UMDNJ-Robert Wood Johnson Medical School Strain Differences in Stem/Progenitor Cell Proliferation and Survival During Adult Neurogenesis in the Dentate Gyrus Richard S. Nowakowski Nancy L. Hayes UMDNJ-Robert Wood Johnson Medical School Piscataway, New Jersey

Why do we need stem cells?

Adult Neuronogenesis in the Mouse Two proliferative populations in adult brain Dentate gyrus (intrahilar) Subventricular zone (olfactory bulb) Let’s examine the properties of the proliferative cells in the dentate gyrus in vivo. Some new cells become neurons and seem to become integrated into the circuitry of the dentate gyrus, i.e., exactly what we need stem cells to do.

0.5 hours

12 hours

24 hours

36 hours

12 days

Productive Capacity of a Proliferating Population Proportional to the number of proliferating cells Inversely proportional to the length of the cell cycle Expansion vs production controlled by cell fate decisions, i.e. re-entry into the cell cycle (P) vs exit to differentiate (Q)

Saturate and Survive Method: Principle

Saturate and Survive Method: Application 12 hour saturate with BUdR + sacrifice Note increase and then decrease in # of labeled cells

Estimated Cell Cycle Parameters in C57Bl/6J

Strain Difference in S-phase Cell Number

A Genetic Difference

BALB/c has fewer proliferating cells (800 vs 1800), but same kinetics as C57

Number of S-phase Cells Differs in Inbred Strains C57Bl/6J is high; others are low

Size of the Hippocampus also varies 129X1/SvJ and DBA/2J are significantly smaller than the others

No correlation between Number of S-phase cells and Size of Hippocampus

Maximum Likelihood Analysis shows 3 Clusters with Greatest Distance between C57BL/6J and 129X1/SvJ 3 Clusters --> at least 2 genes Not different?

Strain Differences in Survival

Strain Differences in S-phase and Survival

Survival/S-phase

NeuN is Neuronal Marker

BUdR + NeuN

Double and Single Labeled Cells

Measure of Neuron Production

Summary of Strain Differences

Neuronogenesis Development vs Adult Transient proliferative population VZ/PVE produces mostly neurons Most neurons persist Cell Cycle length changes Changing P/Q Permanent proliferative population Intrahilar zone produces mixture of cell types Most cells are transient Cell cycle length constant (*to be determined*) Constant P/Q (*to be determined*) P=Q=0.5 or, more accurately, P=Q+D=0.5 Output and/or the behavior of the proliferating cells are affected by drugs, behavior, etc. (Question: How are cell cycle vs P/Q changed?)

Translation: It is now possible to get a provisional map location of the genes producing a phenotype by characterizing that phenotype in several inbred strains of mice.

QTL Candidates: 4, 7, 8, 13 7,12,15, X 2, 8, 13, X

Strain Differences in Stem/Progenitor Cell Proliferation in the Mouse Dentate Gyrus Number of S-phase cells (size of proliferating population) Survival of “new” cells for 5-6 weeks Septo-temporal distribution of the S-phase cells Size of the dentate gyrus Proportion of cells that become neurons? Behavior? Tools for Genetic Mapping: Inbred Strains, Recombinant Inbred Strains, F2 hybrids, SNP's, Sequenced Genomes

Effect of Low (50 ug/g) vs High (300 ug/g) Dosage

Conclusion: Low dose is more controlled and avoids issue of toxicity.

NeuN Montage

NeuN in Neocortex

Through-focus series