The role of homeobox transcription factor NKX2.1 in nervous system development

Kennesaw State University received a $540K Project Grant from the National Institute of Neurological Disorders and Stroke (NINDS) under the Extramural Research Programs in the Neurosciences and Neurological Disorders program (CFDA 93.853), effective September 1, 2025, through August 31, 2028. The award funds basic neuroscience research investigating the role of the homeobox transcription factor NKX2.1 in nervous system development, with particular emphasis on understanding genetic mechanisms underlying Attention Deficit/Hyperactivity Disorder (ADHD) and Autism Spectrum Disorder (ASD). Using the nematode Caenorhabditis elegans as a model organism, the research examines transcription factor function and cell fate specification during neural development, leveraging the organism's invariant cell lineage and established genetic tools to advance fundamental knowledge of gene regulation defects implicated in these prevalent neurological conditions.

The research directly supports NINDS's mission to seek fundamental knowledge about the brain and nervous system to reduce the burden of neurological disease. By investigating how multiple ADHD- and ASD-associated genes interact within transcriptional cascades, this project addresses gaps in understanding the polygenic nature of these highly prevalent neurological disorders and may inform future therapeutic and diagnostic approaches for affected populations.

Abstract

Attention deficit/hyperactivity disorder (ADHD) affects almost 5% of children and persists into 2.5% of adults. There is a strong genetic component to ADHD. For example, heterozygosity in the homeobox transcription factor NKX2.1 causes a syndromic variant of ADHD, which is frequently associated with autism-like symptoms. In addition, mutations in RFX family transcription factors cause autosomal dominant ADHD with concomitant autism spectrum disorder symptoms in many cases. Expanding on this, recent genome-wide association studies and meta-analyses identified 27 ADHD-associated loci. Of these genes, one third were associated with gene transcription in some way, either as transcription factors, transcriptional CO-regulators, RNA splicing factors, transcription terminators, or histone modification enzymes. These data strongly implicate defects in gene regulation as underlying these highly prevalent neurological conditions, and point to their polygenic nature. This brings up a question; how do these multiple ADHD and ASD associated genes relate to each other and how does that impact the neurological disorder in question? Transcription factors frequently function in cascades, with one factor switching on the gene expression of another transcription factor, etc. To ultimately define the battery of genes expressed in a terminal fate cell. Many transcription factors, including NKX2.1, are deeply conserved across species. This allows US to examine transcription factor function in genetically tractable model organisms such as nematodes and flies. This proposal aims to take advantage of this, using the nematode caenorhabditis elegans to investigate NKX2.1 function during nervous system development. C. Elegans has an invariant cell lineage, making IT ideal for investigating questions of cell fate specification and function. In addition, there are powerful genetic tools available to aid investigation. Finally, C. Elegans is a transparent organism, so individual neurons can be highlighted using genetically encoded reporter genes such as green fluorescent protein. C. Elegans contains a single NKX2.1 ortholog, CEH-27. Our preliminary data reveals that CEH-27 null mutations are 100% embryonic lethal, showing that this gene is absolutely required for normal embryonic development. The proposed studies will 1) investigate CEH-27'S role in cell fate specification during nervous system development; 2) investigate its relationship with other proneural transcription factors such as NGN-1/neurogenin; and 3) use state-of-the-art single-cell RNA sequencing approaches to identify CEH-27 downstream targets. The high sequence conservation of CEH-27 across species means that our data can be used to investigate human orthologs of C. Elegans CEH-27/NKX2.1 targets, to better understand their roles in neurological disorders such ADHD and ASD. This project directly addresses fundamental mechanisms of nervous system development and gene regulation. In addition, the proposed experiments are technically straight forward and are comfortably within the grasp of undergraduate and master's students. As such, this project will accomplish both broad and specific area program goals, including enhancing kennesaw state university's research environment and exposing students to high quality research through direct participation. PHS 398/2590 (REV. 11/07) page 1 continuation format page