A fundamental challenge in neuroscience and regenerative medicine is to understand how specific neuron subtypes are generated in appropriate numbers, at defined times, and targeted to precise locations. Programs enacted to coordinate these events have been defined and advances made through studies of early-stage spinal neural tube. Less clear is how such events are regulated in germinal zones distributing neuron cohorts long distances through tangential migration modes and which typically do so at later developmental stages. Yet such germinal zones contribute substantially to the diversity of neurons in the mature brain, and likely do so in novel ways given their unique properties and the distinct milieu of late-gestation neural tissue. We are addressing key aspects of this problem through studies of the lower rhombic lip (LRL) - an essential germinal zone in the dorsal hindbrain that, through our recent work and that of others, has become accessible via molecular genetic means. Building on transplantation results in avian systems, our studies in the mouse show that the LRL gives rise to a diverse set of brainstem cell types including the major afferent systems for the cerebellum, numerous auditory brainstem neuron subtypes, and choroid plexus epithelial cells. Through application of genetic fate mapping techniques, we have not only proven spatial and molecular origin for this array of cell types, but have begun to discern that this diversity in mature fates is reflected in a precisely defined molecular subregionalization of the LRL: Gene expression microdomains predictive for cell fate distribute along the DV and AP axes of the LRL. Finer resolution mapping, with evermore restricted LRL genes/drivers will now help clarify how much can be explained by originating position versus subsequent morphogenetic processes. Another critical next step is to delineate how this LRL subregionalization is regulated. Our data identify the transcription factor Pax6 as one such regulator. While acting earlier to influence subtype identity in the ventral hindbrain, our findings reveal a later dorsal role for Pax6 in the LRL. Surprisingly, this later role may involve potentiating bone morphogenetic protein (BMP) signaling. Such co-opting dorsally of a previously excluded cell fate regulator, like Pax6, may represent a general strategy by which a germinal zone that remains active late in embryogenesis is able to produce unique progenitor cell states in order to continue generating unique cell fates. Extending this view, we have localized Sonic hedgehog (Shh), the archetypal “ventralizing” morphogen, to choroid plexus epithelium (CPe), the late-acting dorsal organizing center of the hindbrain which itself is a LRL derivative. Because of its resident proximity, CPe-produced Shh may influence (directly or indirectly) the cellular output and/or molecular organization of the LRL; it may also have the capacity to influence choroid plexus development itself, including morphogenesis of its extensive, specialized vasculature. Our present goals are to extend our molecular fate map of the murine LRL in the form of a gene expression grid in which we link highly resolved DV:AP coordinate positions within this molecular and spatial grid to later-generated cell fates and to place on this map the action of molecules which regulate this molecular grid and thus likely also the production of specific cell types.
