The broad goal of this research is to understand the cellular and molecular mechanisms by which brain nuclei of the appropriate size, shape and neuronal cell-type constituency are generated during development. We study this problem in the midbrain, a principal division of the vertebrate brain. During the period of neurogenesis, the chick ventral midbrain is transiently organized into regularly spaced, longitudinal zones distinguished by morphology and histochemistry. Our hypothesis is that these zones, called midbrain arcs, are a patterning mechanism by which neurons are allocated to different nuclear fates. The aims of our research are (1) to test a prediction of this hypothesis, that the midbrain arcs differ in their fiber-connections, and (2) to discover the molecular mechanisms by which arcs are generated during midbrain development. In the initial funding period, we established that the most medial arc gives rise to two nuclear structures, the oculomotor complex and the red nucleus. We also demonstrated that a complete set of midbrain arcs could be elicited by an ectopic source of the morphogen Sonic Hedgehog (SHH). In the proposed experiments we will extend our test of the arcs-as-pronuclei hypothesis to lateral midbrain, and we will address whether a SHH source patterns the arcs directly, as a long-range morphogen, or acts in part through bone morphogenetic protein (BMP) intermediaries. The first set of experiments studies the anatomy of the lateral midbrain arcs with anterograde and retrograde tract-tracing methods. A finding of that the lateral arcs have characteristic neuronal connections would not only support the pronuclear hypothesis, but could serve to identify particular arcs as specific pronuclei. The second set of experiments tests the competency of SHH to specify midbrain arc cell-types directly, as a long-range morphogen. These experiments will be carried out in wild-type and talpid(2) mutant chick embryos using in ovo electroporation and explant tissue culture methods. Our finding that ectopic SHH induces BMP gene expression raises, however, the possibility that BMP2 and BMP7 may participate in long-range signaling by a SHH source. The third set of experiments tests the contributions of BMP signaling to midbrain arc patterning.

The ventral midbrain is a frequent site of pathology in stroke and other neurological disorders. Understanding its developmental biology should facilitate research into therapeutics, including those based on tissue transplants. More generally, ventral midbrain development provides a model for understanding how brain nuclei form. Insight into these mechanisms may suggest novel approaches to neurological disorders due to nuclear ablations or disconnections.

R01NS035680

1998-08-01

2006-11-30