Neuraxial patterning is a continuous process that extends over a protracted period of development. During gastrulation a crude anteroposterior pattern, detectable by molecular markers, is conferred on the neuroectoderm by signals from the endomesoderm that are largely inseparable from those of neural induction itself. This coarse-grained pattern is subsequently reinforced and refined by diverse, locally acting mechanisms. Segmentation and long-range signaling from organizing centers are prominent among the emerging principles governing regional pattern.

Identification of specific neuronal populations and their projections in the developing hindbrain reveals a segmental organization in which pairs of metameric epithelial units cooperate to generate the repeating sequence of cranial branchiomotor nerves. Neurogenesis also follows a two-segment repeat, suggesting parallels with insect pattern formation.

In the chick embryo hindbrain, morphological segmentation into rhombomeres is matched by metameric patterns of early neuronal differentiation and axonogenesis. Boundaries between rhombomeres coincide with boundaries of expression of murine regulatory genes. By clonal analysis using intracellular marking, we show here that the rhombomere boundaries are partitions across which cells do not move. When a parent cell is marked before the appearance of rhombomere boundaries, the resulting clone is able to spread into the neighbouring rhombomere. When marked after boundary appearance, the clone still expands freely within the rhombomere of origin, but it is now restricted at the boundaries. Rhombomeres in the chick embryo thus behave like polyclonal units, raising the possibility that they are analogous to the compartments of insects.

Fifteen years ago, cell lineage restriction boundaries were discovered in the embryonic vertebrate hindbrain, subdividing it into a series of cell-tight compartments (known as rhombomeres). Compartition, together with segmentally reiterative neuronal architecture and the nested expression of Hox genes, indicates that the hindbrain has a truly metameric organization. This finding initiated a search for compartments in other regions of the developing brain. The results of recent studies have clarified where compartment boundaries exist, have shed light on molecular mechanisms that underlie their formation and have revealed an important function of these boundaries: the positioning and stabilization of local signalling centres.

Segmentation of the vertebrate hindbrain into rhombomeres is important for the anterior-posterior arrangement of cranial motor nuclei and efferent nerves. Underlying this reiterated organization, Hox genes display segmentally restricted domains of expression, such as expression of Hoxb-1 (refs 5, 6) in rhombomere 4 (r4). Here we report that absence of Hoxb-1 leads to changes in r4 identity. In mutant mouse embryos, molecular markers indicate that patterning of r4 is initiated properly but not maintained. Cellular analysis by DiI tracing reveals that the r4-specific facial branchiomotor (FBM) and contralateral vestibuloacoustic efferent (CVA) neurons are incorrectly specified. In wild-type mice CVA neurons migrate from r4 into the contralateral side, and we found in lineage analysis that FBM neurons migrate from r4 into r5. In mutants, motor neurons differentiate but the CVA and FBM neurons fail to migrate into their proper positions. Instead, they form a motor nucleus which migrates atypically, and there is a subsequent loss of the facial motor nerve. These results demonstrate that, as a part of its role in maintaining rhombomere identity, Hoxb-1 is involved in controlling migratory properties of motor neurons in the hindbrain.

In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon guidance depends partly on the recognition of cell-surface and extracellular matrix cues derived from cells along the pathways. It has also been proposed that neuronal growth cones are guided by gradients of chemoattractant molecules emanating from their intermediate or final cellular targets. Although there is evidence that the axons of some peripheral neurons in vertebrates are guided by chemotropism and the directed growth of some central axons to their targets is consistent with such a mechanism, it remains to be determined whether chemotropism operates in the central nervous system. During development of the spinal cord, commissural axons are deflected towards a specialized set of midline neural epithelial cells, termed the floor plate, which could reflect guidance by substrate cues or by diffusible chemoattractant molecules. Here we provide evidence in support of chemotropic guidance by demonstrating that the rat floor-plate cells secrete a diffusible factor(s) that influences the pattern and orientation of commissural axon growth in vitro without affecting other embryonic spinal cord axons. These findings support the hypothesis that chemotropic mechanisms guide developing axons to their intermediate targets in the vertebrate CNS.

The pattern of skeletal structures and muscles in the branchial region of the head is profoundly influenced by the neural crest, whose cells arise at discrete segmental levels of the chick hindbrain: specifically, rhombomeres (r)1+2, r4 and r6, whereas r3 and r5 are crest-depleted. We have demonstrated that an interaction between even-numbered rhombomeres and r3/r5 effects this depletion of neural crest, resulting in the sculpting of discrete migratory streams of neural crest. This mechanism acts through increased expression of msx2 and the induction of apoptosis in dorsal cells of r3 and r5 (ref. 3) (Fig. 1A). Here we demonstrate that the signalling molecule Bmp4 is expressed in r3 and r5 and is dependent on the neighbouring rhombomeres. Addition of recombinant BMP4 protein to explant cultures of r3 or r5, which produce neural crest when isolated from their neighbouring rhombomeres, upregulates msx2 and reinstates apoptosis in the neural crest population.

The zona limitans intrathalamica (ZLI), a narrow compartment in the vertebrate forebrain that bisects the diencephalon transversely, expresses the secreted factor sonic hedgehog (Shh). Because genetic disruption of Shh in mouse causes severe early developmental defects, this strategy has not been useful in identifying a ZLI-specific role for this gene. To modulate Shh signaling in a spatiotemporally restricted manner, we carried out gain- and loss-of-function experiments in chick embryos using in ovo electroporation and found that Shh signaling is required for region-specific gene expression in thalamus and prethalamus, the major diencephalic brain areas flanking the ZLI. We further show that differential competence of thalamic and prethalamic primordia in responding to Shh signaling is regulated by the transcription factor Irx3. We show that, through the release of Shh, the ZLI functions as a local signaling center that regulates the acquisition of identity for these important diencephalic regions.