In the developing hindbrain, the functional loss of individual Hox genes has revealed some of their roles in specifying rhombomere (r) identity. However, it is unclear how Hox genes act in concert to confer the unique identity to multiple rhombomeres. Moreover, it remains to be elucidated how these genes interact with other transcriptional programs to specify distinct neuronal lineages within each rhombomere. We demonstrate that in r5, the combined mutation of Hoxa3 and Hoxb3 result in a loss of Pax6- and Olig2-expressing progenitors that give rise to somatic motoneurons of the abducens nucleus. In r6, the absence of any combination of the Hox3 paralogous genes results in ectopic expression of the r4-specific determinant Hoxb1. This ectopic expression in turn results in the differentiation of r4-like facial branchiomotoneurons within this rhombomere. These studies reveal that members of the Hox1 and Hox3 paralogous groups participate in a 'Hox code' that is necessary for coordinating both suppression and activation mechanisms that ensure distinction between the multiple rhombomeres in the developing hindbrain.

Planar cell polarity (PCP) describes the polarization of cell structures and behaviors within the plane of a tissue. PCP is essential for the generation of tissue architecture during embryogenesis and for postnatal growth and tissue repair, yet how it is oriented to coordinate cell polarity remains poorly understood [1]. In Drosophila, PCP is mediated via the Frizzled-Flamingo (Fz-PCP) and Dachsous-Fat (Fat-PCP) pathways [1-3]. Fz-PCP is conserved in vertebrates, but an understanding in vertebrates of whether and how Fat-PCP polarizes cells, and its relationship to Fz-PCP signaling, is lacking. Mutations in human FAT4 and DCHS1, key components of Fat-PCP signaling, cause Van Maldergem syndrome, characterized by severe neuronal abnormalities indicative of altered neuronal migration [4]. Here, we investigate the role and mechanisms of Fat-PCP during neuronal migration using the murine facial branchiomotor (FBM) neurons as a model. We find that Fat4 and Dchs1 are expressed in complementary gradients and are required for the collective tangential migration of FBM neurons and for their PCP. Fat4 and Dchs1 are required intrinsically within the FBM neurons and extrinsically within the neuroepithelium. Remarkably, Fat-PCP and Fz-PCP regulate FBM neuron migration along orthogonal axes. Disruption of the Dchs1 gradients by mosaic inactivation of Dchs1 alters FBM neuron polarity and migration. This study implies that PCP in vertebrates can be regulated via gradients of Fat4 and Dchs1 expression, which establish intracellular polarity across FBM cells during their migration. Our results also identify Fat-PCP as a novel neuronal guidance system and reveal that Fat-PCP and Fz-PCP can act along orthogonal axes.

The vertebrate cranial neural crest cells give rise to many complex derivatives of the head, neck, and face, including neuronal and glial cells that act in concert for proper development of the anterior-peripheral nervous system. Several genes have been implicated in the processes of neural crest specification, migration, and differentiation; among these are the hox gene clusters. To determine the fates of hox-expressing cranial neural crest, we describe the results of a genetic lineage analysis by using the Cre/loxP system to drive the activation of different ROSA26 reporter alleles under the regulation of the hoxb1 locus. By targeting the 3 untranslated region of the hoxb1 gene, we have preserved endogenous gene activity and have been able to accurately follow the fates of the cells derived from the hoxb1 expression domain. Emphasis was placed on identifying the cell and tissue types that arise from the rhombomere 4-derived neural crest. Our results demonstrate that, in addition to forming much of the cartilage, bones, and muscle of the ears and neck, a significant population of rhombomere 4-derived neural crest is fated to generate the glial component of the seventh cranial nerve.

Formation of neuronal circuits in the head requires the coordinated development of neurons within the central nervous system (CNS) and neural crest-derived peripheral target tissues. Hoxb1, which is expressed throughout rhombomere 4 (r4), has been shown to be required for the specification of facial branchiomotor neuron progenitors that are programmed to innervate the muscles of facial expression. In this study, we have uncovered additional roles for Hoxb1-expressing cells in the formation and maintenance of the VIIth cranial nerve circuitry. By conditionally deleting the Hoxb1 locus in neural crest, we demonstrate that Hoxb1 is also required in r4-derived neural crest to facilitate and maintain formation of the VIIth nerve circuitry. Genetic lineage analysis revealed that a significant population of r4-derived neural crest is fated to generate glia that myelinate the VIIth cranial nerve. Neural crest cultures show that the absence of Hoxb1 function does not appear to affect overall glial progenitor specification, suggesting that a later glial function is critical for maintenance of the VIIth nerve. Taken together, these results suggest that the molecular program governing the development and maintenance of the VIIth cranial nerve is dependent upon Hoxb1, both in the neural crest-derived glia and in the facial branchiomotor neurons.

The parasympathetic reflex circuit is controlled by three basic neurons. In the vertebrate head, the sensory, and pre-and postganglionic neurons that comprise each circuit have stereotypic positions along the anteroposterior (AP) axis, suggesting that the circuit arises from a common developmental plan. Here, we show that precursors of the VIIth circuit are initially aligned along the AP axis, where the placodederived sensory neurons provide a critical ''guidepost'' through which preganglionic axons and their neural crest-derived postganglionic targets navigate before reaching their distant target sites. In the absence of the placodal sensory ganglion, preganglionic axons terminate and the neural crest fated for postganglionic neurons undergo apoptosis at the site normally occupied by the placodal sensory ganglion. The stereotypic organization of the parasympathetic cranial sensory-motor circuit thus emerges from the initial alignment of its precursors along the AP axis, with the placodal sensory ganglion coordinating the formation of the motor pathway.

Neurogenesis during development depends on the coordinated regulation of self-renewal and differentiation of neural precursor cells. Chromatin regulation is a key step in self-renewal activity and fate decision of neural precursor cells. However, the molecular mechanism(s) of this regulation is not fully understood. Here, we demonstrate for the first time that MRG15, a chromatin regulator, is important for proliferation and neural fate decision of neural precursor cells. Neuroepithelia from Mrg15 deficient embryonic brain are much thinner than those from control, and apoptotic cells increase in this region. We isolated neural precursor cells from Mrg15 deficient and wild-type embryonic whole brains and produced neurospheres to measure the self-renewal and differentiation abilities of these cells in vitro. Neurospheres culture from Mrg15 deficient embryo grew less-efficiently than those from wild-type. Measurement of proliferation, using BrdU incorporation, revealed that Mrg15 deficient neural precursor cells have reduced proliferation ability and apoptotic cells do not increase during in vitro culture. The reduced proliferation of Mrg15 deficient neural precursor cells most likely accounts for the thinner neuroepithelia in Mrg15 deficient embryonic brain. Moreover, we also demonstrate Mrg15 deficient neural precursor cells are defective in differentiation into neurons in vitro. Our results demonstrate that MRG15 has more than one function in neurogenesis and defines a novel role for this chromatin regulator that integrates proliferation and cell-fate determination in neurogenesis during development.

The perception of environmental stimuli is mediated through a diverse group of first-order sensory relay interneurons located in stereotypic positions along the dorsoventral (DV) axis of the neural tube. These interneurons form contiguous columns along the anteroposterior (AP) axis. Like neural crest cells and motoneurons, first-order sensory relay interneurons also require specification along the AP axis. Hox genes are prime candidates for providing this information. In support of this hypothesis, we show that distinct combinations of Hox genes in rhombomeres (r) 4 and 5 of the hindbrain are required for the generation of precursors for visceral sensory interneurons. As Hoxa2 is the only Hox gene expressed in the anterior hindbrain(r2), disruption of this gene allowed us to also demonstrate that the precursors for somatic sensory interneurons are under the control of Hox genes. Surprisingly, the Hox genes examined are not required for the generation of proprioceptive sensory interneurons. Furthermore, the persistence of some normal rhombomere characteristics in Hox mutant embryos suggests that the loss of visceral and somatic sensory interneurons cannot be explained solely by changes in rhombomere identity. Hox genes may thus directly regulate the specification of distinct first-order sensory relay interneurons within individual rhombomeres. More generally, these findings contribute to our understanding of how Hox genes specifically control cellular diversity in the developing organism

The vestibular nuclear complex (VNC) consists of a collection of sensory relay nuclei that integrates and relays information essential for coordination of eye movements, balance, and posture. Spanning the majority of the hindbrain alar plate, the rhombomere (r) origin and projection pattern of the VNC have been characterized in descriptive works using neuroanatomical tracing. However, neither the molecular identity nor developmental regulation of individual nucleus of the VNC has been determined. To begin to address this issue, we found that Hoxb1 is required for the anterior-posterior (AP) identity of precursors that contribute to the lateral vestibular nucleus (LVN). Using a gene-targeted Hoxb1-GFP reporter in the mouse, we show that the LVN precursors originate exclusively from r4 and project to the spinal cord in the stereotypic pattern of the lateral vestibulospinal tract that provides input into spinal motoneurons driving extensor muscles of the limb. The r4-derived LVN precursors express the transcription factors Phox2a and Lbx1, and the glutamatergic marker Vglut2, which together defines them as dB2 neurons. Loss of Hoxb1 function does not alter the glutamatergic phenotype of dB2 neurons, but alters their stereotyped spinal cord projection. Moreover, at the expense of Phox2a, the glutamatergic determinants Lmx1b and Tlx3 were ectopically expressed by dB2 neurons. Our study suggests that the Hox genes determine the AP identity and diversity of vestibular precursors, including their output target, by coordinating the expression of neurotransmitter determinant and target selection properties along the AP axis.