Differential Regulation of Immune-Related Genes in the Developing Heart.

In many congenital heart defects, it can be difficult to ascertain primary pathology from secondary consequences from altered flow through the developing heart. The molecular differences between the growing right and left ventricles (RV and LV, respectively) following the completion of septation and the impact of sex on these mechanisms have not been investigated. We analyzed RNA-seq data derived from twelve RV and LVs, one with Hypoplastic Left Heart Syndrome (HLHS), to compare the transcriptomic landscape between the ventricles during development. Differential gene expression analysis revealed a large proportion of genes unique to either the RV or LV as well as sex bias. Our GO enrichment and network analysis strategy highlighted the differential role of immune functions between the RV and LV in the developing heart. Comparatively, RNA-seq analysis of data from C57Bl6/J mice hearts collected at E14 resulted in the enrichment of similar processes related to T cells and leukocyte migration and activation. Differential gene expression analysis of an HLHS case highlighted significant downregulation of chromatin organization pathways and upregulation of genes involved in muscle organ development. This analysis also identified previously unreported upregulation of genes involved in IL-17 production pathways. In conclusion, differences exist between the gene expression profiles of RV versus LV with the expression of immune-related genes being significantly different between these two chambers. The pathogenesis of HLHS may involve alterations in the expression of chromatin and muscle gene organization as well as upregulation of the IL-17 response pathway.

De novo variants in PLCG1 are associated with hearing impairment, ocular pathology, and cardiac defects.

Phospholipase C isozymes (PLCs) hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol, important signaling molecules involved in many cellular processes. PLCG1 encodes the PLCγ1 isozyme that is broadly expressed. Hyperactive somatic mutations of PLCG1 are observed in multiple cancers, but only one germline variant has been reported. Here we describe three unrelated individuals with de novo heterozygous missense variants in PLCG1 (p.Asp1019Gly, p.His380Arg, and p.Asp1165Gly) who exhibit variable phenotypes including hearing loss, ocular pathology and cardiac septal defects. To model these variants in vivo, we generated the analogous variants in the Drosophila ortholog, small wing (sl). We created a null allele slT2A and assessed the expression pattern. sl is broadly expressed, including in wing discs, eye discs, and a subset of neurons and glia. Loss of sl causes wing size reductions, ectopic wing veins and supernumerary photoreceptors. We document that mutant flies exhibit a reduced lifespan and age-dependent locomotor defects. Expressing wild-type sl in slT2A mutant rescues the loss-of-function phenotypes whereas expressing the variants causes lethality. Ubiquitous overexpression of the variants also reduces viability, suggesting that the variants are toxic. Ectopic expression of an established hyperactive PLCG1 variant (p.Asp1165His) in the wing pouch causes severe wing phenotypes, resembling those observed with overexpression of the p.Asp1019Gly or p.Asp1165Gly variants, further arguing that these two are gain-of-function variants. However, the wing phenotypes associated with p.His380Arg overexpression are mild. Our data suggest that the PLCG1 de novo heterozygous missense variants are pathogenic and contribute to the features observed in the probands.

MerTK is a mediator of alpha-synuclein fibril uptake by human microglia.

Mer tyrosine kinase (MerTK) is a receptor tyrosine kinase that mediates non-inflammatory, homeostatic phagocytosis of diverse types of cellular debris. Highly expressed on the surface of microglial cells, MerTK is of importance in brain development, homeostasis, plasticity and disease. Yet, involvement of this receptor in the clearance of protein aggregates that accumulate with ageing and in neurodegenerative diseases has yet to be defined. The current study explored the function of MerTK in the microglial uptake of alpha-synuclein fibrils which play a causative role in the pathobiology of synucleinopathies. Using human primary and induced pluripotent stem cell-derived microglia, the MerTK-dependence of alpha-synuclein fibril internalization was investigated in vitro. Relevance of this pathway in synucleinopathies was assessed through burden analysis of MERTK variants and analysis of MerTK expression in patient-derived cells and tissues. Pharmacological inhibition of MerTK and siRNA-mediated MERTK knockdown both caused a decreased rate of alpha-synuclein fibril internalization by human microglia. Consistent with the non-inflammatory nature of MerTK-mediated phagocytosis, alpha-synuclein fibril internalization was not observed to induce secretion of pro-inflammatory cytokines such as IL-6 or TNF, and downmodulated IL-1ß secretion from microglia. Burden analysis in two independent patient cohorts revealed a significant association between rare functionally deleterious MERTK variants and Parkinson's disease in one of the cohorts (P = 0.002). Despite a small upregulation in MERTK mRNA expression in nigral microglia from Parkinson's disease/Lewy body dementia patients compared to those from non-neurological control donors in a single-nuclei RNA-sequencing dataset (P = 5.08 × 10-21), no significant upregulation in MerTK protein expression was observed in human cortex and substantia nigra lysates from Lewy body dementia patients compared to controls. Taken together, our findings define a novel role for MerTK in mediating the uptake of alpha-synuclein fibrils by human microglia, with possible involvement in limiting alpha-synuclein spread in synucleinopathies such as Parkinson's disease. Upregulation of this pathway in synucleinopathies could have therapeutic values in enhancing alpha-synuclein fibril clearance in the brain.

Sweat gland development requires an eccrine dermal niche and couples two epidermal programs.

Eccrine sweat glands are indispensable for human thermoregulation and, similar to other mammalian skin appendages, form from multipotent epidermal progenitors. Limited understanding of how epidermal progenitors specialize to form these vital organs has precluded therapeutic efforts toward their regeneration. Herein, we applied single-nucleus transcriptomics to compare the expression content of wild-type, eccrine-forming mouse skin to that of mice harboring a skin-specific disruption of Engrailed 1 (En1), a transcription factor that promotes eccrine gland formation in humans and mice. We identify two concurrent but disproportionate epidermal transcriptomes in the early eccrine anlagen: one that is shared with hair follicles and one that is En1 dependent and eccrine specific. We demonstrate that eccrine development requires the induction of a dermal niche proximal to each developing gland in humans and mice. Our study defines the signatures of eccrine identity and uncovers the eccrine dermal niche, setting the stage for targeted regeneration and comprehensive skin repair.

A Spatiotemporal and Machine-Learning Platform Accelerates the Manufacturing of hPSC-derived Esophageal Mucosa.

Human pluripotent stem cell-derived tissue engineering offers great promise in designer cell-based personalized therapeutics. To harness such potential, a broader approach requires a deeper understanding of tissue-level interactions. We previously developed a manufacturing system for the ectoderm-derived skin epithelium for cell replacement therapy. However, it remains challenging to manufacture the endoderm-derived esophageal epithelium, despite both possessing similar stratified structure. Here we employ single cell and spatial technologies to generate a spatiotemporal multi-omics cell atlas for human esophageal development. We illuminate the cellular diversity, dynamics and signal communications for the developing esophageal epithelium and stroma. Using the machine-learning based Manatee, we prioritize the combinations of candidate human developmental signals for in vitro derivation of esophageal basal cells. Functional validation of the Manatee predictions leads to a clinically-compatible system for manufacturing human esophageal mucosa. Our approach creates a versatile platform to accelerate human tissue manufacturing for future cell replacement therapies to treat human genetic defects and wounds.

Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.

Single-cell census of human tooth development enables generation of human enamel.

Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.

Human microglia maturation is underpinned by specific gene regulatory networks.

Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage- and disease-specific phenotypes.

Lipid nanoparticle-mediated delivery of mRNA into the mouse and human retina and other ocular tissues.

Purpose: Lipid nanoparticles (LNPs) show promise in their ability to introduce mRNA to drive protein expression in specific cell types of the mammalian eye. Here, we examined the ability of mRNA encapsulated in lipid nanoparticles (LNPs) with two distinct formulations to drive gene expression in mouse and human retina and other ocular tissues. Methods: We introduced mRNA carrying LNPs into two biological systems. Intravitreal injections were tested to deliver LNPs into the mouse eye. Human retinal pigment epithelium (RPE) and retinal explants were used to assess mRNA expression in human tissue. We analyzed specificity of expression using histology, immunofluorescence, and imaging. Results: In mice, mRNAs encoding GFP and ciliary neurotrophic factor (CNTF) were specifically expressed by Müller glia and retinal pigment epithelium (RPE). Acute inflammatory changes measured by microglia distribution (Iba-1) or interleukin-6 (IL-6) expression were not observed 6 hours post-injection. Human RPE also expressed high levels of GFP. Human retinal explants expressed GFP in cells with apical and basal processes consistent with Müller glia and in perivascular cells consistent with macrophages. Conclusions: We demonstrated the ability to reliably transfect subpopulations of retinal cells in mice eye tissues in vivo and in human ocular tissues. Of significance, intravitreal injections were sufficient to transfect the RPE in mice. To our knowledge we demonstrate delivery of mRNA using LNPs in human ocular tissues for the first time.

A Trisomy 21 Lung Cell Atlas.

Trisomy 21 (T21), resulting in Down Syndrome (DS), is the most prevalent chromosomal abnormality worldwide. While pulmonary disease is a major cause of morbidity and mortality in DS, the ontogeny of pulmonary complications remains poorly understood. We recently demonstrated that T21 lung anomalies, including airway branching and vascular lymphatic abnormalities, are initiated in utero. Here, we aimed to describe molecular changes at the single cell level in prenatal T21 lungs. Our results demonstrate differences in the proportion of cell populations and detail changes in gene expression at the time of initiation of histopathological abnormalities. Notably, we identify shifts in the distribution of alveolar epithelial progenitors, widespread induction of key extracellular matrix molecules in mesenchymal cells and hyper-activation of IFN signaling in endothelial cells. This single cell atlas of T21 lungs greatly expands our understanding of antecedents to pulmonary complications and should facilitate efforts to mitigate respiratory disease in DS.

Unified rhombic lip origins of group 3 and group 4 medulloblastoma.

Medulloblastoma, a malignant childhood cerebellar tumour, segregates molecularly into biologically distinct subgroups, suggesting that a personalized approach to therapy would be beneficial1. Mouse modelling and cross-species genomics have provided increasing evidence of discrete, subgroup-specific developmental origins2. However, the anatomical and cellular complexity of developing human tissues3-particularly within the rhombic lip germinal zone, which produces all glutamatergic neuronal lineages before internalization into the cerebellar nodulus-makes it difficult to validate previous inferences that were derived from studies in mice. Here we use multi-omics to resolve the origins of medulloblastoma subgroups in the developing human cerebellum. Molecular signatures encoded within a human rhombic-lip-derived lineage trajectory aligned with photoreceptor and unipolar brush cell expression profiles that are maintained in group 3 and group 4 medulloblastoma, suggesting a convergent basis. A systematic diagnostic-imaging review of a prospective institutional cohort localized the putative anatomical origins of group 3 and group 4 tumours to the nodulus. Our results connect the molecular and phenotypic features of clinically challenging medulloblastoma subgroups to their unified beginnings in the rhombic lip in the early stages of human development.

A unique cardiovascular presentation of Marfan syndrome.

We report a neonate with severe Marfan syndrome (MS), prenatally identified to have persistent atrial tachycardia, biventricular dysfunction, and an unusual structure within the atria. Detailed postnatal echocardiographic evaluation and cross-sectional imaging confirmed congenital pseudoaneurysm of the mitral-aortic intervalvular fibrosa. Emergent testing by next-generation sequencing identified a FBN1 pathological variant, key to establishing goals of care. To our knowledge, this is the first reported case of a congenital pseudoaneurysm of the mitral-aortic intervalvular fibrosa in MS.

Cellular resolution anatomical and molecular atlases for prenatal human brains.

Increasing interest in studies of prenatal human brain development, particularly using new single-cell genomics and anatomical technologies to create cell atlases, creates a strong need for accurate and detailed anatomical reference atlases. In this study, we present two cellular-resolution digital anatomical atlases for prenatal human brain at postconceptional weeks (PCW) 15 and 21. Both atlases were annotated on sequential Nissl-stained sections covering brain-wide structures on the basis of combined analysis of cytoarchitecture, acetylcholinesterase staining, and an extensive marker gene expression dataset. This high information content dataset allowed reliable and accurate demarcation of developing cortical and subcortical structures and their subdivisions. Furthermore, using the anatomical atlases as a guide, spatial expression of 37 and 5 genes from the brains, respectively, at PCW 15 and 21 was annotated, illustrating reliable marker genes for many developing brain structures. Finally, the present study uncovered several novel developmental features, such as the lack of an outer subventricular zone in the hippocampal formation and entorhinal cortex, and the apparent extension of both cortical (excitatory) and subcortical (inhibitory) progenitors into the prenatal olfactory bulb. These comprehensive atlases provide useful tools for visualization, segmentation, targeting, imaging, and interpretation of brain structures of prenatal human brain, and for guiding and interpreting the next generation of cell census and connectome studies.

Rescuing human fetal tissue research in the United States: A call for additional regulatory reform.

Research using human fetal tissue has saved millions of lives through vaccines and other advances, but was markedly restricted by federal regulations in 2019. Although the restrictions were partially reversed in 2021, additional regulatory changes are needed to prevent further damage to essential research programs while preserving protection for human subjects.

Evidence of disrupted rhombic lip development in the pathogenesis of Dandy-Walker malformation.

Dandy-Walker malformation (DWM) and Cerebellar vermis hypoplasia (CVH) are commonly recognized human cerebellar malformations diagnosed following ultrasound and antenatal or postnatal MRI. Specific radiological criteria are used to distinguish them, yet little is known about their differential developmental disease mechanisms. We acquired prenatal cases diagnosed as DWM and CVH and studied cerebellar morphobiometry followed by histological and immunohistochemical analyses. This was supplemented by laser capture microdissection and RNA-sequencing of the cerebellar rhombic lip, a transient progenitor zone, to assess the altered transcriptome of DWM vs control samples. Our radiological findings confirm that the cases studied fall within the accepted biometric range of DWM. Our histopathological analysis points to reduced foliation and inferior vermian hypoplasia as common features in all examined DWM cases. We also find that the rhombic lip, a dorsal stem cell zone that drives the growth and maintenance of the posterior vermis is specifically disrupted in DWM, with reduced proliferation and self-renewal of the progenitor pool, and altered vasculature, all confirmed by transcriptomics analysis. We propose a unified model for the developmental pathogenesis of DWM. We hypothesize that rhombic lip development is disrupted through either aberrant vascularization and/or direct insult which causes reduced proliferation and failed expansion of the rhombic lip progenitor pool leading to disproportionate hypoplasia and dysplasia of the inferior vermis. Timing of insult to the developing rhombic lip (before or after 14 PCW) dictates the extent of hypoplasia and distinguishes DWM from CVH.

Spatial and cell type transcriptional landscape of human cerebellar development.

The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.

Charting human development using a multi-endodermal organ atlas and organoid models.

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

Two Missense CACNA1A Variants in a Single Family with Variable Neurobehavioral, Cerebellar, Epileptic, and Oculomotor Features.

We describe two novel missense variants in CACNA1A segregating in a family with variable severity of ataxia/oculomotor dysfunction, neurobehavioral impairments, and epilepsy. The most severe outcome occurred in a compound heterozygous proband, which could represent variable expression of the paternal allele or biallelic modulation of calcium channel function. Acetazolamide and lamotrigine were effective for seizure control.