The Hatten Lab Projects


Specification of early precursors of granule neurons within the rhombic lip of the midbrain/hindbrain region.



The mechanisms underlying the specification of the diverse array of neurons in the CNS constitutes a second core area of our research. Insights from studies on the development of the body plan in invertebrate systems have suggested that a coordinate grid of positional information may control the specification of cell types in brain. To examine the specification of cerebellar granule cells, we have recently carried out studies on precursor cells isolated from an early embryonic structure, called the rhombic lip (see below), the source of granule cell progenitors. The model that we are currently testing is that the rhombic lip is a dorsal structure. By analogy to the spinal cord, the action of peptide growth factors, such as the BMPs would be anticipated to signal the dorsalization of cells within the rhombic lip, and thereby restrict their fate. In vitro studies with cells purified from the rhombic lip support the general conclusion that granule cell fate is set early, between E9-E13, as dorsalizing signals act. This general time frame is similar to that for other cerebellar cell classes, eg. Purkinje cells. However, unlike Purkinje cells, precursors of the granule cell continue proliferation well into the early postnatal period, after formation of a displaced germinal zone, the EGL.
Formation of the murine cerebellar anlage. During embryonic phases of the development of the cerebellar primordium, a gap in the dorsal neural tube combined with the bending of the pontine flexure results in the formation of a mouth-like structure at the IVth ventricle. Precursors of the granule neurons arise from rhombic lip (RL) at the posterior edge of the anlage (shaded dark gray), adjacent to the IVth ventricle (light gray). At E13-E14, cells of the RL begin to move (black arrows) over the surface of the anlage to form the EGL, also shown in cross section in the inset. The choroid plexus (CP) extends from the RL. The RL precursors undergo clonal expansion with the EGL until the early postnatal period, when differentiation begins. By contrast, precursors of the Purkinje cells arise in the ventricular zone (VZ; medium gray). These cells cease proliferation at E14 and begin to differentiate. Postmitotic Purkinje cell precursors migrate radially, up through the wall of the anlage (white arrows). A, anterior; D, dorsal; P,posterior; V, ventral.
Rhombic Lip Cells Can Differentiate into Mature Granule Cells when Implanted into the EGL of a Host P6 Cerebellum. (A and B) E14 rhombic lip cells were triturated to a single cell suspension and labeled with the lipophilic dye PKH-26 before being implanted into the EGL of P6 mice. 3-5 days later, the animals were sacrificed and their brains examined for the location and morphology of the labeled cells. The labeled cells migrated passed the Purkinje cell layer to the IGL. The ascending axon of the labeled neuron formed a "T-shape" characteristic of mature granule cells. Within the IGL, the cell extended several short dendritic processes. Bar = 50 µm.

Cells purified from the early postnatal EGL are restricted to a granule cell fate. When cells are isolated from the EGL of the early postnatal cerebellum and implanted into early postnatal EGL, all of the cells undergo the differentiation program of granule neurons. This suggests that granule cell identity is specified in the embryonic period, with signals that induce differentiation appearing in the late embyronic and early postnatal periods. Identification of the genes that control this intial step in differentiation constitute a major research effort in the lab.
Granule neurons, the most abundant class of CNS neurons, are generated within a discrete domain that comprises the dorsal border of the mesencephalon and metencephalon called the rhombic lip. In the mouse embryo, cells in the developing rhombic lip express a number of granule neuron markers, notably the bHLH transcription factor Math1. Dorsal midline cells adjacent to the incipient rhombic lip express Bmp6, Bmp7 and Gdf7, three genes that encode peptide growth factors of the TGFß superfamily. Each of these BMP family members can induce the expression of markers of the granule neuron lineage in neural cells in vitro. Moreover, BMP-treated ventral neural cells form mature granule neurons after transplantation into the early postnatal cerebellum, suggesting that BMPs initiate the program of granule cell specification in the dorsal mesencephalon / metencephalon (Alder et al, 1999).
Treatment of ventral cells with BMP and reimplementation into early postnatal cerebellar cortex leads to granule neurons.
The Unc51.1 serine-threonine kinase and the Discodin domain receptor I (DDRI) receptor kinase function in the early steps of granule cell differentiation.

In the early postnatal cerebellum, granule neuron outgrowth is a key step toward establishing connections with Purkinje neurons, the principal neuron in the region. During a search for genes that function in this process, we identified a a muirine homnolog of the C. elegans gene UNC51 and a receptor tyrosine kinase discoidin domain receptor I (DDRI) that are expressed in granule cells throughout their development. Overexpression of a dominant negative form of Unc51.1 or of DDRI in immature granule cells results in severe reduction of axon growth in primary culture, and in vivo, in organotypic slices of neonatal cerebellum. For the Unc51.1 gene, this happens before expression of any markers of differentiation yet after withdrawal from the cell cycle. For DDRI granule cells that fail to extend neurites are positive for differentiation markers such as TAG-1 and the neuron-specific class III b tubulin, suggesting that development is affected after granule cells commit to terminal differentiation. Our results therefore indicate that Unc51.1 is one of the earliest signals for axon extension and that collagen-DDRI signaling is essential for the maintenance of granule cell axon formation. Work is in progress to study animals with targeted deletions of these genes and BAC transgenic animals expressing these genes.
Hatten Lab Projects
Hatten Lab Home Page