Ptf1a determines GABAergic over glutamatergic neuronal cell fate in the spinal cord dorsal horn

Mutations in the human and mouse PTF1A/Ptf1a genes result in permanent diabetes mellitus and cerebellar agenesis. We show that Ptf1a is present in precursors to GABAergic neurons in spinal cord dorsal horn as well as the cerebellum. A null mutation in Ptf1a reveals its requirement for the dorsal horn GABAergic neurons. Specifically, Ptf1a is required for the generation of early-born (dI4, E10.5) and late-born (dIL(A), E12.5) dorsal interneuron populations identified by homeodomain factors Lhx1/5 and Pax2. Furthermore, in the absence of Ptf1a, the dI4 dorsal interneurons trans-fate to dI5 (Lmx1b(+)), and the dIL(A) to dIL(B) (Lmx1b(+);Tlx3(+)). This mis-specification of neurons results in a complete loss of inhibitory GABAergic neurons and an increase in the excitatory glutamatergic neurons in the dorsal horn of the spinal cord by E16.5. Thus, Ptf1a function is essential for GABAergic over glutamatergic neuronal cell fates in the developing spinal cord, and provides an important genetic link between inhibitory and excitatory interneuron development.     

GM1 ganglioside as a marker for neuronal differentiation in mouse cerebellum

The distribution of GM1 ganglioside in developing mouse cerebellum was monitored by indirect immunofluorescent detection of choleragenoid receptors. In frozen sections of cerebellum from mice 5 to 10 days old, fluorescence is observed on granule cells in the inner rows of the external granular layer, in the growing molecular layer, the Purkinje cell layer, and the internal granular layer. In sections of adult mice, fluorescence is restricted to the bodies of Purkinje and internal granule neurons. The percentage of fluorescent dissociated or cultured cerebellar cells increases with the postnatal age of the mouse or the duration of time in vitro. No fluorescence is observed in the absence of choleragenoid or if the test material is extracted with chloroform:methanol. To determine whether the expression of surface GM1 ganglioside in culture is a reflection of a developmental program, mice are injected at particular times with [³H]thymidine and cerebellar cultures processed for simultaneous autoradiography and immunofluorescence. Granule cells from 8-day-old mice having cholera toxin receptors at 20 hr in vitro are a distinct population born 1 day or earlier prior to sacrifice. Cells synthesizing DNA on the day of sacrifice are not fluorescent at 20 hr in vitro. This observation correlates well with immunohistological results showing a lack of fluorescence in the outer proliferative rows of the external granular layer. Therefore GM1 ganglioside is not present on granule cell precursors but is expressed at some time after the cells become postmitotic. GM1 ganglioside is detected on growing parallel fibers in situ and neurites in vitro but not on adult axons, suggesting differential localization at a later stage of development.     

Regulation of neuronal lineage decisions by the HES-related bHLH protein REF-1

Members of the HES subfamily of bHLH proteins play crucial roles in neural patterning via repression of neurogenesis. In C. elegans, loss-of-function mutations in ref-1, a distant nematode-specific member of this subfamily, were previously shown to cause ectopic neurogenesis from postembryonic lineages. However, while the vast majority of the nervous system in C. elegans is generated embryonically, the role of REF-1 in regulating these neural lineage decisions is unknown. Here, we show that mutations in ref-1 result in the generation of multiple ectopic neuron types derived from an embryonic neuroblast. In wild-type animals, neurons derived from this sublineage are present in a left/right symmetrical manner. However, in ref-1 mutants, while the ectopically generated neurons exhibit gene expression profiles characteristic of neurons on the left, they are present only on the right side. REF-1 functions in a Notch-independent manner to regulate this ectopic lineage decision. We also demonstrate that loss of REF-1 function results in defective differentiation of an embryonically generated serotonergic neuron type. These results indicate that REF-1 functions in both Notch-dependent and independent pathways to regulate multiple developmental decisions in different neuronal sublineages.