Identification and characterization of intermediate states in mammalian neural crest cell epithelial to mesenchymal transition and delamination.

Epithelial to mesenchymal transition (EMT) is a cellular process that converts epithelial cells to mesenchymal cells with migratory potential in developmental and pathological processes. Although originally considered a binary event, EMT in cancer progression involves intermediate states between a fully epithelial and a fully mesenchymal phenotype, which are characterized by distinct combinations of epithelial and mesenchymal markers. This phenomenon has been termed epithelial to mesenchymal plasticity (EMP), however, the intermediate states remain poorly described and it's unclear whether they exist during developmental EMT. Neural crest cells (NCC) are an embryonic progenitor cell population that gives rise to numerous cell types and tissues in vertebrates, and their formation and delamination is a classic example of developmental EMT. However, whether intermediate states also exist during NCC EMT and delamination remains unknown. Through single-cell RNA sequencing of mouse embryos, we identified intermediate NCC states based on their transcriptional signature and then spatially defined their locations in situ in the dorsolateral neuroepithelium. Our results illustrate the importance of cell cycle regulation and functional role for the intermediate stage marker Dlc1 in facilitating mammalian cranial NCC delamination and may provide new insights into mechanisms regulating pathological EMP.

Identification and characterization of intermediate states in mammalian neural crest cell epithelial to mesenchymal transition and delamination.

Epithelial to mesenchymal transition (EMT) is a cellular process that converts epithelial cells to mesenchymal cells with migratory potential in both developmental and pathological processes. Although originally considered a binary event, EMT in cancer progression involves intermediate states between a fully epithelial and a fully mesenchymal phenotype, which are characterized by distinct combinations of epithelial and mesenchymal markers. This phenomenon has been termed epithelial to mesenchymal plasticity (EMP), however, the intermediate states remain poorly described and it's unclear whether they exist during developmental EMT. Neural crest cells (NCC) are an embryonic progenitor cell population that gives rise to numerous cell types and tissues in vertebrates, and their formation is a classic example of developmental EMT. An important feature of NCC development is their delamination from the neuroepithelium via EMT, following which NCC migrate throughout the embryo and undergo differentiation. NCC delamination shares similar changes in cellular state and structure with cancer cell invasion. However, whether intermediate states also exist during NCC EMT and delamination remains unknown. Through single cell RNA sequencing, we identified intermediate NCC states based on their transcriptional signature and then spatially defined their locations in situ in the dorsolateral neuroepithelium. Our results illustrate the progressive transcriptional and spatial transitions from premigratory to migratory cranial NCC during EMT and delamination. Of note gene expression and trajectory analysis indicate that distinct intermediate populations of NCC delaminate in either S phase or G2/M phase of the cell cycle, and the importance of cell cycle regulation in facilitating mammalian cranial NCC delamination was confirmed through cell cycle inhibition studies. Additionally, transcriptional knockdown revealed a functional role for the intermediate stage marker Dlc1 in regulating NCC delamination and migration. Overall, our work identifying and characterizing the intermediate cellular states, processes, and molecular signals that regulate mammalian NCC EMT and delamination furthers our understanding of developmental EMP and may provide new insights into mechanisms regulating pathological EMP.

Dynamic changes in ocular shape during human development and its implications for retina fovea formation.

The human fovea is known for its distinctive pit-like appearance, which results from the displacement of retinal layers superficial to the photoreceptors cells. The photoreceptors are found at high density within the foveal region but not the surrounding retina. Efforts to elucidate the mechanisms responsible for these unique features have ruled out cell death as an explanation for pit formation and changes in cell proliferation as the cause of increased photoreceptor density. These findings have led to speculation that mechanical forces acting within and on the retina during development underly the formation of foveal architecture. Here we review eye morphogenesis and retinal remodeling in human embryonic development. Our meta-analysis of the literature suggests that fovea formation is a protracted process involving dynamic changes in ocular shape that start early and continue throughout most of human embryonic development. From these observations, we propose a new model for fovea development.

Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.

rRNA transcription is integral to phase separation and maintenance of nucleolar structure.

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.

Morphological changes and two Nodal paralogs drive left-right asymmetry in the squamate veiled chameleon (C. calyptratus).

The ancestral mode of left-right (L-R) patterning involves cilia in the L-R organizer. However, the mechanisms regulating L-R patterning in non-avian reptiles remains an enigma, since most squamate embryos are undergoing organogenesis at oviposition. In contrast, veiled chameleon (Chamaeleo calyptratus) embryos are pre-gastrula at oviposition, making them an excellent organism for studying L-R patterning evolution. Here we show that veiled chameleon embryos lack motile cilia at the time of L-R asymmetry establishment. Thus, the loss of motile cilia in the L-R organizers is a synapomorphy of all reptiles. Furthermore, in contrast to avians, geckos and turtles, which have one Nodal gene, veiled chameleon exhibits expression of two paralogs of Nodal in the left lateral plate mesoderm, albeit in non-identical patterns. Using live imaging, we observed asymmetric morphological changes that precede, and likely trigger, asymmetric expression of the Nodal cascade. Thus, veiled chameleons are a new and unique model for studying the evolution of L-R patterning.

POLR1A variants underlie phenotypic heterogeneity in craniofacial, neural, and cardiac anomalies.

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.