De novo missense variants in exon 9 of SEPHS1 cause a neurodevelopmental condition with developmental delay, poor growth, hypotonia, and dysmorphic features.

Selenophosphate synthetase (SEPHS) plays an essential role in selenium metabolism. Two mammalian SEPHS paralogues, SEPHS1 and SEPHS2, share high sequence identity and structural homology with SEPHS. Here, we report nine individuals from eight families with developmental delay, growth and feeding problems, hypotonia, and dysmorphic features, all with heterozygous missense variants in SEPHS1. Eight of these individuals had a recurrent variant at amino acid position 371 of SEPHS1 (p.Arg371Trp, p.Arg371Gln, and p.Arg371Gly); seven of these variants were known to be de novo. Structural modeling and biochemical assays were used to understand the effect of these variants on SEPHS1 function. We found that a variant at residue Trp352 results in local structural changes of the C-terminal region of SEPHS1 that decrease the overall thermal stability of the enzyme. In contrast, variants of a solvent-exposed residue Arg371 do not impact enzyme stability and folding but could modulate direct protein-protein interactions of SEPSH1 with cellular factors in promoting cell proliferation and development. In neuronal SH-SY5Y cells, we assessed the impact of SEPHS1 variants on cell proliferation and ROS production and investigated the mRNA expression levels of genes encoding stress-related selenoproteins. Our findings provided evidence that the identified SEPHS1 variants enhance cell proliferation by modulating ROS homeostasis. Our study supports the hypothesis that SEPHS1 plays a critical role during human development and provides a basis for further investigation into the molecular mechanisms employed by SEPHS1. Furthermore, our data suggest that variants in SEPHS1 are associated with a neurodevelopmental disorder.

Retinoic acid signaling regulates spatiotemporal specification of human green and red cones.

Trichromacy is unique to primates among placental mammals, enabled by blue (short/S), green (medium/M), and red (long/L) cones. In humans, great apes, and Old World monkeys, cones make a poorly understood choice between M and L cone subtype fates. To determine mechanisms specifying M and L cones, we developed an approach to visualize expression of the highly similar M- and L-opsin mRNAs. M-opsin was observed before L-opsin expression during early human eye development, suggesting that M cones are generated before L cones. In adult human tissue, the early-developing central retina contained a mix of M and L cones compared to the late-developing peripheral region, which contained a high proportion of L cones. Retinoic acid (RA)-synthesizing enzymes are highly expressed early in retinal development. High RA signaling early was sufficient to promote M cone fate and suppress L cone fate in retinal organoids. Across a human population sample, natural variation in the ratios of M and L cone subtypes was associated with a noncoding polymorphism in the NR2F2 gene, a mediator of RA signaling. Our data suggest that RA promotes M cone fate early in development to generate the pattern of M and L cones across the human retina.

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.

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 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 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 mouse 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. Translational Relevance: Ocular gene-replacement therapies using non-viral vector methods are a promising alternative to adeno-associated virus (AAV) vectors. Our studies show that mRNA LNP delivery can be used to transfect retinal cells in both mouse and human tissues without inducing significant inflammation. This methodology could be used to transfect retinal cell lines, tissue explants, mice, or potentially as gene-replacement therapy in a clinical setting in the future.

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.

Developmental control of rod number via a light-dependent retrograde pathway from intrinsically photosensitive retinal ganglion cells.

Photoreception is essential for the development of the visual system, shaping vision's first synapse to cortical development. Here, we find that the lighting environment controls developmental rod apoptosis via Opn4-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Using genetics, sensory environment manipulations, and computational approaches, we establish a pathway where light-dependent glutamate released from ipRGCs is detected via a transiently expressed glutamate receptor (Grik3) on rod precursors within the inner retina. Communication between these cells is mediated by hybrid neurites on ipRGCs that sense light before eye opening. These structures span the ipRGC-rod precursor distance over development and contain the machinery for photoreception (Opn4) and neurotransmitter release (Vglut2 & Syp). Assessment of the human gestational retina identifies conserved hallmarks of an ipRGC-to-rod axis, including displaced rod precursors, transient GRIK3 expression, and ipRGCs with deep-projecting neurites. This analysis defines an adaptive retrograde pathway linking the sensory environment to rod precursors via ipRGCs prior to eye opening.

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.