Ebf2, a new regulator of neuronal differentiation: from gene identification to analysis of the Ebf2 -/- mouse

Corradi, Anna (2001). Ebf2, a new regulator of neuronal differentiation: from gene identification to analysis of the Ebf2 -/- mouse. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f9b6

Abstract

To shed light on molecular mechanisms underlying patterning and cell type specification during the ontogeny of the Central Nervous System (CNS), I set up to isolate new gènes with restricted patterns of expression in the developing mouse brain through a PCR based differential screening technique [1].
Among other genes found with this approach, I isolated Ebf2. This gene belongs to a new subclass of the HLH transcription factors, including several members ranging from rodents to C. elegans. In the mouse three homologs have been cloned by different groups and named Ebf1, Ebf2 and Ebf3 [2-4].
I studied the expression domain of Ebf2 during development at stages from E10.5 to E14.5 and compared it with the expression of other family members. In the region spanning midbrain to spinal cord, the patterns of expression of the three genes are very similar with few salient differences and correlate with neuronal maturation.
In order to analyse the specific function of Ebf2 in neuronal development, we decided to generate a null mutant for this gene. To this end, I have determined the genomic structure at the Ebf2 locus and I finely mapped the position of the first 6 exons in 20Kb of genomic DNA. Based on the map of the locus, I generated a construct containing a 5.2 kb deletion, encompassing the translation initiation site, the first 5 exons and half of the domain encoding the zinc finger. I substituted this region with a LacZ Neo cassette. The construct was electroporated into mouse embryonic stem cells (ES). Out of 1200 clones screened, I found 3 homologous recombinant clones, that we microinjected into C57B1/B6 blastocysts obtaining chimeras and germline transmission.
Despite the similarities observed among the three Ebf genes, suggesting possible redundancy in their functions, Ebf2 -/- mice show an evidently abnormal phenotype. The knocks out animals are viable at birth but show lower sizes than their littermates, as well as a mild ataxic phenotype and problems in movements and coordination. Around 50% of these mice die in the first two months of life.
The analysis of the Ebf2 -/- mutant CNS, even if preliminary, revealed several alterations in the different regions of postnatal brain. We observed numerous abnormalities in Ebf2-expressing neural structures, namely the cerebellum, the septum and the olfactory bulb; we found that other telencephalic structures seem to be affected, including the hippocampus and putamen. Finally, we observed problems in the spinal cord and an impairment of peripheral nerve myelination.