Hox Paralog Group 2 Genes Control the Migration of Mouse Pontine Neurons through Slit-Robo Signaling

Abstract

The pontine neurons (PN) represent a major source of mossy fiber projections to the cerebellum. During mouse hindbrain development, PN migrate tangentially and sequentially along both the anteroposterior (AP) and dorsoventral (DV) axes. Unlike DV migration, which is controlled by the Netrin-1/Dcc attractive pathway, little is known about the molecular mechanisms guiding PN migration along the AP axis. Here, we show that Hoxa2 and Hoxb2 are required both intrinsically and extrinsically to maintain normal AP migration of subsets of PN, by preventing their premature ventral attraction towards the midline. Moreover, the migration defects observed in Hoxa2 and Hoxb2 mutant mice were phenocopied in compound Robo1;Robo2, Slit1;Slit2, and Robo2;Slit2 knockout animals, indicating that these guidance molecules act downstream of Hox genes to control PN migration. Indeed, using chromatin immunoprecipitation assays, we further demonstrated that Robo2 is a direct target of Hoxa2 in vivo and that maintenance of high Robo and Slit expression levels was impaired in Hoxa2 mutant mice. Lastly, the analysis of Phox2b-deficient mice indicated that the facial motor nucleus is a major Slit signaling source required to prevent premature ventral migration of PN. These findings provide novel insights into the molecular control of neuronal migration from transcription factor to regulation of guidance receptor and ligand expression. Specifically, they address the question of how exposure to multiple guidance cues along the AP and DV axes is regulated at the transcriptional level and in turn translated into stereotyped migratory responses during tangential migration of neurons in the developing mammalian brain. In the developing central nervous system, neurons migrate sometimes over long distances from their birthplace to their final location, where they condense in specific nuclei. The precise positioning of migrating neurons is critical to the building of ordered connectivity with their target partners. Little is known about how exposure of migrating neurons to simultaneous attractive and repulsive guidance cues may be integrated at the transcriptional level and in turn translated into directional migratory responses specific for each neuronal population. Here, we focus on the molecular mechanisms regulating the directionality of long-distance migration of pontine neurons in the mouse brainstem. Such neurons belong to the so-called precerebellar system, which is essential for coordinated motor activity, and provide the principal input to the cerebellum. We provide evidence for the implication of homeodomain transcription factors of the Hox gene family in the control of pontine neuron migration along the brain rostrocaudal axis. We identify the guidance receptor Robo2 as a direct target gene of the Hoxa2 gene. We further show that repulsive signaling mediated through the Robo2 receptor expressed in migrating neurons and its ligand Slit2 secreted from the facial motor nucleus are key components of the molecular guidance system that maintains caudorostral migration and prevents premature attraction towards the brainstem ventral midline. Our data provide a conceptual framework to understand how transcriptional regulation of the response to environmental guidance cues controls stereotyped neuronal migratory behavior in the developing mammalian brain.