A multidisciplinary investigation into the function of NFXL1
Human speech and language, unique to our species, are essential for communication, cognitive development, and societal function. Disorders affecting these abilities are associated with learning disabilities, reduced academic achievement, and social challenges. Despite their significance, the molecular genetic basis of these disorders remains poorly understood. NFXL1, identified as a candidate gene for Developmental Language Disorder (DLD) in a Chilean population, is predicted to be a transcriptional repressor that is highly expressed in the cerebellum and conserved across evolution to Drosophila melanogaster. However, its cellular function and downstream targets remain elusive. This thesis investigates NFXL1 function using Drosophila, human and mouse models to uncover its role in neurodevelopment. Evolutionary analyses revealed conserved functional domains, including the RING finger and NFX1-type zinc finger motifs, supporting its role in transcriptional regulation and protein stability. Immunohistochemistry showed that CG15011 (the Drosophila NFXL1 ortholog) is expressed maternally and in the salivary glands of late-stage embryos and third instar larvae. Expression was also localised throughout the central nervous system (CNS) of third instar larvae, specifically in the cortex, astrocyte-like, and ensheathing glial cells. Its nuclear and cytoplasmic distribution within these cells suggests diverse roles in nervous system development. Functional studies demonstrated that maternal knockdown of CG15011 reduces egg hatch rate and fertility, indicating a key developmental role. Glial-specific CRISPR-mediated knockout revealed its importance for female survival, lifespan, and larval locomotion. Genome-wide profiling using an existing ChIP-seq dataset in embryos and NanoDam in larval glial cells identified CG15011-binding sites enriched for cytoskeletal organisation and neuronal projection morphogenesis. Weighted gene co-expression network analyses (WGCNA) in human, mouse, and Drosophila datasets revealed conserved roles in transcriptional regulation, RNA processing, and protein modification. Cell type-specific roles were observed in mice, with Nfxl1 implicated in mitochondrial regulation in mouse Purkinje cells and chromatin organisation in granule cells. The integration of NanoDam and WGCNA datasets identified 26 conserved candidate genes, linking CG15011/NFXL1 to RNA splicing, cytoskeletal regulation, and neurodevelopmental pathways. Together, these findings establish a conserved role for CG15011/NFXL1 in regulating neural and glial development across species, with implications for understanding neurodevelopmental disorders, including those affecting speech and language. Future studies could validate candidate target genes, explore sex-specific effects, and investigate mechanisms of transcriptional regulation and nuclear-cytoplasmic shuttling.
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