LMX1B missense-perturbation of regulatory element footprints disrupts serotonergic forebrain axon arborization
Pathogenic coding mutations are prevalent in human neuronal transcription factors (TFs) but how they disrupt development is poorly understood. Lmx1b is a master transcriptional regulator of postmitotic Pet1 neurons that give rise to mature serotonin (5-HT) neurons; over two hundred pathogenic heterozygous mutations have been discovered in human LMX1B, yet their impact on brain development has not been investigated. Here, we developed mouse models with different LMX1B DNA-binding missense mutations. Missense heterozygosity broadly altered Pet1 neuron transcriptomes, but expression changes converged on axon and synapse genes. Missense heterozygosity effected highly specific deficits in the postnatal maturation of forebrain serotonin axon arbors, primarily in the hippocampus and motor cortex, which was associated with spatial memory defects. Digital genomic footprinting (DGF) revealed that missense heterozygosity caused complete loss of Lmx1b motif protection and chromatin accessibility at sites enriched for a distal active enhancer/active promoter histone signature and homeodomain binding motifs; at other bound Lmx1b motifs, varying levels of losses, gains or no change in motif binding and accessibility were found. The spectrum of footprint changes was strongly associated with synapse and axon genes. Further, Lmx1b missense heterozygosity caused wide disruption of Lmx1b-dependent GRNs comprising diverse TFs expressed in Pet1 neurons. These findings reveal an unanticipated continuum of Lmx1b missense-forced perturbations on Pet1 neuron regulatory element TF binding and accessibility. Our work illustrates the power of DGF for gaining unique insight into how TF missense mutations interfere with developing neuronal GRNs. Significance StatementWe modeled human LMX1B missense mutations in mice to explore how they disrupt brain serotonin neuron development. Missense heterozygosity selectively impaired postnatal formation of serotonin axon arbors throughout the forebrain, notably in the hippocampus and motor cortex. DGF revealed that Lmx1b missense heterozygosity exerted a continuum of footprint changes associated with synapse and axon gene expression. Footprint changes ranged from total eliminations to partial losses and gains within the Pet1 neuronal epigenome. LMX1B missense mutations may cause human brain pathogenesis by selectively disrupting cis regulatory elements controlling 5-HT axon arbor formation thus impairing 5-HT delivery to presynaptic release sites.