The Hatten Lab Projects


Development of the Cerebellum - Granule Cell Specification and Proliferation Pathways




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.


Within the rhombic lip, granule cell precursors are specified by locally acting peptides that are members of the TGFb family, BMP6, BMP7 and GDF7. Experimental analysis of the role of BMPs in granule cell specification provide evidence for a critical role for BMPs. (Alder et al, 1999).

Expression studies show that Dorsal midline cells adjacent to the incipient rhombic lip express Bmp6, Bmp7 and Gdf7, three genes that encode peptide growth factors of the TGFb 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).


Generating Granule Neurons from Stem Cells
Work is now ongoing to direct the differentiation of ES cells toward a granule cell fate by following the pathways discovered for normal granule cell specification. Preliminary evidence indicates that this strategy provides cells that are EN1/Math1+ and which, upon implantation into early postnatal cerebellum, give rise to granule neurons.

Cloning of the Dreher Gene: Lmx1a is Required for Roof Plate Formation
As a genetic approach to understanding early cerebellar development, we cloned the gene affected in the naturally occurring neurological mutant mouse dreher, showing that it encoded Lmx1a, a gene required for roof plate formation (Millonig et al, 2000). Dreher was of special interest, because dorsal cell classes along the entire neuroaxis had developmental defects. This suggested that dreher was in a signaling pathway that controlled dorsal patterning. In the dreher spinal cord, shown the drawing below and in panel b, the roof plate fails to form. In the absence of the roof plate, dorsal cell populations that express Math1 spread across the top of the spinal cord (panel e) and fail to project axons along their normal pathways (g) (wild type, panels b, d, f, for details see Millonig et al, 2000.


The absence of the roof plate in dreher embryos is illustrated below. On the left, a wild type embryo is shown. On the right, Dreher lacks a roof plate as shown by MAF staining.

In more recent studies we have shown that Lmx1a is first expressed at E8.5 in a small number of cells in the lateral neural plate. As the neural tube closes, Lmx1a expression is restricted to the roof plate. In drJ/drJ, although Lmx1a is expressed at E8.5-E9.5, expression is lost in the roof plate by E10.5. Coincident with the loss of Lmx1a expression, Bmp expression fails, and the generation and differentiation of the dorsal D1 interneurons is abnormal. In drJ/drJ embryos, defects are evident in the number of D1 progenitors, as well as in their migration to form the lateral and medial nuclei, and axon patterning, through mechanisms that apparently involve defects in early steps of neuronal polarity. Consistent with the general hypothesis that a failure of roof plate formation and function results in deficits in dorsal patterning of the neural tube, the dreher decifit is restricted to the D1 interneuron population,his was followed by a detailed study of the expression of Lmx1a and the early developmental defects in the dreher spinal cord (Millen et al, 2004, Develop Biol, in press).

Transcription Factor Expression in the Embryonic Cerebellar Anlage
To define the molecular mechanisms that specify the three primary classes of neurons in the emerging cerebellar anlagen - the cells of the deep nuclei, the EGL and the Purkinje cells, we are examining expression patterns of transcription factors, including LIM homeodomain (Lhx), basic helix-loop-helix protein (bHLH), Iroquois homeodomain (Irx) and Meis homeodomain factors. By combined in situ analysis, immunocytochemistry and EGFP expression in BAC transgenic mice, we have examined transcription factor expression from E9, when cell proliferation first generates postmitotic precursor cells through the early neonatal period. Our results indicate that Irx3, Meis1, Lhx2 and Lhx9 expression define prospective deep nuclei cells and allow tracing of the migration patterns of these cells from the surface of the anlage to the zones that give rise to the three deep nuclei. Expression domains of Math1, Pax6 and Meis1 can be used to track the EGL precursor cells from the rhombic lip over the surface of the anlagen to form the EGL. Finally, Lhx1 marks Purkinje cell precursors as they exit the ventricular zone and migration inward. By E14.5 in the mouse, Lhx1 positive cells co-express Calbindin, a marker of differentiating Purkinje cells. Together these findings reveal a spatiotemporal map of gene expression that marks progenitors of the major cerebellar neuronal classes as well as their migratory paths in the embryonic cerebellum. (Morales and Hatten, in preparation).

Notch2 Controls Granule Cell Proliferation
From the early embryonic period, when EGL precursors first emerge until the second neonatal week, proliferation continues to expand the pool of EGL progenitor cells. This prolonged period of proliferation generates a massive population of granule cells such that the total population of granule neurons in the cerebellum is greater than the population of neurons in the cerebral cortex. Work by others demonstrated the importance of the SHH pathway to this proliferation as well as to the generation of tumors, the medulloblastoma from these cells. We recently showed that the Notch2 pathway and its ligand Jagged1 are nearly as potent as SHH in driving proliferation. Our work on EGL cells showed that the downstream gene Hes1 controls the proliferative effects of Notch2. As Hes1 is also downstream of Shh signaling as well as Math1 signaling, it is likely that these genes act in concert to drive the proliferation of EGL precursor cells (Solecki et al, 2001).
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