Layden Michael J, Johnston Hereroa, Amiel Aldine R, Havrilak Jamie, Steinworth Bailey, Chock Taylor, Röttinger Eric, Martindale Mark Q
Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA.
Université Nice Sophia Antipolis UMR 7284, CNRS UMR 7284, INSERM U1081, Institute for Research on Cancer and Aging, Nice, France.
BMC Biol. 2016 Aug 1;14:61. doi: 10.1186/s12915-016-0282-1.
The nerve net of Nematostella is generated using a conserved cascade of neurogenic transcription factors. For example, NvashA, a homolog of the achaete-scute family of basic helix-loop-helix transcription factors, is necessary and sufficient to specify a subset of embryonic neurons. However, positive regulators required for the expression of neurogenic transcription factors remain poorly understood.
We show that treatment with the MEK/MAPK inhibitor U0126 severely reduces the expression of known neurogenic genes, Nvath-like, NvsoxB(2), and NvashA, and known markers of differentiated neurons, suggesting that MAPK signaling is necessary for neural development. Interestingly, ectopic NvashA fails to rescue the expression of neural markers in U0126-treated animals. Double fluorescence in situ hybridization and transgenic analysis confirmed that NvashA targets represent both unique and overlapping populations of neurons. Finally, we used a genome-wide microarray to identify additional patterning genes downstream of MAPK that might contribute to neurogenesis. We identified 18 likely neural transcription factors, and surprisingly identified ~40 signaling genes and transcription factors that are expressed in either the aboral domain or animal pole that gives rise to the endomesoderm at late blastula stages.
Together, our data suggest that MAPK is a key early regulator of neurogenesis, and that it is likely required at multiple steps. Initially, MAPK promotes neurogenesis by positively regulating expression of NvsoxB(2), Nvath-like, and NvashA. However, we also found that MAPK is necessary for the activity of the neurogenic transcription factor NvashA. Our forward molecular approach provided insight about the mechanisms of embryonic neurogenesis. For instance, NvashA suppression of Nvath-like suggests that inhibition of progenitor identity is an active process in newly born neurons, and we show that downstream targets of NvashA reflect multiple neural subtypes rather than a uniform neural fate. Lastly, analysis of the MAPK targets in the early embryo suggests that MAPK signaling is critical not only to neurogenesis, but also endomesoderm formation and aboral patterning.
星状海葵的神经网是通过保守的神经源性转录因子级联反应产生的。例如，NvashA是碱性螺旋-环-螺旋转录因子achaete-scute家族的同源物，对于指定胚胎神经元的一个子集是必需且充分的。然而，神经源性转录因子表达所需的正向调节因子仍知之甚少。
我们发现，用MEK/MAPK抑制剂U0126处理会严重降低已知神经源性基因Nvath-like、NvsoxB(2)和NvashA以及分化神经元的已知标志物的表达，这表明MAPK信号传导对于神经发育是必需的。有趣的是，异位表达的NvashA无法挽救U0126处理动物中神经标志物的表达。双荧光原位杂交和转基因分析证实，NvashA的靶标代表神经元的独特和重叠群体。最后，我们使用全基因组微阵列来鉴定MAPK下游可能有助于神经发生的其他模式形成基因。我们鉴定出18个可能的神经转录因子，并且令人惊讶地鉴定出约40个在反口区域或动物极表达的信号基因和转录因子，这些区域在囊胚晚期产生内胚层。
总之，我们的数据表明MAPK是神经发生的关键早期调节因子，并且可能在多个步骤中发挥作用。最初，MAPK通过正向调节NvsoxB(2)、Nvath-like和NvashA的表达来促进神经发生。然而，我们还发现MAPK对于神经源性转录因子NvashA的活性是必需的。我们的正向分子方法为胚胎神经发生的机制提供了见解。例如，NvashA对Nvath-like的抑制表明，抑制祖细胞身份在新生神经元中是一个活跃的过程，并且我们表明NvashA的下游靶标反映了多种神经亚型，而不是统一的神经命运。最后，对早期胚胎中MAPK靶标的分析表明，MAPK信号传导不仅对神经发生至关重要，而且对内胚层形成和反口模式形成也至关重要。
