Fall 2007
Characterization of mash1-expressing neurons in the mature embryonic mouse spinal cord
Jane E Johnson Lab
Purpose:
We are trying to find what kinds of cells are derived from mash1-expressing progenitors in the mature spinal cord structure.
To study mash1 derived cells in the spinal cord, spinal cords have been dissected from mash1 creERtm X RYFP mice, and stored in the -80. In this conditional knockout system, Cre-ERtm construct has only transcribed where mash1 gene is normally expressed, by crossing of mash1 Cre-ERtm mouse with RYFP mouse, heterozygote embryos have been produced. Later, activity of Cre has been induced by giving double injection of tamoxifen at e10.5, e11.5, e12.5 and e17.5. By using YFP, activity of Cre, mash1 Cre-ERtm construct and cross has been determined.
Strategy and Methodology:
I have used several procedures during my work such as cryosectioning, immunostaining, confocal microscopy, mouse handling and sacrifice. Firstly, I started with training on cryosectioning of spinal cord and I have prepared one sample from each progeny. Then, I have stained sections whether they are YFP positive or not using confocal microscopy. I used NeuN antibody as a general marker for neuron cells. I have found opportunity to practice mouse perfusion assay to take out brain.
Microscopy:
I have tried to determine localization of mash1-expressing cells. We knew that location of cells can give us clue about the function of the stained cells such as pain, movement etc. We are planning to determine, first of all, the location, distribution, percentage of some distinct populations if we can identify, structure of cells, any similarity with known cells, then we would look for whether stained cells are gabaergic or glutamatergic using Pax2 antibody as an inhibitory gabaergic marker and Tlx3 antibody as an excitatory glutamatergic marker.
Marker List: List of markers I have used
Marker Type Purpose
NeuN Neuronal General neural marker to stain neurons
Pax2 Neuronal To stain inhibitory-GABAergic neurons
Tlx3 Neuronal To stain excitatory Glutamatergic neurons.
Olig2 Oligodendrocyte To stain oligodentrocytes
Mash1 is affecting dI3 and dI5 formation, possibly dILA ¬and DILB. We can use Lbx1 and Tlx3 as a marker for dILB (known as inhibitory neurons) and Lhx1/5 or Pax2 as a marker for dILA(known as excitatory). Thx3 would also be marker for dILB. For dI3, Islet1 would be good markers and for dI5, we can use Lmx1b as a marker. We also can use Islet1 as a negative marker to distinguish dI5 form dI3 because Islet3 does not stain dI5 cells.
Characterization of neurons:
To determine what kind of neurons or cells are derived from mash1-expressing cells, we can further explore using adult neural markers, especially dorsal markers, and other markers such as calcium binding proteins; parvalbumin and calretinin, then we can check if they overlap with mash1 staining. In addition, we can investigate interneuronal/dorsal groups that show selective expression of certain combinations of transcription factors, and then identify groups of interneurons. We have to be sure about they are really neurons so that it is better to stain again and check. Staining of cells might be different, and density of cell types or some group of cell may differ with position and time. Thus, it will be good idea to take into consideration density of cells and intensity of staining.
To further investigate characteristics of cell types, we can use markers showing neurotransmitter phenotype, and we can check calcium-buffering protein expression, axon-distribution, time dependent changes at neuronal staining.
Future directions:
After identification of the specific mash1-expressing derived neuron cells, we can ask if mash1 is really required for them or not. In addition, we might look at their physiological function and importance of these neurons. I would be great if could say mash1 is certainly of the important determinant of certain functions in neural system.
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Exploration of the cis-acting elements of proteins that are possibly related to Math1 activity or affected by math1
We are trying to determine enhancers or inhibitor elements affecting gene activity of proteins that are possibly related to Math1 activity or affected by math1 binding and expression.
Primary target proteins:
Lhx2, Barhl1, Klf7, Tle4, and Lhx9
Approach:
Using ECR browser (http://ecrbrowser.dcode.org/), we selected primarily conserved regions in different organism to increase our chance to hunt desired sequence. We have looked at the 10 Kb upstream and downstream of the genes. Then, I have classified and selected mostly conserved regions to make constructs up to 3Kb. Then, I got the sequence from ensembll.org and I have checked the possible BACs that we can use. Using the Web Primer website (http://seq.yeastgenome.org/cgi-bin/web-primer), I designed primers to amplify the region from BACs. As a key software, I used ApE A plasmid editor(http://www.biology.utah.edu/jorgensen/wayned/ape/) to determine possible restriction enzyme sites to design a plasmid that can be ligated with two different sticky ends. Thus, we could able to know the direction of our insert into BgnEGFP vector. This vector contains multiple RE sites and GFP protein. By insertion of our PCR products into multiple RE site, we hope to see expression of GFP when we electroporate in chick embryos. Then, we plan to coinject these constructs with Math1 containing vector to see whether math1 binds to target sequence and regulates the activity of GFP expression.
Regions, Primers, and BACs:
I already determined the regions, primers, and BACs for four of the target proteins; Lhx2, Barhl1, Klf7, and Tle4. They are in my folder for further information.
For example;
Lhx2+10Kb target sequences
Construct Lhx2-A : target region chr2: 38163602-38166253= 2651kb long
Construct Lhx2-B : target region chr2: 38168090-38170566= 2476 kb long
Construct Lhx2-C : target region chr2: 38190826-38191755= 929 kb long
And so on
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Sinir sisteminin nasil gelistigi, hangi hucrelerin zamanla hangi hucrelere donustugu omurga ile ilgili rahatsizliklarin tedavi edilmesinde, onlara tedavi gelistirilmesinde onem arz ediyor. Bu calismada omuga gelisimini, oncu hucrelerin zamanla hangi hucrelere donustugunu gozlemlemeye calisiyor, kok hucreler ve olgun sinir hucrelerinin olusumunu nelerin tetikledigini anlamaya calisiyoruz. Fareler uzerinde calisiyor, onlarin genlerine yerlestirilen genleri takip etmemizi saglayan bir strateji , CRE proteinlerini kullaniyoruz. Ozellikle Olig2, NeuN, Pax2 ve Tlx3 uzerinde yogunlasiyoruz.