Biology subjects

Hamid, F.

Publications and source records attributed to Hamid, F..

12 recordsLinked to original sources

bioRxiv2026

An integrated pipeline to count individual transcripts with single-cell resolution

Quantifying transcript abundance at single-cell resolution is important for understanding gene regulation in intact multicellular organisms. In Caenorhabditis elegans, RNA fluorescence in situ hybridization has been widely used to visualize transcripts, but conventional smFISH approaches can be limited by low signal-to-noise, poor performance with short transcripts, and workflows that do not readily support absolute transcript counting in identified cells. Here, we present an integrated experimental and computational pipeline for quantitative transcript analysis in whole-mount C. elegans embryos, larvae, and adults. The pipeline combines Hybridization Chain Reaction (HCR), confocal microscopy, RS-FISH spot detection, manual cell annotation in FIJI, and custom MATLAB-based spot assignment to quantify individual transcripts within defined cells. We show that this approach enables sensitive, specific, and multiplexed detection of transcripts, including short insulin-like peptide mRNAs, with single-cell resolution. Known spatial expression patterns were resolved in embryos, larvae, and adults, and probe specificity was validated using deletion mutants that remove HCR probe-binding sites. Applying this pipeline to ins-4, ins-6, and daf-28 in ASI and ASJ sensory neurons revealed cell-specific and context-dependent regulatory relationships across multiple mutant backgrounds. This workflow provides an accessible method for absolute transcript counting in anatomically intact C. elegans and should support mechanistic studies of gene regulation, cellular heterogeneity, and transcriptional network function.

molecular biology↗

bioRxiv2026

Distinct signaling center and progenitor identity dynamics initiate human forebrain patterning

At which developmental stage does the human brain start to differ from other mammals? We uncover human features of telencephalic patterning as early as the fourth post-conception week, comparing the early mouse and human forebrains using single-cell transcriptomics and 3D spatial multi-transcript imaging. In comparison to mice, the human telencephalon is delayed in ventral SHH signaling, accompanied by a human signature of anterior FGF signaling. These observations correlate with a less-resolved patterning and reduced progenitor diversity along the dorsoventral and anteroposterior axes, as well as a human early neurogenic signature. Our complementary approaches reveal a human divergence in allocation of telencephalic progenitor identities, propelled by temporal and qualitative differences in ventro-anterior signals following neural tube closure in the developing forebrain.

developmental biology↗

bioRxiv2026

Cryptic Last Exon splicing is a conserved tuning mechanism during neural development

Cryptic last exons (CLEs) are unannotated terminal exons that arise from intronic sequence and have predominantly been associated with RNA-binding-protein dysfunction and neurodegenerative disease. Whether CLEs also contribute to physiological gene regulation remained unclear. Here, we identify thousands of CLE-containing transcripts across wild-type zebrafish, mouse and human transcriptomes. CLE sequences are poorly conserved, yet CLE occurrence recurs in orthologous genes and, less frequently, orthologous introns, suggesting that gene architecture creates recurrent opportunities for CLE formation. A subset of CLEs is developmentally regulated and CLE usage is unusually sensitive to perturbation of neural RNA-binding proteins. CLE-containing transcripts associate with ribosomes, and endogenous tagging demonstrates that ephA4b-CLE produces a stable truncated protein during normal development. Loss of ephA4b-CLE impairs retinal ganglion cell axon growth, whereas disruption of GAN-CLE increases motor-axon extension and terminal branching. Moreover, independently arising human and zebrafish EphA4 CLEs converge on similar predicted ephrin-binding ectodomains despite lacking sequence conservation. These findings establish CLEs as a physiological source of transcript diversity within which individual isoforms can acquire regulated and gene-specific developmental functions.

neuroscience↗

bioRxiv2024

A cleaved cytosolic FOXG1 promotes excitatory neurogenesis by modulation of mitochondrial translation - a new therapeutic target for brain disorders.

The modulation of mitochondrial function is core to cell fate decisions and tissue homeostasis, yet the mechanisms regulating these processes remain poorly understood. Here, we demonstrate that mitochondrial OXPHOS activity is controlled in a tissue-specific manner through a non-canonical cytoplasmic function of the transcription factor FOXG1. A temporally regulated short cytoplasmic FOXG1 interacts with mitochondrial ribosomal proteins and enhances translation of mt-DNA-encoded OXPHOS proteins. Zebrafish and human models of early nonsense FOXG1 syndrome mutations unexpectedly produce a short C-terminal peptide. Expression of this truncated protein drives an overproduction of excitatory neurons and induces a structural, functional, and translational phenotype in mutant mitochondria. We demonstrate that this activity is a gain of function, normally carried out by a cleaved FOXG1 in wildtype. Both normal and mutant peptides are transported to the mitochondria, interact with mito-ribosomal proteins to enhance translation, thereby stimulating neurogenesis. Adjusting the dosage of the mutant peptide rescues the excitatory aspect of the FOXG1 syndrome. Our study demonstrates a novel role for cytoplasmic Foxg1 in promoting neurogenesis via the tuning of mitochondrial translation and provides the first evidence for direct tissue-specific control of OXPHOS activity. This study opens a novel therapeutic avenue for the treatment of disorders associated with mitochondrial hypoactivity. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/607559v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1b8806forg.highwire.dtl.DTLVardef@1f81e3forg.highwire.dtl.DTLVardef@89d9e1org.highwire.dtl.DTLVardef@3f7a72_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Recurrent disruption of tumour suppressor genes in cancer by somatic mutations in cleavage and polyadenylation signals

The expression of eukaryotic genes relies on the precise 3-terminal cleavage and polyadenylation of newly synthesized pre-mRNA transcripts. Defects in these processes have been associated with various diseases, including cancer. While cancer-focused sequencing studies have identified numerous driver mutations in protein-coding sequences, noncoding drivers - particularly those affecting the cis-elements required for pre-mRNA cleavage and polyadenylation - have received less attention. Here, we systematically analysed cancer somatic mutations affecting 3UTR polyadenylation signals using the Pan-Cancer Analysis of Whole Genomes (PCAWG) dataset. We found a striking enrichment of cancer-specific somatic mutations that disrupt strong and evolutionarily conserved cleavage and polyadenylation signals within tumour suppressor genes. Further bioinformatics and experimental analyses conducted as a part of our study suggest that these mutations have a profound capacity to downregulate the expression of tumour suppressor genes. Thus, this work uncovers a novel class of noncoding somatic mutations with significant potential to drive cancer progression.

cancer biology↗

bioRxiv2024

A postnatal molecular switch drives the activity-dependent maturation of parvalbumin interneurons

Cortical neurons are specified during embryonic development but often only acquire their mature properties at relatively late stages of postnatal development. This delay in terminal differentiation is particularly prominent for fast-spiking parvalbumin-expressing (PV+) interneurons, which play critical roles in regulating the function of the cerebral cortex. We found that the maturation of PV+ interneurons is triggered by neuronal activity and mediated by the transcriptional cofactor peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1). Developmental loss of PGC-1 prevents PV+ interneurons from acquiring unique structural, electrophysiological, synaptic, and metabolic features and disrupts their diversification into distinct subtypes. PGC-1 exerts its function as a master regulator of the differentiation of PV+ interneurons by directly controlling gene expression through a transcriptional complex that includes ERR{gamma} and Mef2c. Our results uncover a molecular switch that translates neural activity into a specific transcriptional program promoting the maturation of PV+ interneurons at the appropriate developmental stage.

neuroscience↗

bioRxiv2023

Cadherins orchestrate specific patterns of perisomatic inhibition onto distinct pyramidal cell populations

In the mammalian neocortex, excitatory pyramidal cells are assembled into distinct subnetworks, which project to different brain areas. GABAergic interneurons were long thought to connect promiscuously and extensively to pyramidal neurons, but recent evidence supports the existence of a cell type-specific inhibitory connectome. How and when interneurons establish such a precise connectivity pattern among intermingled populations of excitatory neurons remains enigmatic. Here, we investigated the molecular rules shaping cell type- and input-specific inhibitory connectivity in different Layer 5 (L5) pyramidal cell populations. We found that neighboring L5 intra- (L5 IT) and extra-telencephalic (L5 ET) neurons receive different combinations of inhibitory perisomatic inputs from Parvalbumin- (PV+) and Cholecystokynine-positive (CCK+) basket cells. We also identified Cdh12 and Cdh13, two cadherin superfamily members, as critical mediators of L5 pyramidal cell type-specific inhibitory connectivity. Our data revealed a minimal overlap between L5 IT and L5 ET presynaptic inhibitory networks, and suggests that different populations PV+ basket cells innervate distinct L5 pyramidal cell types. Altogether, our work unravels the contribution of cadherins in shaping cortical interneuron wiring and provides new insights into the development of inhibitory microcircuits.

neuroscience↗

bioRxiv2023

Alternative splicing coupled to nonsense-mediated decay coordinates downregulation of non-neuronal genes in developing neurons

The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However, the systems-level effects of AS-NMD in this context are poorly understood. We developed an R package, factR2, which offers a comprehensive suite of AS-NMD analysis functions. Using this tool, we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovered hundreds of AS-NMD events with significant potential to regulate gene expression. Notably, this regulation was significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation was detected for alternative cassette exons stimulating NMD (NS-CEs) upon their inclusion into mature mRNA. By combining bioinformatics analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NS-CEs regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provide evidence that the NS-CE activity is temporally coordinated with NMD-independent gene repression mechanisms. Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes.

molecular biology↗

bioRxiv2023

Regulation of parvalbumin interneuron plasticity by neuropeptide-encoding genes

Neuronal activity is regulated in a narrow permissive band for the proper operation of neural networks. Changes in synaptic connectivity and network activity, for example, during learning, might disturb this balance, eliciting compensatory mechanisms to maintain network function. In the neocortex, excitatory pyramidal cells and inhibitory interneurons exhibit robust forms of stabilising plasticity. However, while neuronal plasticity has been thoroughly studied in pyramidal cells, little is known about how interneurons adapt to persistent changes in their activity. Here we uncover the critical cellular and molecular mechanisms through which cortical parvalbumin-expressing (PV+) interneurons adapt to changes in their activity levels. We found that changes in the activity of PV+ interneurons drive cell-autonomous, bi-directional compensatory adjustments of the number and strength of inhibitory synapses received by these cells, specifically from other PV+ interneurons. High-throughput profiling of ribosome-associated mRNA revealed that increasing the activity of PV+ interneurons leads to the cell-autonomous upregulation of two genes encoding multiple secreted neuropeptides, Vgf and Scg2. Functional experiments demonstrated that VGF is critically required for the activity-dependent scaling of inhibitory PV+ synapses onto PV+ interneurons. Our findings reveal an instructive role for neuropeptide-encoding genes in regulating synaptic connections among PV+ interneurons in the adult mouse neocortex.

neuroscience↗

bioRxiv2021

Seroprevalence and associated risk factors of Toxoplasma gondii infections among pregnant women in Makassar, Indonesia

The protozoan parasite, Toxoplasma gondii is estimated to infect one-third of the worlds population. Infection in pregnant women can cause severe conditions for their babies. Until now, there is no data regarding Toxoplasma infection from Makassar pregnant mothers. This study aims to obtain information on Toxoplasma specific antibodies and to measure the risk factor associate with parasite infection. This cross-sectional study conducted in 9 of 47 primary health centres (Puskesmas) in Makassar. Blood samples and questionnaires were collected from 184 pregnant women aged 15-42 years old from September to October 2020. ELISA technique was used to examine the IgG and IgM antibodies. Univariable and multivariable analyses were carried out to measure factors that independently associate with Toxoplasma antibody positivity. Our result showed the range of Toxoplasma IgM and IgG are 0.06-1.01 and 0.09-3.01, respectively. While no one of our participants has an acute Toxoplasma gondii infection (IgM positive), we found 32,6% pregnant mothers are exposed to parasite (positive IgG). Contact with cats [OR(95CI): 8.04 (3.09-20.87)], consume chicken satay [OR(95CI): 7.46(3.06-18.21)] and consume un-boiled water [OR(95CI): 5.23(1.57-17.38)] are independently associate with positive Toxoplasma IgG antibody. Based on the result, we conclude that pregnant women in Makassar are exposed to T. gondii and the cyst of parasite contaminates food and water in Makassar.

microbiology↗

bioRxiv2020

Cytoplasmic pool of spliceosome protein SNRNP70 regulates the axonal transcriptome and development of motor connectivity

Regulation of pre-mRNA splicing and polyadenylation plays a profound role in neurons by diversifying the proteome and modulating gene expression during development and in response to physiological cues. Although most pre-mRNA processing reactions are thought to occur in the nucleus, numerous splicing regulators are also found in neurites. Here, we show that U1-70K/SNRNP70, a major spliceosomal component, localizes in RNA-associated granules in axons. We identify the cytoplasmic pool of SNRNP70 as an important local regulator of motor axonal growth, nerve-dependent acetylcholine receptor (AChR) clustering and neuromuscular synaptogenesis. This cytoplasmic pool has a protective role for a limited number of axonal transcripts preventing them from degradation. Moreover, non-nuclear SNRNP70 is able to locally regulates splice variants of transcripts such as agrin, thereby locally controlling formation of synapses. Our results point to an unexpected, yet essential, function of local SNRNP70 in axonal development and indicate a role of splicing factors in local RNA metabolism during establishment and maintenance of neuronal connectivity.

neuroscience↗

bioRxiv2020

A conserved role for SFPQ in repression of pathogenic cryptic last exons

The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including ALS, FTLD, and Alzheimers Disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These CLEs appear preferentially in long introns of genes with neuronal functions and dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a new gene regulation mechanism with wide relevance to human pathologies.

neuroscience↗