Abstract
BackgroundThe homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, particularly in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis. Because of its roles in regulating cerebellar development and evidence of cerebellar pathology in autism spectrum disorder (ASD), we previously examined an ENGRAILED2 association and found evidence to support EN2 as a susceptibility gene, a finding replicated by several other investigators. However, its functions at the cell biological level remain undefined. In the mouse, En2 gene is expressed in granule neuron precursors (GNPs) just as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may modulate these developmental processes.MethodsTo define En2 functions, we examined proliferation, differentiation and signaling pathway activation in En2 knockout (KO) and wild-type (WT) GNPs in response to a variety of extracellular growth factors and following En2 cDNA overexpression in cell culture. In vivo analyses of cerebellar GNP proliferation as well as responses to insulin-like growth factor-1 (IGF1) treatment were also conducted.ResultsProliferation markers were increased in KO GNPs in vivo and in 24-h cultures, suggesting En2 normally serves to promote cell cycle exit. Significantly, IGF1 stimulated greater DNA synthesis in KO than WT cells in culture, a finding associated with markedly increased phospho-S6 kinase activation. Similarly, there was three-fold greater DNA synthesis in the KO cerebellum in response to IGF1 in vivo. On the other hand, KO GNPs exhibited reduced neurite outgrowth and differentiation. Conversely, En2 overexpression increased cell cycle exit and promoted neuronal differentiation.ConclusionsIn aggregate, our observations suggest that the ASD-associated gene En2 promotes GNP cell cycle exit and differentiation, and modulates IGF1 activity during postnatal cerebellar development. Thus, genetic/epigenetic alterations of EN2 expression may impact proliferation, differentiation and IGF1 signaling as possible mechanisms that may contribute to ASD pathogenesis.
Highlights
The homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis
En2 gene expression is increased in granule neuron precursors (GNP) as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may participate in these developmental processes [14,15,24]
Proliferation is enhanced in vivo in the absence of En2 Cerebellar GNPs increase expression of En2 postnatally in the inner external germinal layer (EGL), concurrent with their cell cycle exit and early differentiation; En2 may be an important regulator of these processes
Summary
The homeobox transcription factor Engrailed (En2) has been studied extensively in neurodevelopment, in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis. Because of its roles in regulating cerebellar morphogenesis and Purkinje neuron development, and evidence of cerebellar pathology in human disease [3,4,5,6], we previously examined ENGRAILED2’s association with human autism spectrum disorder (ASD) and found evidence to support EN2 as an ASD susceptibility gene. These results, initially observed in 167 families, were subsequently replicated in two additional data sets (518 families; P = 0.00000035), and six other groups have demonstrated EN2 association with ASD [7,8,9,10,11,12]. We explore the function of En2 during GNP development by comparing wild-type (WT) and knockout (KO) GNPs both in vivo and in culture, as well as by using En2 overexpression constructs
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