Abnormalities in genes that regulate early brain development are known risk factors for neurodevelopmental disorders. Engrailed-2 (En2) is a homeodomain transcription factor with established roles in cerebellar patterning. En2 is highly expressed in the developing mid-hindbrain region, and En2 knockout (KO) mice exhibit major deficits in mid-hindbrain structures. However, En2 is also expressed in forebrain regions including the hippocampus, but its function is unknown. Previous studies have shown that the hippocampus of En2-KO mice exhibits reductions in its volume and cell numbers due to aberrant neurogenesis. Aberrant neurogenesis is due, in part, to noncell autonomous effects, specifically, reductions of innervating norepinephrine fibers from the locus coeruleus. In this study, we investigate possible cell autonomous roles of En2 in hippocampal neurogenesis. We examine proliferation, survival, and differentiation using cultures of hippocampal neurospheres of P7 wild-type (WT) and En2-KO hippocampal neural progenitor cells (NPCs). At 7 days, En2-KO neurospheres were larger on average than WT spheres and exhibited 2.5-fold greater proliferation and 2-fold increase in apoptotic cells, similar to in vivo KO phenotype. Further, En2-KO cultures exhibited 40% less cells with neurite projections, suggesting decreased differentiation. Lastly, reestablishing En2 expression in En2-KO NPCs rescued excess proliferation. These results indicate that En2 functions in hippocampal NPCs by inhibiting proliferation and promoting survival and differentiation in a cell autonomous manner. More broadly, this study suggests that En2 impacts brain structure and function in diverse regions outside of the mid-hindbrain.

中文翻译：

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Engrailed-2 是培养的海马神经干细胞中神经发生的细胞自主调节剂

调节早期大脑发育的基因异常是神经发育障碍的已知危险因素。Engrailed-2 ( En2 ) 是同源域转录因子，在小脑模式中具有既定作用。En2在发育中的中后脑区域高度表达，并且En2敲除 (KO) 小鼠在中后脑结构中表现出主要缺陷。然而，En2也在包括海马在内的前脑区域中表达，但其功能尚不清楚。以往的研究表明，En2的海马-KO 小鼠由于异常的神经发生而表现出其体积和细胞数量的减少。异常的神经发生部分是由于非细胞自主效应，特别是来自蓝斑的神经支配去甲肾上腺素纤维的减少。在这项研究中，我们研究了En2在海马神经发生中可能的细胞自主作用。我们使用 P7 野生型 (WT) 和En2 -KO 海马神经祖细胞 (NPC) 的海马神经球培养物检查增殖、存活和分化。在第 7 天，En2 -KO 神经球平均比 WT 球大，并且表现出 2.5 倍的增殖和 2 倍的凋亡细胞增加，类似于体内 KO 表型。此外，En2-KO 培养物的神经突突起细胞减少了 40%，表明分化减少。最后，在En2 -KO NPC中重新建立En2表达挽救了过度增殖。这些结果表明，En2在海马 NPCs 中通过以细胞自主方式抑制增殖并促进存活和分化而发挥作用。更广泛地说，这项研究表明，En2会影响中后脑以外不同区域的大脑结构和功能。