Origin of Climbing Fiber Neurons and Their Developmental Dependence onPtf1a

Abstract

Climbing fiber (CF) neurons in the inferior olivary nucleus (ION) extend their axons to Purkinje cells, playing a crucial role in regulating cerebellar function. However, little is known about their precise place of birth and developmental molecular machinery. Here, we describe the origin of the CF neuron lineage and the involvement of Ptf1a ( pancreatic transcription factor 1a) in CF neuron development. Ptf1a protein was found to be expressed in a discrete dorsolateral region of the embryonic caudal hindbrain neuroepithelium. Because expression of Ptf1a is not overlapping other transcription factors such as Math1 (mouse atonal homolog 1) and Neurogenin1, which are suggested to define domains within caudal hindbrain neuroepithelium (Landsberg et al., 2005), we named the neuroepithelial region the Ptf1a domain. Analysis of mice that express ␤-galactosidase from the Ptf1a locus revealed that CF neurons are derived from the Ptf1a domain. In contrast, retrograde labeling of precerebellar neurons indicated that mossy fiber neurons are not derived from Ptf1a-expressing progenitors. We could observe a detailed migratory path of CF neurons from the Ptf1a domain to the ION during embryogenesis. In Ptf1a null mutants, putative immature CF neurons produced from this domain were unable to migrate or differentiate appropriately, resulting in a failure of ION formation. Apoptotic cells were observed in the mutant hindbrain. Furthermore, the fate of some cells in the Ptf1a lineage were changed to mossy fiber neurons in Ptf1a null mutants. These findings clarify the precise origin of CF neurons and suggest that Ptf1a controls their fate, survival, differentiation, and migration during development.

Ptf1a Directly Controls Expression of Immunoglobulin Superfamily Molecules Nephrin and Neph3 in the Developing Central Nervous System

“…Antibodies-Anti-Ptf1a antibody was described previously (9,30). Anti-FLAG (M2, Sigma), anti-Myc (9E10, Santa Cruz Biotechnology), anti-HA (3F10, Roche Applied Science), alkaline phosphatase (AP)-conjugated anti-mouse IgG (Bio-Rad), AP-conjugated anti-rat IgG (Jackson Laboratories), AP-conjugated anti-digoxigenin (DIG, Roche Applied Science), peroxidase-conjugated anti-DIG (Roche Applied Science), anti-GFP (Molecular Probes), and biotin-conjugated anti-rabbit IgG (Jackson Laboratories) antibodies were obtained commercially.…”

“…Endogenous Ptf1a was immunoprecipitated with an anti-Ptf1a antibody (9). The amount of co-precipitated DNA fragments was analyzed by quantitative PCR using a LightCycler (Roche Applied Science).…”

“…In the Ptf1a-null cerebellum, Ptf1a-positive progenitors, which are normally fated to inhibitory neurons, trans-fate to excitatory granule cells (8). Cell fate changes in Ptf1a-null mice were also reported in the retina, hindbrain, and spinal cord (5)(6)(7)9). Conversely, ectopic expression of Ptf1a in neural progenitors of the dorsal telencephalon, which are normally fated to glutamatergic excitatory neurons, confers inhibitory GABA characteristics to these neurons (4).…”

<em>In vitro</em> Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

“…The LRL has been characterized as possessing unique progenitor domains characterized by the differential expression of proneural transcription factors (reviewed in 1 ). A subset of these factors (Mash1, Math1, Ngn1, and Ptf1a) were chosen for analysis due to their proposed and/or characterized roles in the specification of precerebellar neural subtypes in the LRL, a subject of future studies [16][17][18] . All four proteins have highly characteristic expression domains in the caudal hindbrain at E11.5 [16][17][18] .…”

“…A subset of these factors (Mash1, Math1, Ngn1, and Ptf1a) were chosen for analysis due to their proposed and/or characterized roles in the specification of precerebellar neural subtypes in the LRL, a subject of future studies [16][17][18] . All four proteins have highly characteristic expression domains in the caudal hindbrain at E11.5 [16][17][18] . We observed that embryos that were placed cultures failed to increase in size and also failed to initiate production of choroid plexus epithelium and invagination of the LRL and roofplate, morphological events Figure 1A.…”