Maintenance of pluripotency-like signature in the entire ectoderm leads to neural crest stem cell potential.

The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like derivatives is obtained. Here, we monitor transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. We show maintenance of pluripotency-like signature (Nanog, Oct4/PouV, Klf4-positive) in undecided pan-ectodermal stem-cells spanning the entire ectoderm late during neurulation with ectodermal patterning completed only at the end of neurulation when the pluripotency-like signature becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency-like signature is found at multiple axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond "ectodermal-capacity" in chick and mouse embryos.

A ubiquitin-based effector-to-inhibitor switch coordinates early brain, craniofacial, and skin development.

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-ßPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.

Maintenance of pluripotency in the entire ectoderm enables neural crest formation.

The ability of the pluripotent epiblast to contribute progeny to all three germ layers is thought to be lost after gastrulation. The later-forming neural crest (NC) rises from ectoderm and it remains poorly understood how its exceptionally high stem-cell potential to generate mesodermal- and endodermal-like cells is obtained. We monitored transcriptional changes from gastrulation to neurulation using single-cell-Multiplex-Spatial-Transcriptomics (scMST) complemented with RNA-sequencing. Unexpectedly, we find maintenance of undecided Nanog/Oct4-PouV/Klf4-positive pluripotent-like pan-ectodermal stem-cells spanning the entire ectoderm late in the neurulation process with ectodermal patterning completed only at the end of neurulation when pluripotency becomes restricted to NC, challenging our understanding of gastrulation. Furthermore, broad ectodermal pluripotency is found at all axial levels unrelated to the NC lineage the cells later commit to, suggesting a general role in stemness enhancement and proposing a mechanism by which the NC acquires its ability to form derivatives beyond "ectodermal-capacity" in chick and mouse embryos.

ShapeMetrics: A userfriendly pipeline for 3D cell segmentation and spatial tissue analysis.

The demand for single-cell level data is constantly increasing within life sciences. In order to meet this demand, robust cell segmentation methods that can tackle challenging in vivo tissues with complex morphology are required. However, currently available cell segmentation and volumetric analysis methods perform poorly on 3D images. Here, we generated ShapeMetrics, a MATLAB-based script that segments cells in 3D and, by performing unbiased clustering using a heatmap, separates the cells into subgroups according to their volumetric and morphological differences. The cells can be accurately segregated according to different biologically meaningful features such as cell ellipticity, longest axis, cell elongation, or the ratio between cell volume and surface area. Our machine learning based script enables dissection of a large amount of novel data from microscope images in addition to the traditional information based on fluorescent biomarkers. Furthermore, the cells in different subgroups can be spatially mapped back to their original locations in the tissue image to help elucidate their roles in their respective morphological contexts. In order to facilitate the transition from bulk analysis to single-cell level accuracy, we emphasize the user-friendliness of our method by providing detailed step-by-step instructions through the pipeline hence aiming to reach users with less experience in computational biology.

Enhanced expression of MycN/CIP2A drives neural crest toward a neural stem cell-like fate: Implications for priming of neuroblastoma.

Neuroblastoma is a neural crest-derived childhood tumor of the peripheral nervous system in which MycN amplification is a hallmark of poor prognosis. Here we show that MycN is expressed together with phosphorylation-stabilizing factor CIP2A in regions of the neural plate destined to form the CNS, but MycN is excluded from the neighboring neural crest stem cell domain. Interestingly, ectopic expression of MycN or CIP2A in the neural crest domain biases cells toward CNS-like neural stem cells that express Sox2. Consistent with this, some forms of neuroblastoma have been shown to share transcriptional resemblance with CNS neural stem cells. As high MycN/CIP2A levels correlate with poor prognosis, we posit that a MycN/CIP2A-mediated cell-fate bias may reflect a possible mechanism underlying early priming of some aggressive forms of neuroblastoma. In contrast to MycN, its paralogue cMyc is normally expressed in the neural crest stem cell domain and typically is associated with better overall survival in clinical neuroblastoma, perhaps reflecting a more "normal" neural crest-like state. These data suggest that priming for some forms of aggressive neuroblastoma may occur before neural crest emigration from the CNS and well before sympathoadrenal specification.