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Molecular and cellular analysis of hindbrain boundary cells

Periodic Report Summary - MCAHBC (Molecular and cellular analysis of hindbrain boundary cells)

The central nervous system (CNS) is divided in different compartment structures: forebrain, midbrain and hindbrain. The hindbrain in turn is subdivided in rhombomeres: compartmental structures separated by boundaries. It has been described that another CNS compartment borders are organising centres, but it's unknown if the hindbrain boundaries have this role and which are the molecular mechanisms that specify this territory, to be distinguished from the remaining hindbrain cells. Nevertheless, there are cues that suggest that hindbrain boundaries act like organising centres: activation of signalling pathways and stereotypic organisation of neuronal groups.

Our questions are:
(i) What are the mechanisms that induce boundary cells at the segment interfaces?
(ii) What is the behaviour and fate of boundary cells? And,
(iii) What is the role of boundaries in the zebrafish hindbrain?
To answer them we have mapped the regulatory region of rfng, a hindbrain boundary marker, in order to know the region that drives its expression only in boundaries.

Through this approach we follow two objectives:
(1) To know which genes regulate its expression, and
(2) to obtain transgenic lines, which express reporters in boundaries.
These tools will help us, through the use of time-lapse microscopy and hystochemistry to address the boundary cell fate, proliferation, and gene expression. Both approaches will help us to understand the regulatory complex that drives the boundary specification and differentiation and also to know if the rhombomere boundaries act like organising centres.

My studies during the last three years in the laboratory of Dr David Wilkinson (NIMR/London) have focused in the identification of the key regulators necessary for the maintenance of the undifferentiated state in boundary cells. In a soon to be published work we make a thorough description of the wild type boundary cell phenotype.

Briefly our observations are the following:
i) boundary cells are only part of the Eph receptor rhombomere interface being absent from the ephrin rhombomere. That suggests that the boundary cell specification is only dependant on Eph/ephrin forward signalling. It also shows, through the expression profile of DeltaD, the absence of proneural activity within the rfng+ cells;
ii) the expression of sox3, a marker of non-differentiated neuroepithelial cells, is maintained in rhombomeric boundary cells meanwhile its expression is downregulated in the rest of the rhombomere with the onset of neuronal differentiation;
iii) boundary cells are still actively proliferating by the mean of a wide set of proliferation markers.
These three results let us conclude that boundary cells are still in undifferentiated state, and suggest their entry into differentiation depends on Eph/ephrin forward signalling, possibly in collaboration with Notch.

We confirmed the role of Eph/ephrin forward signalling in the activation of rfng in boundaries, and showed that Rfng is necessary for Notch signalling to repress proneural genes specifically in boundary cells. Very interestingly, given its implication in the maintenance of different stem cell niches during mouse embryonic development, we found the signalling complex Jak2/Stat3 to be phosphorilated in boundary cells by Eph/ephrin forward signalling, mediating the activation of rfng. These results unveil a novel crosstalk between Eph/ephrin and Notch signalling through Jak2/Stat3.

All these events drive together the repression of proneural genes in boundary cells and the maintenance of this cell population as undifferentiated (Terriente et al., manuscript in preparation). Therefore, boundary cells are a promising biological model to study the dynamics between stemcellness and differentiation during CNS development.