Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced ∼35% in forebrain, they were increased 40 -: 75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5 -: 15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of β-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders.

Sleep is an essential behavioral state of rest that is regulated by homeostatic drives to ensure a balance of sleep and activity, as well as independent arousal mechanisms in the central brain. Dopamine has been identified as a critical regulator of both sleep behavior and arousal. Here, we present results of a genetic screen that selectively restored the Dopamine Receptor (DopR/DopR1/dumb) to specific neuroanatomical regions of the adult Drosophila brain to assess requirements for DopR in sleep behavior. We have identified subsets of the mushroom body that utilizes DopR in daytime sleep regulation. These data are supported by multiple examples of spatially restricted genetic rescue data in discrete circuits of the mushroom body, as well as immunohistochemistry that corroborates the localization of DopR protein within mushroom body circuits. Independent loss of function data using an inducible RNAi construct in the same specific circuits also supports a requirement for DopR in daytime sleep. Additional circuit activation of discrete DopR+ mushroom body neurons also suggests roles for these subpopulations in sleep behavior. These conclusions support a new separable function for DopR in daytime sleep regulation within the mushroom body. This daytime regulation is independent of the known role of DopR in nighttime sleep, which is regulated within the Fan-Shaped Body (FSB). This study provides new neuroanatomical loci for exploration of dopaminergic sleep functions in Drosophila, and expands our understanding of sleep regulation during the day vs. night.