Probes for INS

During development, retinal progenitors navigate a complex landscape of fate decisions to generate the major cell classes necessary for proper vision. Transcriptional regulation is critical to generate diversity within these major cell classes. Here, we aim to provide the resources and techniques required to identify transcription factors necessary to generate and maintain diversity in photoreceptor subtypes, which are critical for vision. First, we generate a key resource: a high-quality and deep transcriptomic profile of each photoreceptor subtype in adult zebrafish. We make this resource openly accessible, easy to explore, and have integrated it with other currently available photoreceptor transcriptomic datasets. Second, using our transcriptomic profiles, we derive an in-depth map of expression of transcription factors in photoreceptors. Third, we use efficient CRISPR-Cas9 based mutagenesis to screen for null phenotypes in F0 larvae (F0 screening) as a fast, efficient, and versatile technique to assess the involvement of candidate transcription factors in the generation of photoreceptor subtypes. We first show that known phenotypes can be easily replicated using this method: loss of S cones in foxq2 mutants and loss of rods in nr2e3 mutants. We then identify novel functions for the transcription factor Tbx2, demonstrating that it plays distinct roles in controlling the generation of all photoreceptor subtypes within the retina. Our study provides a roadmap to discover additional factors involved in this process. Additionally, we explore four transcription factors of unknown function (Skor1a, Sall1a, Lrrfip1a, and Xbp1), and find no evidence for their involvement in the generation of photoreceptor subtypes. This dataset and screening method will be a valuable way to explore the genes involved in many other essential aspects of photoreceptor biology.

Sex differences occur in the structure and function of the rat cerebral cortex and hippocampus, which can change from the juvenile period through old age. Although the evidence is incomplete, it appears that in at least some portions of the cortex these differences develop due to the rise of ovarian hormones at puberty and are potentially not dependent on the perinatal rise in testosterone, which is essential for sexual differentiation of the hypothalamus and sexual behavior. During aging of female rats, the presence of continued ovarian hormone secretion after cessation of the estrous cycle also influences sex differences in neuroanatomical structure and cognitive behavior, resulting in nullification or reversal of sex differences seen in younger adults. Sex differences can be altered by experience in a stimulating environment during the juvenile/adolescent period, and sex differences in performance even can be affected by the parameters of a task. Thus, broad generalizations about differences such as “spatial ability” are to be avoided. It is clear that to understand how the brain produces behavior, sex and hormones have to be taken into account.

Proper development of the cerebral cortex relies on asymmetric divisions of neural precursor cells (NPCs) to produce a recurring NPC and a differentiated neuron. Asymmetric divisions are promoted by the differential localization of cell-fate determinants, such as mRNA, between daughter cells. Staufen 1 (Stau1) is an RNA-binding protein known to localize mRNA in mature hippocampal neurons. Its expression pattern and role in the developing mammalian cortex remains unknown.Both stau1 mRNA and Stau1 protein were found to be expressed in all cells of the developing murine cortex. Stau1 protein expression was characterized spatially and temporally throughout cortical development and found to be present in all stages investigated. We observed expression in the nucleus, cytoplasm and distal processes of both NPCs and newly born neurons and found it to shuttle between the nucleus and the cytoplasm. Upon shRNA-mediated knock-down of Stau1 in primary cultures of the developing cortex, we did not observe any phenotype in NPCs. They were able to both self-renew and generate neurons in the absence of Stau1 expression.We propose that Stau1 is either dispensable for the development of the cerebral cortex or that its paralogue, Stau2, is able to compensate for its loss.

Embryonic ethanol exposure in zebrafish and rats, while stimulating hypothalamic hypocretin/orexin (Hcrt) neurons along with alcohol consumption and related behaviors, increases the chemokine receptor Cxcr4 that promotes neuronal migration and may mediate ethanol's effects on neuronal development. Here we performed a more detailed anatomical analysis in zebrafish of ethanol's effects on the Cxcl12a/Cxcr4b system throughout the entire brain as it relates to Hcrt neurons developing within the anterior hypothalamus (AH) where they are normally located. We found that ethanol increased these Hcrt neurons only in the anterior part of the AH and induced ectopic Hcrt neurons further anterior in the preoptic area, and these effects along with ethanol-induced behaviors were completely blocked by a Cxcr4 antagonist. Analysis of cxcl12a transcripts and internalized Cxcr4b receptors throughout the brain showed they both exhibited natural posterior-to-anterior concentration gradients, with levels lowest in the posterior AH and highest in the anterior telencephalon. While stimulating their density in all areas and maintaining these gradients, ethanol increased chemokine expression only in the more anterior and ectopic Hcrt neurons, effects blocked by the Cxcr4 antagonist. These findings demonstrate how increased chemokine expression acting along natural gradients mediates ethanol-induced anterior migration of ectopic Hcrt neurons and behavioral disturbances.

Astrocytes negatively impact neuronal development in many models of neurodevelopmental disorders (NDs); however, how they do this, and if mechanisms are shared across disorders, is not known. In this study, we developed a cell culture system to ask how astrocyte protein secretion and gene expression change in three mouse models of genetic NDs (Rett, Fragile X and Down syndromes). ND astrocytes increase release of Igfbp2, a secreted inhibitor of insulin-like growth factor (IGF). IGF rescues neuronal deficits in many NDs, and we found that blocking Igfbp2 partially rescues inhibitory effects of Rett syndrome astrocytes, suggesting that increased astrocyte Igfbp2 contributes to decreased IGF signaling in NDs. We identified that increased BMP signaling is upstream of protein secretion changes, including Igfbp2, and blocking BMP signaling in Fragile X and Rett syndrome astrocytes reverses inhibitory effects on neurite outgrowth. This work provides a resource of astrocyte-secreted proteins in health and ND models and identifies novel targets for intervention in diverse NDs.

IRSp53 (or BAIAP2) is an abundant excitatory postsynaptic scaffolding/adaptor protein that is involved in actin regulation and has been implicated in autism spectrum disorders, schizophrenia, and attention-deficit/hyperactivity disorder. IRSp53 deletion in mice leads to enhanced NMDA receptor (NMDAR) function and social deficits that are responsive to NMDAR inhibition. However, it remains unclear whether IRSp53 re-expression in the adult IRSp53-mutant mouse brain after the completion of brain development could reverse these synaptic and behavioral dysfunctions. Here we employed a brain-blood barrier (BBB)-penetrant adeno-associated virus (AAV) known as PHP.eB to drive adult IRSp53 re-expression in IRSp53-mutant mice. The adult IRSp53 re-expression normalized social deficits without affecting hyperactivity or anxiety-like behavior. In addition, adult IRSp53 re-expression normalized NMDAR-mediated excitatory synaptic transmission in the medial prefrontal cortex. Our results suggest that adult IRSp53 re-expression can normalize synaptic and behavioral deficits in IRSp53-mutant mice and that BBB-penetrant adult gene re-expression has therapeutic potential.

Phospholipids in the membrane consist of diverse pairs of fatty acids bound to a glycerol backbone. The biological significance of the diversity, however, remains mostly unclear. Part of this diversity is due to lysophospholipid acyltransferases (LPLATs), which introduce a fatty acid into lysophospholipids. The human genome has 14 LPLATs and most of them are highly conserved in vertebrates. Here, we analyzed the function of one of these enzymes, lysophosphatidylglycerol acyltransferase 1 (Lpgat1), in zebrafish. We found that the reproduction of heterozygous (lpgat1+/-) male mutants was abnormal. Crosses between heterozygous males and wild-type females produced many eggs with no obvious cleavage, whereas eggs produced by crosses between heterozygous females and wild-type males cleaved normally. Consistent with this, spermatozoa from heterozygous males had reduced motility and abnormal morphology. We also found that the occurrence of lpgat1 homozygous (lpgat1-/-) mutants was far lower than expected. In addition, downregulation of lpgat1 by morpholino antisense oligonucleotides resulted in severe developmental defects. Lipidomic analysis revealed that selective phospholipid species with stearic acid and docosahexaenoic acid were reduced in homozygous larvae and spermatozoa from heterozygotes. These results suggest that the specific phospholipid molecular species produced by Lpgat1 have an essential role in sperm fertilization and in embryonic development.

SP7/Osterix is a transcription factor critical for osteoblast maturation and bone formation. Homozygous loss-of-function mutations in SP7 cause osteogenesis imperfecta type XII, but neomorphic (gain-of-new-function) mutations of SP7 have not been reported in humans. Here we describe a de novo dominant neomorphic missense variant (c.926 C > G:p.S309W) in SP7 in a patient with craniosynostosis, cranial hyperostosis, and long bone fragility. Histomorphometry shows increased osteoblasts but decreased bone mineralization. Mice with the corresponding variant also show a complex skeletal phenotype distinct from that of Sp7-null mice. The mutation alters the binding specificity of SP7 from AT-rich motifs to a GC-consensus sequence (typical of other SP family members) and produces an aberrant gene expression profile, including increased expression of Col1a1 and endogenous Sp7, but decreased expression of genes involved in matrix mineralization. Our study identifies a pathogenic mechanism in which a mutation in a transcription factor shifts DNA binding specificity and provides important in vivo evidence that the affinity of SP7 for AT-rich motifs, unique among SP proteins, is critical for normal osteoblast differentiation.

Placozoa is a phylum of non-bilaterian marine animals. These small, flat organisms adhere to the substrate via their densely ciliated ventral epithelium, which mediates mucociliary locomotion and nutrient uptake. They have only six morphological cell types, including one, fiber cells, for which functional data is lacking. Fiber cells are non-epithelial cells with multiple processes. We used electron and light microscopic approaches to unravel the roles of fiber cells in Trichoplax adhaerens, a representative member of the phylum. Three-dimensional reconstructions of serial sections of Trichoplax showed that each fiber cell is in contact with several other cells. Examination of fiber cells in thin sections and observations of live dissociated fiber cells demonstrated that they phagocytose cell debris and bacteria. In situ hybridization confirmed that fiber cells express genes involved in phagocytic activity. Fiber cells also are involved in wound healing as evidenced from microsurgery experiments. Based on these observations we conclude that fiber cells are multi-purpose macrophage-like cells. Macrophage-like cells have been described in Porifera, Ctenophora, and Cnidaria and are widespread among Bilateria, but our study is the first to show that Placozoa possesses this cell type. The phylogenetic distribution of macrophage-like cells suggests that they appeared early in metazoan evolution.

Ventilatory support, such as supplemental oxygen, used to save premature infants impairs the growth of the pulmonary microvasculature and distal alveoli, leading to bronchopulmonary dysplasia (BPD). Although lung cellular composition changes with exposure to hyperoxia in neonatal mice, most human BPD survivors are weaned off oxygen within the first weeks to months of life, yet they may have persistent lung injury and pulmonary dysfunction as adults. We hypothesized that early-life hyperoxia alters the cellular landscape in later life and predicts long-term lung injury. Using single-cell RNA sequencing, we mapped lung cell subpopulations at postnatal day (pnd)7 and pnd60 in mice exposed to hyperoxia (95% O2) for 3 days as neonates. We interrogated over 10,000 cells and identified a total of 45 clusters within 32 cell states. Neonatal hyperoxia caused persistent compositional changes in later life (pnd60) in all five type II cell states with unique signatures and function. Premature infants requiring mechanical ventilation with different durations also showed similar alterations in these unique signatures of type II cell states. Pathologically, neonatal hyperoxic exposure caused alveolar simplification in adult mice. We conclude that neonatal hyperoxia alters the lung cellular landscape in later life, uncovering neonatal programing of adult lung dysfunction.

Skeletal muscle experiences a decline in lean mass and regenerative potential with age, in part due to intrinsic changes in progenitor cells. However, it remains unclear how age-related changes in progenitors manifest across a differentiation trajectory. Here, we perform single-cell RNA sequencing (RNA-seq) on muscle mononuclear cells from young and aged mice and profile muscle stem cells (MuSCs) and fibro-adipose progenitors (FAPs) after differentiation. Differentiation increases the magnitude of age-related change in MuSCs and FAPs, but it also masks a subset of age-related changes present in progenitors. Using a dynamical systems approach and RNA velocity, we find that aged MuSCs follow the same differentiation trajectory as young cells but stall in differentiation near a commitment decision. Our results suggest that differentiation reveals latent features of aging and that fate commitment decisions are delayed in aged myogenic cells in vitro.

Communication between neurons and glia has an important role in establishing and maintaining higher-order brain function1. Astrocytes are endowed with complex morphologies, placing their peripheral processes in close proximity to neuronal synapses and directly contributing to their regulation of brain circuits2-4. Recent studies have shown that excitatory neuronal activity promotes oligodendrocyte differentiation5-7; whether inhibitory neurotransmission regulates astrocyte morphogenesis during development is unclear. Here we show that inhibitory neuron activity is necessary and sufficient for astrocyte morphogenesis. We found that input from inhibitory neurons functions through astrocytic GABAB receptor (GABABR) and that its deletion in astrocytes results in a loss of morphological complexity across a host of brain regions and disruption of circuit function. Expression of GABABR in developing astrocytes is regulated in a region-specific manner by SOX9 or NFIA and deletion of these transcription factors results in region-specific defects in astrocyte morphogenesis, which is conferred by interactions with transcription factors exhibiting region-restricted patterns of expression. Together, our studies identify input from inhibitory neurons and astrocytic GABABR as universal regulators of morphogenesis, while further revealing a combinatorial code of region-specific transcriptional dependencies for astrocyte development that is intertwined with activity-dependent processes.