Nitrogen sensing and regulatory networks: it's about time and space.

A plant's response to external and internal nitrogen signals/status relies on sensing and signaling mechanisms that operate across spatial and temporal dimensions. From a comprehensive systems biology perspective, this involves integrating nitrogen responses in different cell types and over long distances to ensure organ coordination in real time and yield practical applications. In this prospective review, we focus on novel aspects of nitrogen (N) sensing/signaling uncovered using temporal and spatial systems biology approaches, largely in the model Arabidopsis. The temporal aspects span: transcriptional responses to N-dose mediated by Michaelis-Menten kinetics, the role of the master NLP7 transcription factor as a nitrate sensor, its nitrate-dependent TF nuclear retention, its "hit-and-run" mode of target gene regulation, and temporal transcriptional cascade identified by "network walking." Spatial aspects of N-sensing/signaling have been uncovered in cell type-specific studies in roots and in root-to-shoot communication. We explore new approaches using single-cell sequencing data, trajectory inference, and pseudotime analysis as well as machine learning and artificial intelligence approaches. Finally, unveiling the mechanisms underlying the spatial dynamics of nitrogen sensing/signaling networks across species from model to crop could pave the way for translational studies to improve nitrogen-use efficiency in crops. Such outcomes could potentially reduce the detrimental effects of excessive fertilizer usage on groundwater pollution and greenhouse gas emissions.

Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina.

The basic motif-leucine zipper (bZIP) transcription factor neural retina leucine zipper (NRL) determines rod photoreceptor cell fate during retinal development, and its loss leads to cone-only retina in mice. NRL works synergistically with homeodomain protein Cone-Rod Homeobox and other regulatory factors to control the transcription of most genes associated with rod morphogenesis and functional maturation, which span over a period of several weeks in the mammalian retina. We predicted that NRL gradually establishes rod cell identity and function by temporal and dynamic regulation of stage-specific transcriptional targets. Therefore, we mapped the genomic occupancy of NRL at four stages of mouse photoreceptor differentiation by CUT&RUN analysis. Dynamics of NRL binding revealed concordance with the corresponding changes in transcriptome of the developing rods. Notably, we identified c-Jun proto-oncogene as one of the targets of NRL, which could bind to specific cis-elements in the c-Jun promoter and modulate its activity in HEK293 cells. Coimmunoprecipitation studies showed the association of NRL with c-Jun, also a bZIP protein, in transfected cells as well as in developing mouse retina. Additionally, shRNA-mediated knockdown of c-Jun in the mouse retina in vivo resulted in altered expression of almost 1000 genes, with reduced expression of phototransduction genes and many direct targets of NRL in rod photoreceptors. We propose that c-Jun-NRL heterodimers prime the NRL-directed transcriptional program in neonatal rod photoreceptors before high NRL expression suppresses c-Jun at later stages. Our study highlights a broader cooperation among cell-type restricted and widely expressed bZIP proteins, such as c-Jun, in specific spatiotemporal contexts during cellular differentiation.

Multiomics analyses reveal early metabolic imbalance and mitochondrial stress in neonatal photoreceptors leading to cell death in Pde6brd1/rd1 mouse model of retinal degeneration.

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multiomics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations. Transcriptome profiling of neonatal and developing rods from the rd1 retina revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial overactivation and related metabolic alterations as determinants of early pathology and implicate aberrant calcium signaling as an initiator of higher mitochondrial stress. Our studies thus provide a mechanistic framework with mitochondrial damage and metabolic disruptions as early drivers of photoreceptor cell death in retinal degeneration.

Biological nitrogen fixation maintains carbon/nitrogen balance and photosynthesis at elevated CO2.

Understanding crop responses to elevated CO2 is necessary to meet increasing agricultural demands. Crops may not achieve maximum potential yields at high CO2 due to photosynthetic downregulation, often associated with nitrogen limitation. Legumes have been proposed to have an advantage at elevated CO2 due to their ability to exchange carbon for nitrogen. Here, the effects of biological nitrogen fixation (BNF) on the physiological and gene expression responses to elevated CO2 were examined at multiple nitrogen levels by comparing alfalfa mutants incapable of nitrogen fixation to wild-type. Elemental analysis revealed a role for BNF in maintaining shoot carbon/nitrogen (C/N) balance under all nitrogen treatments at elevated CO2, whereas the effect of BNF on biomass was only observed at elevated CO2 and the lowest nitrogen dose. Lower photosynthetic rates at were associated with the imbalance in shoot C/N. Genome-wide transcriptional responses were used to identify carbon and nitrogen metabolism genes underlying the traits. Transcription factors important to C/N signalling were identified from inferred regulatory networks. This work supports the hypothesis that maintenance of C/N homoeostasis at elevated CO2 can be achieved in plants capable of BNF and revealed important regulators in the underlying networks including an alfalfa (Golden2-like) GLK ortholog.

Are Rural Areas Holdouts in the Second Demographic Transition? Evidence From Canada and the United States.

A central premise of the first demographic transition theory is that demographic change would occur more slowly in rural than urban areas. Few studies, however, have investigated whether rural areas remain holdouts during the second demographic transition. To address this gap, this study (1) examines trends among rural and urban families in Canada and the United States over a 30-year period and (2) determines whether compositional differences in demographic, socioeconomic, and religious factors explain current differences between rural and urban families. We find that rural Canadian women continue to have, on average, 0.6 more children than urban women. However, rural families do not trail behind urban families on any other indicator of family change. In fact, rural women in both countries are now significantly more likely to cohabit and roughly 10 percentage points more likely to have children outside of marriage than urban women. These differences are largely explained by lower levels of education and income among rural American women and fewer immigrants in rural Canada. Examining family change through a rural-urban lens fills important empirical gaps and yields novel insights into current debates on the fundamental causes of ongoing family change in high-income countries.

Nitrate in 2020: Thirty Years from Transport to Signaling Networks.

Nitrogen (N) is an essential macronutrient for plants and a major limiting factor for plant growth and crop production. Nitrate is the main source of N available to plants in agricultural soils and in many natural environments. Sustaining agricultural productivity is of paramount importance in the current scenario of increasing world population, diversification of crop uses, and climate change. Plant productivity for major crops around the world, however, is still supported by excess application of N-rich fertilizers with detrimental economic and environmental impacts. Thus, understanding how plants regulate nitrate uptake and metabolism is key for developing new crops with enhanced N use efficiency and to cope with future world food demands. The study of plant responses to nitrate has gained considerable interest over the last 30 years. This review provides an overview of key findings in nitrate research, spanning biochemistry, molecular genetics, genomics, and systems biology. We discuss how we have reached our current view of nitrate transport, local and systemic nitrate sensing/signaling, and the regulatory networks underlying nitrate-controlled outputs in plants. We hope this summary will serve not only as a timeline and information repository but also as a baseline to define outstanding questions for future research.

Persistent disparities in affordable rental housing among America's ethnoracial groups.

Housing and residential outcomes in the United States are significantly stratified by ethnoracial group, but the extent to which disparities exist in affordable renting over time is less clear. In this study, I explore affordable housing disparities among White, Black, Hispanic, and Asian renters and test hypotheses regarding variation by education, local ethnoracial composition, and chosen measurement of affordability. Chiefly, I find that White households have higher rates of affordable housing than Black and Hispanic households with disparities remaining nearly identical between 2005 and 2019 and become larger when considering household's ability to afford other basic needs. Nevertheless, returns to education are not uniformly larger for White renters, in that Black and Asian renters experience larger marginal increases in residual income based affordable housing at higher levels of education. The effects of county ethnoracial composition effects are consistent with all groups-including White households-experiencing declining affordability when living in counties with large coethnic populations.

ConnecTF: A platform to integrate transcription factor-gene interactions and validate regulatory networks.

Deciphering gene regulatory networks (GRNs) is both a promise and challenge of systems biology. The promise lies in identifying key transcription factors (TFs) that enable an organism to react to changes in its environment. The challenge lies in validating GRNs that involve hundreds of TFs with hundreds of thousands of interactions with their genome-wide targets experimentally determined by high-throughput sequencing. To address this challenge, we developed ConnecTF, a species-independent, web-based platform that integrates genome-wide studies of TF-target binding, TF-target regulation, and other TF-centric omic datasets and uses these to build and refine validated or inferred GRNs. We demonstrate the functionality of ConnecTF by showing how integration within and across TF-target datasets uncovers biological insights. Case study 1 uses integration of TF-target gene regulation and binding datasets to uncover TF mode-of-action and identify potential TF partners for 14 TFs in abscisic acid signaling. Case study 2 demonstrates how genome-wide TF-target data and automated functions in ConnecTF are used in precision/recall analysis and pruning of an inferred GRN for nitrogen signaling. Case study 3 uses ConnecTF to chart a network path from NLP7, a master TF in nitrogen signaling, to direct secondary TF2s and to its indirect targets in a Network Walking approach. The public version of ConnecTF (https://ConnecTF.org) contains 3,738,278 TF-target interactions for 423 TFs in Arabidopsis, 839,210 TF-target interactions for 139 TFs in maize (Zea mays), and 293,094 TF-target interactions for 26 TFs in rice (Oryza sativa). The database and tools in ConnecTF will advance the exploration of GRNs in plant systems biology applications for model and crop species.

SDG8-Mediated Histone Methylation and RNA Processing Function in the Response to Nitrate Signaling.

Chromatin modification has gained increased attention for its role in the regulation of plant responses to environmental changes, but the specific mechanisms and molecular players remain elusive. Here, we show that the Arabidopsis (Arabidopsis thaliana) histone methyltransferase SET DOMAIN GROUP8 (SDG8) mediates genome-wide changes in H3K36 methylation at specific genomic loci functionally relevant to nitrate treatments. Moreover, we show that the specific H3K36 methyltransferase encoded by SDG8 is required for canonical RNA processing, and that RNA isoform switching is more prominent in the sdg8-5 deletion mutant than in the wild type. To demonstrate that SDG8-mediated regulation of RNA isoform expression is functionally relevant, we examined a putative regulatory gene, CONSTANS, CO-like, and TOC1 101 (CCT101), whose nitrogen-responsive isoform-specific RNA expression is mediated by SDG8. We show by functional expression in shoot cells that the different RNA isoforms of CCT101 encode distinct regulatory proteins with different effects on genome-wide transcription. We conclude that SDG8 is involved in plant responses to environmental nitrogen supply, affecting multiple gene regulatory processes including genome-wide histone modification, transcriptional regulation, and RNA processing, and thereby mediating developmental and metabolic processes related to nitrogen use.

Unequal From the Start? Poverty Across Immigrant Generations of Hispanic Children.

Recent cohorts of U.S. children increasingly consist of immigrants or the immediate descendants of immigrants, a demographic shift that has been implicated in high rates of child poverty. Analyzing data from the 2014-2018 Current Population Survey and using the U.S. Census Bureau's Supplemental Poverty Measure, we describe differences in child poverty rates across immigrant generations and assess how these disparities are rooted in generational differences in the prevalence and impact of key poverty risk factors. Our estimates show that poverty rates among Hispanic children are very high, particularly among first-generation children and second-generation children with two foreign-born parents. Low family employment is the most significant risk factor for poverty, but the prevalence of this risk varies little across immigrant generations. Differences in parental education account for the greatest share of observed intergenerational disparities in child poverty. Supplemental comparisons with third+-generation non-Hispanic White children underscore the disadvantages faced by all Hispanic children, highlighting the continued salience of race and ethnicity within the U.S. stratification system. Understanding the role of immigrant generation vis-à-vis other dimensions of inequality has significant policy implications given that America's population continues to grow more diverse along multiple social axes.

Temporal transcriptional logic of dynamic regulatory networks underlying nitrogen signaling and use in plants.

This study exploits time, the relatively unexplored fourth dimension of gene regulatory networks (GRNs), to learn the temporal transcriptional logic underlying dynamic nitrogen (N) signaling in plants. Our "just-in-time" analysis of time-series transcriptome data uncovered a temporal cascade of cis elements underlying dynamic N signaling. To infer transcription factor (TF)-target edges in a GRN, we applied a time-based machine learning method to 2,174 dynamic N-responsive genes. We experimentally determined a network precision cutoff, using TF-regulated genome-wide targets of three TF hubs (CRF4, SNZ, and CDF1), used to "prune" the network to 155 TFs and 608 targets. This network precision was reconfirmed using genome-wide TF-target regulation data for four additional TFs (TGA1, HHO5/6, and PHL1) not used in network pruning. These higher-confidence edges in the GRN were further filtered by independent TF-target binding data, used to calculate a TF "N-specificity" index. This refined GRN identifies the temporal relationship of known/validated regulators of N signaling (NLP7/8, TGA1/4, NAC4, HRS1, and LBD37/38/39) and 146 additional regulators. Six TFs-CRF4, SNZ, CDF1, HHO5/6, and PHL1-validated herein regulate a significant number of genes in the dynamic N response, targeting 54% of N-uptake/assimilation pathway genes. Phenotypically, inducible overexpression of CRF4 in planta regulates genes resulting in altered biomass, root development, and 15NO3- uptake, specifically under low-N conditions. This dynamic N-signaling GRN now provides the temporal "transcriptional logic" for 155 candidate TFs to improve nitrogen use efficiency with potential agricultural applications. Broadly, these time-based approaches can uncover the temporal transcriptional logic for any biological response system in biology, agriculture, or medicine.

Association of Electronic Health Record Vendors With Hospital Financial and Quality Performance: Retrospective Data Analysis.

BACKGROUND: Electronic health records (EHRs) are a central feature of care delivery in acute care hospitals; however, the financial and quality outcomes associated with system performance remain unclear. OBJECTIVE: In this study, we aimed to evaluate the association between the top 3 EHR vendors and measures of hospital financial and quality performance. METHODS: This study evaluated 2667 hospitals with Cerner, Epic, or Meditech as their primary EHR and considered their performance with regard to net income, Hospital Value-Based Purchasing Total Performance Score (TPS), and the unweighted subdomains of efficiency and cost reduction; clinical care; patient- and caregiver-centered experience; and patient safety. We hypothesized that there would be a difference among the 3 vendors for each measure. RESULTS: None of the EHR systems were associated with a statistically significant financial relationship in our study. Epic was positively associated with TPS outcomes (R2=23.6%; ß=.0159, SE 0.0079; P=.04) and higher patient perceptions of quality (R2=29.3%; ß=.0292, SE 0.0099; P=.003) but was negatively associated with patient safety quality scores (R2=24.3%; ß=-.0221, SE 0.0102; P=.03). Cerner and Epic were positively associated with improved efficiency (R2=31.9%; Cerner: ß=.0330, SE 0.0135, P=.01; Epic: ß=.0465, SE 0.0133, P<.001). Finally, all 3 vendors were associated with positive performance in the clinical care domain (Epic: ß=.0388, SE 0.0122, P=.002; Cerner: ß=.0283, SE 0.0124, P=.02; Meditech: ß=.0273, SE 0.0123, P=.03) but with low explanatory power (R2=4.2%). CONCLUSIONS: The results of this study provide evidence of a difference in clinical outcome performance among the top 3 EHR vendors and may serve as supportive evidence for health care leaders to target future capital investments to improve health care delivery.

Competency assessment of an undergraduate program using a third-party, objective pre-post examination.

BACKGROUND: Assessing competencies or program learning outcomes in educational programs is often a leadership challenge. This case study reports medical education program's efforts to document undergraduate competency attainment using a pre-post, third-party, objective testing service that allows for inter-university comparison, a testing service that is being adopted by some certification and accrediting bodies. METHODS: Students completed a pre-test after program acceptance and a post-test at the end of the last didactic semester (1.5 years later) just prior to their required internships. Scores and subscores were evaluated using t-tests (Holm-adjusted p-values). MANOVA models of sub-competency difference scores were also evaluated. RESULTS: Results indicate competency improvement for each of the 12 areas based on the n = 55 student sample, (p < .001 for all scores). These improvements were independent of ethnicity, age, gender, and grades. The average student improved by 12.85 points (95% CI of 10.52 to 15.18) with the largest improvements in strategic planning and leadership competency areas (21.30 and 18.33 percentage points, respectively). CONCLUSIONS: The third-party pre-post has some face validity given that student performance improved after completing a related curriculum as would be expected. Congruent with earlier studies, we find that repeated testing helps document competency attainment and that a single method for assessment is insufficient. We further document limitations of this 3d-party exam.

Emotion Regulation Mediates Posttraumatic Growth and Cluster B Personality Traits After Childhood Trauma.

OBJECTIVES: Cluster B personality disorder traits and positive psychological change, known as posttraumatic growth (PTG), are both possible outcomes following childhood trauma. However, existing research has not yet explored whether emotion regulation difficulties can simultaneously explain these negative and positive changes. METHOD: A sample of childhood trauma survivors (N = 223) provided responses to an online survey, with findings assessed using structural equation modeling techniques. RESULTS: Emotion regulation difficulties were found to mediate between childhood trauma severity and cluster B traits (ab cs = -.05), and between childhood trauma severity and PTG (abcs = .13), with small to medium indirect effects. The final model accounted for more variance in cluster B traits (56%) than PTG (10%). CONCLUSIONS: Emotion regulation is therefore a key mediator of positive and negative psychological changes and should be the focus of intervention efforts among childhood trauma survivors.

Patient iPSC-derived neural stem cells exhibit phenotypes in concordance with the clinical severity of mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) is caused by deficiency of α-l-iduronidase (IDUA), a lysosomal enzyme involved in the breakdown and recycling of glycosaminoglycans (GAGs). Although enzyme replacement therapy is available, the efficacy of the treatment for neuropathic manifestations is limited. To facilitate drug discovery and model disease pathophysiology, we generated neural stem cells (NSCs) from MPS I patient-derived induced pluripotent stem cells (iPSCs). The NSCs exhibited characteristic disease phenotypes with deficiency of IDUA, accumulation of GAGs and enlargement of lysosomes, in agreement with the severity of clinical subgroups of MPS I. Transcriptome profiling of NSCs revealed 429 genes that demonstrated a more extensive change in expression in the most severe Hurler syndrome subgroup compared to the intermediate Hurler-Scheie or the least severe Scheie syndrome subgroups. Clustering and pathway analysis revealed high concordance of the severity of neurological defects with marked dysregulation of GAG biosynthesis, GAG degradation, lysosomal function and autophagy. Gene ontology (GO) analysis identified a dramatic upregulation of the autophagy pathway, especially in the Hurler syndrome subgroup. We conclude that GAG accumulation in the patient-derived cells disrupts lysosomal homeostasis, affecting multiple related cellular pathways in response to IDUA deficiency. These dysregulated processes likely lead to enhanced autophagy and progressively severe disease states. Our study provides potentially useful targets for clinical biomarker development, disease diagnosis and prognosis, and drug discovery.

County Reclassifications and Rural-Urban Mortality Disparities in the United States (1970-2018).

Objectives. To demonstrate how inferences about rural-urban disparities in age-adjusted mortality are affected by the reclassification of rural and urban counties in the United States from 1970 to 2018.Methods. We compared estimates of rural-urban mortality disparities over time, produced through a time-varying classification of rural and urban counties, with counterfactual estimates of rural-urban disparities, assuming no changes in rural-urban classification since 1970. We evaluated mortality rates by decade of reclassification to assess selectivity in reclassification.Results. We found that reclassification amplified rural-urban mortality disparities and accounted for more than 25% of the rural disadvantage observed from 1970 to 2018. Mortality rates were lower in counties that reclassified from rural to urban than in counties that remained rural.Conclusions. Estimates of changing rural-urban mortality differentials are significantly influenced by rural-urban reclassification. On average, counties that have remained classified as rural over time have elevated mortality. Longitudinal research on rural-urban health disparities must consider the methodological and substantive implications of reclassification.Public Health Implications. Attention to rural-urban reclassification is necessary when evaluating or justifying policy interventions focusing on geographic health disparities.

Engineering the lutein epoxide cycle into Arabidopsis thaliana.

Although sunlight provides the energy necessary for plants to survive and grow, light can also damage reaction centers of photosystem II (PSII) and reduce photochemical efficiency. To prevent damage, plants possess photoprotective mechanisms that dissipate excess excitation. A subset of these mechanisms is collectively referred to as NPQ, or nonphotochemical quenching of chlorophyll a fluorescence. The regulation of NPQ is intrinsically linked to the cycling of xanthophylls that affects the kinetics and extent of the photoprotective response. The violaxanthin cycle (VAZ cycle) and the lutein epoxide cycle (LxL cycle) are two xanthophyll cycles found in vascular plants. The VAZ cycle has been studied extensively, owing in large part to its presence in model plant species where mutants are available to aid in its characterization. In contrast, the LxL cycle is not found in model plants, and its role in photosynthetic processes has been more difficult to define. To address this challenge, we introduced the LxL cycle into Arabidopsis thaliana and functionally isolated it from the VAZ cycle. Using these plant lines, we showed an increase in dark-acclimated PSII efficiency associated with Lx accumulation and demonstrated that violaxanthin deepoxidase is responsible for the light-driven deepoxidation of Lx. Conversion of Lx to L was reversible during periods of low light and occurred considerably faster than rates previously described in nonmodel species. Finally, we present clear evidence of the LxL cycle's role in modulating a rapid component of NPQ that is necessary to prevent photoinhibition in excess light.

Molecular codes for cell type specification in Brn3 retinal ganglion cells.

Visual information is conveyed from the eye to the brain by distinct types of retinal ganglion cells (RGCs). It is largely unknown how RGCs acquire their defining morphological and physiological features and connect to upstream and downstream synaptic partners. The three Brn3/Pou4f transcription factors (TFs) participate in a combinatorial code for RGC type specification, but their exact molecular roles are still unclear. We use deep sequencing to define (i) transcriptomes of Brn3a- and/or Brn3b-positive RGCs, (ii) Brn3a- and/or Brn3b-dependent RGC transcripts, and (iii) transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation, and synaptogenesis. We reveal a combinatorial code of TFs, cell surface molecules, and determinants of neuronal morphology that is differentially expressed in specific RGC populations and selectively regulated by Brn3a and/or Brn3b. This comprehensive molecular code provides a basis for understanding neuronal cell type specification in RGCs.

REEP6 mediates trafficking of a subset of Clathrin-coated vesicles and is critical for rod photoreceptor function and survival.

In retinal photoreceptors, vectorial transport of cargo is critical for transduction of visual signals, and defects in intracellular trafficking can lead to photoreceptor degeneration and vision impairment. Molecular signatures associated with routing of transport vesicles in photoreceptors are poorly understood. We previously reported the identification of a novel rod photoreceptor specific isoform of Receptor Expression Enhancing Protein (REEP) 6, which belongs to a family of proteins involved in intracellular transport of receptors to the plasma membrane. Here we show that loss of REEP6 in mice (Reep6-/-) results in progressive retinal degeneration. Rod photoreceptor dysfunction is observed in Reep6-/- mice as early as one month of age and associated with aberrant accumulation of vacuole-like structures at the apical inner segment and reduction in selected rod phototransduction proteins. We demonstrate that REEP6 is detected in a subset of Clathrin-coated vesicles and interacts with the t-SNARE, Syntaxin3. In concordance with the rod degeneration phenotype in Reep6-/- mice, whole exome sequencing identified homozygous REEP6-E75K mutation in two retinitis pigmentosa families of different ethnicities. Our studies suggest a critical function of REEP6 in trafficking of cargo via a subset of Clathrin-coated vesicles to selected membrane sites in retinal rod photoreceptors.