| Abstract |
Transcription factors of the bHLH family play a major role in cellular
determination and differentiation. bHLH proteins have been classified in seven
categories. Class I HLH proteins like ME1a, E2 and daughterless, are expressed in many
tissues and are capable of forming either homo- or heterodimers. Class II HLH proteins,
which include members such as Mash1, Math1, NeuroD and NSCL1, show a tissuerestricted
expression pattern. With few exceptions, they cannot form homodimers and
preferentially heterodimerize with the class I proteins.
In the present study, the chick Nscl1 gene has been isolated with the aim of
performing functional experiments in ovo. Chick NSCL1 consists of 130 aminoacids and
shows a significant conservation with the human and mouse homologs, which consist of
133 aminoacids. They differ just in one aminoacid in the bHLH region, reaching 98%
homology.
Our experiments showed that cNscl1 is strictly expressed in neurons of the CNS
and the PNS, like mouse and human homologs. Particularly, we observed that the Nscl1
is expressed in almost all the tissues of the nervous system such as the neural tube, the
cranial and sensory ganglia, the hippocampus, the spinal cord, the diencephalon, the
hindbrain and the cerebellum. In situ hybridization experiments showed that cNscl1 is
only expressed in differentiating postmitotic neurons of the brain and the neural tube,
while it is expressed in the cerebellum, in mitotic neuroblasts and postmitotic
premigratory neurons of the external granule layer (EGL).
In Hoxb1-/- mice we observed that Nscl1 expression is ceased in facial
branchiomotor neurons (fbm). In Nscl1-/- mice a delay on the expression pattern is
observed for Hoxb1, Hoxb2, Plzf and Engrailed-1 in the hindbrain, possibly due to a
slowing down of differentiation of this region.
Our results reveal novel sites of Nscl1 expression in mouse and chick. Nscl1 is
expressed in rhombomeres, rhombomere boundaries and regenerated boundaries that are
formed when an even numbered rhombomere is grafted next to an odd numbered
rhombomere. Grafting an even (odd) numbered rhombomere next to another even (odd)
numbered rhombomere, results in a single enlarged rhombomere that lacks the
interrhombomeric boundary, and no Nscl1 expression is detected.
In ovo injection and electroporation of the replication competent retrovirus
RCAS(BP)-cNSCL1, and subsequent cNscl1 misexpression, results in abnormal brain
development and small eye production.
We furthermore studied the transcriptional activity of NSCL1 as a homodimer or
as a heterodimeric complex with ME1a protein. We found out that homodimers of
cNSCL1 fused to GAL4-DBD resulted in a strong reporter gene repression. On the
contrary, heterodimers of mNSCL1 with ME1a bind to specific E-boxes and behave as
transcriptional activators in reporter gene assays. The transcriptional capacity of NSCL1
should be localized in a basic region (b’) upstream of the bHLH domain.
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Ανίχνευση, κλωνοποίηση, μελέτη τπυ προτύπου έκφρασης και λειτουργικός ρόλος του γονιδίου chick Nscl1
