey). We find that lamprey have a klf2/4 and a klf17 gene, but that only klf17 is expressed in blastula and neural crest stem cells. Moreover, ectopic expression of lamprey klf17 in Xenopus embryos phenocopies Xenopus klf17 activity. These data suggest that klf17, rather than klf4, may have been the ancestral klf factor that functioned in these GRNs in stem vertebrates.

developmental biology↗

bioRxiv2023

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.

evolutionary biology↗

bioRxiv2023

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.

developmental biology↗

bioRxiv2022

Quantitative Analysis of Transcriptome Dynamics Provides Novel Insights into Developmental State Transitions

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. 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 Waddingtons landscape. Our findings shed light on 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. Together these analyses provide quantitative insights into the logic and dynamics of developmental decision making in early embryos.

developmental biology↗

bioRxiv2018

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 is 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 subfamilies of Sox transcription factors; SoxB1 factors play central roles in the pluripotency of naive 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.

developmental biology↗