User:Etchevers/Notebook/Conference notes/2010/06/27

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Notes for seminar by Frederic Causeret (A. Pierani group), May 2010

Brainbow mice – heterogeneity in a homogeneous population cf Jean Livet. Tool.

Developing brain homeobox = Dbx. Alessandra worked on this with Tom Jessell – specifies Vo interneurons.

Cajal-Retzius cells produce reelin – direct radial migration in cortex.

Label with a Dbx promoter in front of lacZ, or the whole gene – IRES – GFP or the whole gene – IRES – Cre and then cross with a Rosa26 – loxP – STOP – loxP – YFP mouse.

Traces indelible over time.

To get cell ablation cross the Dbx1 promoter-driven IRES loxP – STOP – loxP – diphtheria toxin construct mouse, with a generic promoter Cre recombinase mouse. The toxin can not get into neighboring cells. Can also use Emx1-driven or p73-driven Cre.

Affects frontomedial motor cortex by changing the expression of Pax6 / Emx2 / Sp8 domains.

Tbr2+ (Eomes!) progenitors are intermediate and within the SVZ, not the ventricle. Reduction in proliferative divisions and increase of Tbr2+ neurogenic divisions and differentiation, in a Dbx1 -/- background.

The Rosa-YFP labeled cells – sort selectively then microarrays to show which signaling factors are made by Cajal-Retzius cells eg FGF15/17.

Pool of CR cells from the boundary between pallium/subpallium also gives rise to later generated E12-E16 cortical plate, glutaminergic cells, that are transitory.

Then described use of Ngn2-driven Cre (Why?) mice to remove these Dbx1+ cells – this leads to reduction of the upper layers of progenitors through depletion. (Aren't these the Tbr2+ cells?)

When use Pgk-Cre (ubiquitous promoter) cross get truncation of head, just leaving the ears. Like reverse otocephaly. (Re: Otx2? I think I even asked this question and don't remember the answer. How embarrassing.)

Only a ventral bit of the Emx1/Foxg1 domains remaining. Normal eyes but synophthalmic (– implying some effect either on or downstream of Shh pathway / MID1.) Cf: Nestin-Cre; Dbx1 (DTA) – much less severe effect because happens after E10.5

The mutants have a phenotype though already at E9.5 (which mutants?) NT closure at 8-12 ss, E8.5-8.75 NT closed at 15 ss, E9 Start seeing problems at E8.5, early dynamic expression that can change and come on/off within a few hours.

at 20ss, ventral medial telencephalon floorplate cells.

Dorsal expression of Dbx1::Cre;Rosa26-YFP leads to some Sox10++ cells (not a lot, but they're there in the mesenchyme). Specification appears normal as well as a proliferation marker, PH3.

But lots of dorsal apoptosis.

(The idea is that by getting rid of this special neural crest-like population derived from Dbx1+ cells, the way I had done years ago by physical ablations of the neural folds, this is instrumental in causing the phenotype.)

Then did targeted ablations – Wnt1-Cre, Nkx2.1-Cre or FoxG1-Cre. For Nkx2.1-Cre, loss of ventral Shh but also displacement of other signaling centers like Fgf8. Each had a bit of an effect, implying cooperation of different subsets of cell populations. So did the triple Cre vs the Pgk-Cre phenotype; in the former, there is a telencephalon.

(I wrote something about the diencephalon-mesencephalon boundary for some reason.)

Expression analyses – all Shh, Fgfs 7/8/15/17/18 but Wnt1/7a in Dbx1+ cells, however, BMP2/7/Noggin not by Dbx1+ cells themselves, rather in their neighbors, and their levels are affected.

Dependence receptors – withdrawal leads to apoptosis: Ret, DCC, p75, alpha-v-beta-3 integrin, Patched, Unc5h2.

Neogenin is in the neural plate: RGMb in Dbx1+ cells as well as others.

Millicell explants of neural plates in a pretty in vitro assay – try soluble candidates to get Dbx1 cells to survive.