Cell-type Specificity Research Articles

Revisiting reactive oxygen species production in hypoxia.

Cellular responses to hypoxia are crucial in various physiological and pathophysiological contexts and have thus been extensively studied. This has led to a comprehensive understanding of the transcriptional response to hypoxia, which is regulated by hypoxia-inducible factors (HIFs). However, the detailed molecular mechanisms of HIF regulation in hypoxia remain incompletely understood. In particular, there is controversy surrounding the production of mitochondrial reactive oxygen species (ROS) in hypoxia and how this affects the stabilization and activity of HIFs. This review examines this controversy and attempts to shed light on its origin. We discuss the role of physioxia versus normoxia as baseline conditions that can affect the subsequent cellular response to hypoxia and highlight the paucity of data on pericellular oxygen levels in most experiments, leading to variable levels of hypoxia that might progress to anoxia over time. We analyze the different outcomes reported in isolated mitochondria, versus intact cells or whole organisms, and evaluate the reliability of various ROS-detectingtools. Finally, we examine the cell-type and context specificity of oxygen's various effects. We conclude that while recent evidence suggests that the effect of hypoxia on ROS production is highly dependent on the cell type and the duration of exposure, efforts should be made to conduct experiments under carefully controlled, physiological microenvironmental conditions in order to rule out potential artifacts and improve reproducibility in research.

Microglial MyD88-dependent pathways are regulated in a sex-specific manner in the context of HMGB1-induced anxiety.

Chronic stress is a significant risk factor for the development and recurrence of anxiety disorders. Chronic stress impacts the immune system, causing microglial functional alterations in the medial prefrontal cortex (mPFC), a brain region involved in the pathogenesis of anxiety. High mobility group box 1 protein (HMGB1) is an established modulator of neuronal firing and a potent pro-inflammatory stimulus released from neuronal and non-neuronal cells following stress. HMGB1, in the context of stress, acts as a danger-associated molecular pattern (DAMP), instigating robust proinflammatory responses throughout the brain, so much so that localized drug delivery of HMGB1 alters behavior in the absence of any other forms of stress, i.e., social isolation, or behavioral stress models. Few studies have investigated the molecular mechanisms that underlie HMGB1-associated behavioral effects in a cell-specific manner. The aim of this study is to investigate cellular and molecular mechanisms underlying HMGB1-induced behavioral dysfunction with regard to cell-type specificity and potential sex differences. Here, we report that both male and female mice exhibited anxiety-like behavior following increased HMGB1 in the mPFC as well as changes in microglial morphology. Interestingly, our results demonstrate that HMGB1-induced anxiety may be mediated by distinct microglial MyD88-dependent mechanisms in females compared to males. This study supports the hypothesis that MyD88 signaling in microglia may be a crucial mediator of the stress response in adult female mice.

The role of Mce proteins in Mycobacterium avium paratuberculosis infection

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s Disease, a chronic granulomatous enteritis of ruminants. MAP establishes an infection in the host via the small intestine. This requires the bacterium to adhere to, and be internalised by, cells of the intestinal tract. The effector molecules expressed by MAP for this purpose remain to be fully identified and understood. Mammalian cell entry (mce) proteins have been shown to enable other Mycobacterial species to attach to and invade host epithelial cells. Here, we have expressed Mce1A, Mce1D, Mce3C and Mce4A proteins derived from MAP on the surface of a non-invasive Escherichia coli to characterise their role in the initial interaction between MAP and the host. To this end, expression of mce1A was found to significantly increase the ability of the E. coli to attach and survive intracellularly in human monocyte-like THP-1 cells, whereas expression of mce1D was found to significantly increase attachment and invasion of E. coli to bovine epithelial cell-like MDBK cells, implying cell-type specificity. Furthermore, expression of Mce1A and Mce1D on the surface of a previously non-invasive E. coli enhanced the ability of the bacterium to infect 3D bovine basal-out enteroids. Together, our data contributes to our understanding of the effector molecules utilised by MAP in the initial interaction with the host, and may provide potential targets for therapeutic intervention.

A single cell RNA sequence atlas of the early Drosophila larval eye

The Drosophila eye has been an important model to understand principles of differentiation, proliferation, apoptosis and tissue morphogenesis. However, a single cell RNA sequence resource that captures gene expression dynamics from the initiation of differentiation to the specification of different cell types in the larval eye disc is lacking. Here, we report transcriptomic data from 13,000 cells that cover six developmental stages of the larval eye. Our data show cell clusters that correspond to all major cell types present in the eye disc ranging from the initiation of the morphogenetic furrow to the differentiation of each photoreceptor cell type as well as early cone cells. We identify dozens of cell type-specific genes whose function in different aspects of eye development have not been reported. These single cell data will greatly aid research groups studying different aspects of early eye development and will facilitate a deeper understanding of the larval eye as a model system.

Diagnostic and Prognostic Markers for Pancreatitis and Pancreatic Ductal Adenocarcinoma.

Diagnostic markers are desperately needed for the early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity and the differential expression of PDA markers were identified by screening single cell (sc) RNAseq from tumor samples of a mouse model for PDA (KIC) at early and late stages of PDA progression compared to that of a normal pancreas. Candidate genes were identified from three sources: (1) an unsupervised screening of the genes preferentially expressed in mouse PDA tumors; (2) signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes, or genes encoding proteins that were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines; and (3) genes whose expression is associated with poor or better prognoses (PAAD, oncolnc.org). The developmental progression of PDA was detected in the temporal order of gene expression in the cancer cells of the KIC mice. The earliest diagnostic markers were expressed in epithelial cancer cells in early-stage, but not late-stage, PDA tumors. Other early markers were expressed in the epithelium of both early- and late-state PDA tumors. Markers that were expressed somewhat later were first elevated in the epithelial cancer cells of the late-stage tumors, then in both epithelial and mesenchymal cells, or only in mesenchymal cells. Stromal markers were differentially expressed in early- and/or late-stage PDA neoplasia in fibroblast and hematopoietic cells (lymphocytes and/or macrophages) or broadly expressed in cancer and many stromal cell types. Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and the acinar-specific homozygous deletion of differentiation transcription factors (dTFs), were screened for the early expression of all PDA markers identified in the KIC neoplasia. Prognostic markers associated with a more rapid decline were identified and showed differential and cell-type-specific expression in PDA, predominately in late-stage epithelial and/or mesenchymal cancer cells. Select markers were validated by immunohistochemistry in mouse and human samples of a normal pancreas and those with early- and late-stage PDA. In total, we present 2165 individual diagnostic and prognostic markers for disease progression to be tested in humans from pancreatitis to late-stage PDA.

Iterative deep learning-design of human enhancers exploits condensed sequence grammar to achieve cell type-specificity.

An important and largely unsolved problem in synthetic biology is how to target gene expression to specific cell types. Here, we apply iterative deep learning to design synthetic enhancers with strong differential activity between two human cell lines. We initially train models on published datasets of enhancer activity and chromatin accessibility and use them to guide the design of synthetic enhancers that maximize predicted specificity. We experimentally validate these sequences, use the measurements to re-optimize the predictor, and design a second generation of enhancers with improved specificity. Our design methods embed relevant transcription factor binding site (TFBS) motifs with higher frequencies than comparable endogenous enhancers while using a more selective motif vocabulary, and we show that enhancer activity is correlated with transcription factor expression at the single cell level. Finally, we characterize causal features of top enhancers via perturbation experiments and show enhancers as short as 50bp can maintain specificity.

C1q modulation of antibody-dependent enhancement of dengue virus infection in human myeloid cell lines is dependent on cell type and antibody specificity

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is one of the mechanisms contributing to increased severity during heterotypic, secondary infection. The complement protein C1q has been shown to reduce the magnitude of ADE in vitro. Therefore, we investigated the mechanisms of C1q modulation of ADE, focusing on processes of viral entry.Using a model of ADE of DENV-1 infection in human myeloid cell lines in the presence of monoclonal antibodies, 4G2 and 2H2, we found that C1q produced nearly a 40-fold reduction of ADE of DENV-1 in K562 cells, but had no effect in U937 cells. In K562 cells, C1q reduced adsorption of DENV-1/4G2 and exerted a dual inhibitory effect on adsorption and internalization of DENV-1/2H2. Distinct endocytic pathways in the presence of antibody corresponded to conditions where C1q produced a differential action. Also, C1q did not affect the intrinsic cell response mediated by FcγR in human myeloid cells.The modulation of ADE of DENV-1 by C1q is dependent on the FcγR expressed on immune cells and the specificity of the antibody comprising the immune complex. Understanding protective and pathogenic mechanisms in the humoral response to DENV infections is crucial for the successful design of antivirals and vaccines.

CTCF regulates global chromatin accessibility and transcription during rod photoreceptor development.

Chromatin architecture facilitates accurate transcription at a number of loci, but it remains unclear how much chromatin architecture is involved in global transcriptional regulation. Previous work has shown that rapid depletion of the architectural protein CTCF in cell culture strongly alters chromatin organization but results in surprisingly limited gene expression changes. This discrepancy has also been observed when other architectural proteins are depleted, and one possible explanation is that full transcriptional changes are masked by cellular heterogeneity. We tested this idea by performing multi-omics analyses with sorted post-mitotic mouse rods, which undergo synchronized development, and identified CTCF-dependent regulation of global chromatin accessibility and gene expression. Depletion of CTCF leads to dysregulation of ∼20% of the entire transcriptome (>3,000 genes) and ∼41% of genome accessibility (>26,000 sites), and these regions are strongly enriched in euchromatin. Importantly, these changes are highly enriched for CTCF occupancy, suggesting direct CTCF binding and transcriptional regulation at these active loci. CTCF mainly promotes chromatin accessibility of these direct binding targets, and a large fraction of these sites correspond to promoters. At these sites, CTCF binding frequently promotes accessibility and inhibits expression, and motifs of transcription repressors are found to be significantly enriched. Our findings provide different and often opposite conclusions from previous studies, emphasizing the need to consider cell heterogeneity and cell type specificity when performing multi-omics analyses. We conclude that the architectural protein CTCF binds chromatin and regulates global chromatin accessibility and transcription during rod development.

Integrated Systems Analysis Deciphers Transcriptome and Glycoproteome Links in Alzheimer's Disease.

Glycosylation is increasingly recognized as a potential therapeutic target in Alzheimer's disease. In recent years, evidence of Alzheimer's disease-specific glycoproteins has been established. However, the mechanisms underlying their dysregulation, including tissue- and cell-type specificity, are not fully understood. We aimed to explore the upstream regulators of aberrant glycosylation by integrating multiple data sources using a glycogenomics approach. We identified dysregulation of the glycosyltransferase PLOD3 in oligodendrocytes as an upstream regulator of cerebral vessels and found that it is involved in COL4A5 synthesis, which is strongly correlated with amyloid fiber formation. Furthermore, COL4A5 has been suggested to interact with astrocytes via extracellular matrix receptors as a ligand. This study suggests directions for new therapeutic strategies for Alzheimer's disease targeting glycosyltransferases.

A genome-wide meta-analysis of palmoplantar pustulosis implicates TH2 responses and cigarette smoking in disease pathogenesis

Palmoplantar pustulosis (PPP) is an inflammatory skin disorder that mostly affects smokers and manifests with painful pustular eruptions on the palms and soles. Although the disease can present with concurrent plaque psoriasis, TNF and IL-17/IL-23 inhibitors show limited efficacy. There is therefore a pressing need to uncover PPP disease drivers and therapeutic targets. We sought to identify genetic determinants of PPP and investigate whether cigarette smoking contributes to disease pathogenesis. We performed a genome-wide association meta-analysis of 3 North-European cohorts (n= 1,456 PPP cases and 402,050 controls). We then used the scGWAS program to investigate the cell-type specificity of the association signals. We also undertook genetic correlation analyses to examine the similarities between PPP and other immune-mediated diseases. Finally, we applied Mendelian randomization to analyze the causal relationship between cigarette smoking and PPP. We found that PPP is not associated with the main genetic determinants of plaque psoriasis. Conversely, we identified genome-wide significant associations with the FCGR3A/FCGR3B and CCHCR1 loci. We also observed 13 suggestive (P< 5×10-6) susceptibility regions, including the IL4/IL13 interval. Accordingly, we demonstrated a significant genetic correlation between PPP and TH2-mediated diseases such as atopic dermatitis and ulcerative colitis. We also found that genes mapping to PPP-associated intervals were preferentially expressed in dendritic cells and often implicated in T-cell activation pathways. Finally, we undertook a Mendelian randomization analysis, which supported a causal role of cigarette smoking in PPP. The first genome-wide association study of PPP points to a pathogenic role for deregulated TH2 responses and cigarette smoking.

Cell-type specific inference from bulk RNA-sequencing data by integrating single cell reference profiles via EPIC-unmix.

Cell type specific (CTS) analysis is essential to reveal biological insights obscured in bulk tissue data. However, single-cell (sc) or single-nuclei (sn) resolution data are still cost-prohibitive for large-scale samples. Thus, computational methods to perform deconvolution from bulk tissue data are highly valuable. We here present EPIC-unmix, a novel two-step empirical Bayesian method integrating reference sc/sn RNA-seq data and bulk RNA-seq data from target samples to enhance the accuracy of CTS inference. We demonstrate through comprehensive simulations across three tissues that EPIC-unmix achieved 4.6% - 109.8% higher accuracy compared to alternative methods. By applying EPIC-unmix to human bulk brain RNA-seq data from the ROSMAP and MSBB cohorts, we identified multiple genes differentially expressed between Alzheimer's disease (AD) cases versus controls in a CTS manner, including 57.4% novel genes not identified using similar sample size sc/snRNA-seq data, indicating the power of our in-silico approach. Among the 6-69% overlapping, 83%-100% are in consistent direction with those from sc/snRNA-seq data, supporting the reliability of our findings. EPIC-unmix inferred CTS expression profiles similarly empowers CTS eQTL analysis. Among the novel eQTLs, we highlight a microglia eQTL for AD risk gene AP3B2, obscured in bulk and missed by sc/snRNA-seq based eQTL analysis. The variant resides in a microglia-specific cCRE, forming chromatin loop with AP3B2 promoter region in microglia. Taken together, we believe EPIC-unmix will be a valuable tool to enable more powerful CTS analysis.

Pyrogallol intermediates elicit beta-amyloid secretion via radical formation and alterations in intracellular trafficking, distinct from pyrogallol-generated superoxide

This study unveils a novel role of pyrogallol (PG), a recognized superoxide generator, in inducing beta-amyloid (Aβ) secretion in an Alzheimer's disease (AD) cellular model. Contrary to expectations, the analysis of dihydroethidium fluorescence and UV-VIS spectrum scanning reveals that Aβ secretion arises from PG reaction intermediates rather than superoxide or other by-products. Investigation into Aβ secretion mechanisms identifies dynasore-dependent endocytosis and BFA-dependent exocytosis as independent pathways, regulated by tiron, tempol, and superoxide dismutase. Cell-type specificity is observed, with 293sw cells showing both pathways, while H4sw cells and primary astrocytes from an AD animal model exclusively exhibit the Aβ exocytosis pathway. This exploration contributes to understanding PG's chemical reactions and provides insights into the interplay between environmental factors, free radicals, and AD, linking occupational PG exposure to AD risk as reported in the literature.

DeepFace: Deep-learning-based framework to contextualize orofacial-cleft-related variants during human embryonic craniofacial development

Orofacial clefts (OFC) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the ‘casual’ variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson Correlation Coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50 to 0.83. Specifically, our model revealed that SNPs significantly associated with OFC tended to exhibit higher SAD scores across various variant categories compared to less-related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFC. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFC, and the model that can be extended to study other complex diseases or traits.

Kcnma1 alternative splicing in mouse kidney: regulation during development and by dietary K+ intake.

The pore-forming α-subunit of the large-conductance K+ (BK) channel is encoded by a single gene, KCNMA1. BK channel-mediated K+ secretion in the kidney is crucial for overall renal K+ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K+ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K+, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K+ loading in mouse kidney.NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K+.

Origins of tissue and cell-type specificity in mitochondrial DNA (mtDNA) disease.

Mutations of mitochondrial (mt)DNA are a major cause of morbidity and mortality in humans, accounting for approximately two thirds of diagnosed mitochondrial disease. However, despite significant advances in technology since the discovery of the first disease-causing mtDNA mutations in 1988, the comprehensive diagnosis and treatment of mtDNA disease remains challenging. This is partly due to the highly variable clinical presentation linked to tissue-specific vulnerability that determines which organs are affected. Organ involvement can vary between different mtDNA mutations, and also between patients carrying the same disease-causing variant. The clinical features frequently overlap with other non-mitochondrial diseases, both rare and common, adding to the diagnostic challenge. Building on previous findings, recent technological advances have cast further light on the mechanisms which underpin the organ vulnerability in mtDNA diseases, but our understanding is far from complete. In this review we explore the origins, current knowledge, and future directions of research in this area.

Plasma proteome analysis implicates novel proteins as potential therapeutic targets for chronic kidney disease: A proteome-wide association study

Chronic kidney disease (CKD) is prevalent globally with limited therapeutic drugs available. To systemically identify novel proteins involved in the pathogenesis of CKD and possible therapeutic targets, we integrated human plasma proteomes with the genome-wide association studies (GWASs) of CKD, estimated glomerular filtration rate (eGFR) and blood urea nitrogen (BUN) to perform proteome-wide association study (PWAS), Mendelian Randomization and Bayesian colocalization analyses. The single-cell RNA sequencing data of healthy human and mouse kidneys were analyzed to explore the cell-type specificity of identified genes. Functional enrichment analysis was conducted to investigate the involved signaling pathways. The PWAS identified 22 plasma proteins significantly associated with CKD. Of them, the significant associations of three proteins (INHBC, LMAN2, and SNUPN) were replicated in the GWASs of eGFR, and BUN. Mendelian Randomization analyses showed that INHBC and SNUPN were causally associated with CKD, eGFR, and BUN. The Bayesian colocalization analysis identified shared causal variants for INHBC in CKD, eGFR, and BUN (all PP4 > 0.75). The single-cell RNA sequencing revealed that the INHBC gene was sparsely scattered within the kidney cells. This proteomic study revealed that INHBC, LMAN2, and SNUPN may be involved in the pathogenesis of CKD, which represent novel therapeutic targets and warrant further exploration in future research.

The single-cell chromatin landscape in gonadal cell lineage specification

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Conditional Cell Penetration of Masked CPPs by an ADEPT-like Approach.

The intracellular delivery of cargos via cell penetrating peptides (CPPs) holds significant promise as a drug delivery vehicle, but a major issue is their lack of cell type specificity, which can lead to detrimental off-target effects. We use an ADEPT-like concept to introduce conditional and selective activation of cellular uptake by using the lysine-rich, cationic, and amphiphilic L17E peptide as a model CPP. By masking the lysine residues of the L17E peptide with enzyme-cleavable acetyl protecting groups, the delivery of the covalently conjugated fluorophore TAMRA to HeLa cells was diminished. Recovery of cellular uptake could be achieved by deacetylation of the masked acetylated L17E peptide using the NAD-dependent sirtuin 2 (SirT2) deacetylase in vitro. Finally, trastuzumab-SirT2 and anti-B7H3-SirT2 antibody-enzyme conjugates were generated for the conditional and selective delivery of a cryptophycin cytotoxin by the L17E peptide. While the masked peptide still demonstrated some cytotoxicity, selective cell killing mediated by the antibody-enzyme conjugates was observed.

Cell type- and transcription-independent spatial proximity between enhancers and promoters.

Cell type-specific enhancers are critically important for lineage specification. The mechanisms that determine cell-type specificity of enhancer activity, however, are not fully understood. Most current models for how enhancers function invoke physical proximity between enhancer elements and their target genes. Here, we use an imaging-based approach to examine the spatial relationship of cell type-specific enhancers and their target genes with single-cell resolution. Using high-throughput microscopy, we measure the spatial distance from target promoters to their cell type-specific active and inactive enhancers in individual pancreatic cells derived from distinct lineages. We find increased proximity of all promoter-enhancer pairs relative to non-enhancer pairs separated by similar genomic distances. Strikingly, spatial proximity between enhancers and target genes was unrelated to tissue-specific enhancer activity. Furthermore, promoter-enhancer proximity did not correlate with the expression status of target genes. Our results suggest that promoter-enhancer pairs exist in a distinctive chromatin environment but that genome folding is not a universal driver of cell-type specificity in enhancer function.

Experience-dependent, sexually dimorphic synaptic connectivity defined by sex-specific cadherin expression.

We describe here the molecular mechanisms by which juvenile experience defines patterns of sexually dimorphic synaptic connectivity in the adult nervous system of the nematode C. elegans. We show that starvation of juvenile males disrupts serotonin-dependent activation of the CREB transcription factor in a nociceptive sensory neuron, PHB. CREB acts through a cascade of transcription factors to control expression of an atypical cadherin protein, FMI-1/Flamingo. During postembryonic development, FMI-1/Flamingo has the capacity to promote and maintain synaptic connectivity of the PHB nociceptive sensory to a command interneuron, AVA, in both sexes, but the serotonin transcriptional regulatory cassette antagonizes FMI-1/Flamingo expression in males, thereby establishing sexually dimorphic connectivity between PHB and AVA. A critical regulatory node in this process is the CREB-target LIN-29, a Zn finger transcription factor which integrates four different layers of information - sexual specificity, past feeding status, time and cell-type specificity. Our findings provide the mechanistic details of how an early juvenile experience defines sexually dimorphic synaptic connectivity.