Precise extrinsic afferent (visceral sensory) and efferent (sympathetic and parasympathetic) innervation of the gut is fundamental for gut-brain cross talk. Owing to the limitation of intrinsic markers to distinctively visualize the three classes of extrinsic axons, which intimately associate within the gut mesentery, detailed information on the development of extrinsic gut-innervating axons remains relatively sparse. Here, we mapped extrinsic innervation of the gut and explored the relationships among various types of extrinsic axons during embryonic development in mice. Visualization with characterized intrinsic markers revealed that visceral sensory, sympathetic, and parasympathetic axons arise from different anatomic locations, project in close association via the gut mesentery, and form distinctive innervation patterns within the gut from embryonic day (E)10.5 to E16.5. Genetic ablation of visceral sensory trajectories results in the erratic extension of both sympathetic and parasympathetic axons, implicating that afferent axons provide an axonal scaffold to route efferent axons. Coculture assay further confirmed the attractive effect of sensory axons on sympathetic axons. Taken together, our study provides key information regarding the development of extrinsic gut-innervating axons occurring through heterotypic axonal interactions and provides an anatomic basis to uncover neural circuit assembly in the gut-brain axis (GBA).

SIGNIFICANCE STATEMENT Understanding the development of extrinsic innervation of the gut is essential to unravel the bidirectional neural communication between the brain and the gut. Here, with characterized intrinsic markers targeting vagal sensory, spinal sensory, sympathetic, and parasympathetic axons, respectively, we comprehensively traced the spatiotemporal development of extrinsic axons to the gut during embryonic development in mice. Moreover, in line with the somatic nervous system, pretarget sorting via heterotypic axonal interactions is revealed to play critical roles in patterning extrinsic efferent trajectories to the gut. These findings provide basic anatomic information to explore the mechanisms underlying the process of assembling neural circuitry in the gut-brain axis (GBA).

精确的外来传入（内脏感觉）和传出（交感和副交感）神经支配是肠道与大脑相交的基础。由于内在标记物不能显着地可视化与肠系膜密切相关的三类外在轴突，因此关于内在神经的轴突发育的详细信息仍然相对稀疏。在这里，我们绘制了肠道的外在神经支配关系，并探索了小鼠胚胎发育过程中各种类型的外在轴突之间的关系。具有特征性内在标志物的可视化显示，内脏的感觉，交感和副交感神经轴突来自不同的解剖位置，并通过肠系膜紧密相关地投射，从胚胎天（E）10.5到E16.5，在肠道内形成独特的神经支配模式。内脏感觉轨迹的遗传消融导致交感神经轴突和副交感神经轴突的不稳定扩展，暗示传入轴突提供了轴突支架来路由传出轴突。共培养测定进一步证实了感觉轴突对交感轴突的吸引力。两者合计，我们的研究提供有关通过异型轴突相互作用发生的外在肠道神经轴突的发展的关键信息，并提供解剖基础，揭示肠道脑轴（GBA）的神经回路组装。暗示传入轴突提供了轴突支架来路由传出轴突。共培养测定进一步证实了感觉轴突对交感轴突的吸引力。两者合计，我们的研究提供有关通过异型轴突相互作用发生的外在肠道神经轴突的发展的关键信息，并提供解剖基础，揭示肠道脑轴（GBA）的神经回路组装。暗示传入轴突提供了轴突支架以路由传出轴突。共培养测定进一步证实了感觉轴突对交感轴突的吸引力。两者合计，我们的研究提供有关通过异型轴突相互作用发生的外在肠道神经轴突的发展的关键信息，并提供解剖基础，揭示肠道脑轴（GBA）的神经回路组装。

重要声明了解肠道内在神经支配的发展对于解开大脑与肠道之间的双向神经通讯至关重要。在这里，通过分别针对迷走神经感觉，脊髓感觉，交感神经和副交感神经轴突的特征性内在标记，我们全面追踪了小鼠胚胎发育过程中外在轴突到肠道的时空发育。此外，与躯体神经系统一致，通过异型轴突相互作用进行的预靶分选显示出在塑造肠道外源性传出轨迹的模式中起关键作用。这些发现提供了基本的解剖学信息，以探索在肠脑轴（GBA）上组装神经回路的基础机制。