Hi all,
I study a mouse model for a genetic disorder, using a conditional knockout for my gene, which is a transcription factor. Knocking out the gene from principal neurons in the mouse cortex using Nex-Cre (Cre activity starts at ~E12), and analyzing the brains of 1 month-old male mice I measured in my KO significantly lower brain weight, and in the cortex I measured significant reduction in cortical thickness, significantly increased total number of total cells (measured by DAPI), significantly increased neruons number (measured by NeuN), and as a result, significantly higher neuronal density.
Measuring glial cells number by S100B showed trend for increased number of cells in KOs, but not significantly. Similarly for microglia, measured by Iba1.
I am interested in studying what are the molecular reasons for these data, and for that I am running RNAseq from whole cortex.
But to study whether this is a result of less apoptosis/more proliferation/more neurogenesis/anything else, I would like to hear your opinion about how should I tackle this question.
Please let me know what do you think the best way to study that.
Thanks a lot!
Boaz
Hi, this sounds really exciting (right up my alley). I would stain for BLBP at E12.5/E13.5, and combine that with a proliferative marker like Ki67 or PCNA. This would tell you how many radial glial cells/fibers you have, and how many of your BLBP cells are proliferating at the time. Either marker, or Phospho histone 3 would work. I would also look at tbr2+ Transient amplifying cells. TBR2 antibody from abcam is good, millipore used to have a good one (they bought out chemicon and had decided to stop making Tbr2). The transient amplifying (aka intermediate precursor) cells are thought to be important for expanding the number of neurons in the cortex. Looking at proliferating TBR2 cells might be of interest (see my paper with Hanna Stevens on Fgfr2 mutations Stevens et al 2010). You could also stain this time point for Tuj1 to see if there are more neurons, or in situ for Neurogenin. I would also recommend doing an CLDU/IDU experiment were you look at proliferation of radial glial cells and how many became neurons. First you do an injection with cldu, say at E12.5 during neurogenesis, then IDU injection 1 hour before harvesting pups at a second time point, say E14.5 or E16.5. You can look at how many cells are CLDU/IDU double positive (how many stayed as stem cells and devided), vs how many just have CLDU--> exited the cell cycle or just were not dividing at the time. Obviously, location in the cortical plate vs. SVZ IZ is valuable information too. http://www.ncbi.nlm.nih.gov/pubmed/21178965 This should tell you something about how quick the cell cycle is and how many stem cells are undergoing terminal differentiation into neurons.
The thinner cortex, but increased density of neurons is interesting. I've worked on projects with thinner cortex, but these had reductions in neurons. I think you need to look at whether you have an abnormal dendritic tree. This could be done with golgi stain and neuroleucida drawings, or you can look at density of markers like SMI32.
It all sounds very exciting and has implication for ASD. Many Casanova has published post mortem studies of ASD showing increased density of minicolumns in ASD.
Cheers and good luck!
Hi Karen
Thank you very very much for the detailed answer. I really appreciate it, and will now work on trying what you suggested! Many thanks again!
Boaz