1. Somatic, Germ and Stem cells
Distinctions between somatic and germ cells.
Establishment of somatic cell types.
Establishment of the germ line.
Fundamental differences in germ cell establishment between organisms.
Where stem cells fit in….

2. Cell types Two basic classes of cells in animals somatic cells
germ cells
Somatic cells - almost everything we see and use, skin, muscle, blood, etc.
Germ cells - the cells that are responsible for reproduction, propagation of the species.
Stem cell - neither fish nor fowl. Can in some cases do both.

3. Germ vs. Somatic cells
1) These two classes of cells are established in different ways during development.
2) There are two fundamentally different processes by which germ cells are generated in different organisms.

4. Establishment of somatic cells

5. Gastrulation generates the three classes of somatic cells

6. Three classes of somatic cells
Ectoderm (outside)
Endoderm (inside)
Mesoderm (in between)
frequently called the 3 germ layers

7. Establishment of the germline
Two different ways in different organisms
1) Preformation (segregation)
2) Epigenesis (recreation)
Extavour and Akam, Development 130:5896 (2003)

8. Establishment of the germline
Preformation
Germ cells determinants (germ plasm) are set aside before or at fertilization and remain segregated throughout the lifespan of the organism.
C. elegans
Drosophila

9. Germline formation in C. elegans
In worms, the point of sperm entry defines the posterior pole of the egg.
a) A fertilized egg with evenly distributed P-granules
b) At pronuclear migration and fusion, P-granules move to the posterior, where the sperm nucleus was located.
c) After first cell division all P-granules in Posterior cell.
d) At 4-cell stage all P-granules are in Posterior P1 cell

10. Germline formation in C. elegans
6 Founder Cells
+98 die
+14 die
Small posterior cell goes
germline
+1 dies

11. Germline formation in C. elegans
8-cell
DAPI staining of nuclei
Antibodies to P-granules
24-cell

12. Preformation
C. elegans, Drosophila and many other model organisms. Some amphibians, birds.
Segregation of mRNAs and proteins required for germ cell formation and function (germ plasm).
bruno (RNP-type binding protein, regulates translation)
gld-1 (RNA binding protein)
mex-1 (zinc-finger DNA-binding protein)
vasa (DEAD-box RNA helicase, interacts with eIF5b)
germ cell less (transc. repressor, binds E2F)
cycB (B-type cyclin, interacts with CDK1, ubiquitous)
tudor (Tudor domain containing, part of germ granules)

13. Epigenesis of germline in mammals
Major distinction, no preexisting germ plasm in oocytes. Primordial germ cells are generated during development.
Seen in all mammals (so far), many other species.
Shared feature with Preformation: germ cells are "set aside" before other cell types.

14. Epigenesis of germline in mouse
Developmental Cell 2: 537 (2002) Zhao and Garbers, Male Germ Cell Specification and Differentiation
Blue = embryonic
white = extraembryonic
Embryonic
Ectoderm
Embryonic
Mesoderm
Embryonic
Endoderm

15. Epigenesis of germline in mouse
BMP-4, -8b
BMP receptor
BioEssays 27.2, Matsui and Okamura, Mechanisms of germ-cell specification in mouse embryos. 2005

16. Primordial germ cells Generated by epiblast.
Separate from the 3 somatic cell layers.
Migrate to gonadal ridge. (Molyneaux et al. Dev. Biol. 240:488 (2001).
Populate ridge to form germ cells of gonad.
In vitro, PGCs can differentiate into Embryonal Germ Cells (EGC). (Donovan et al. Cell 44:831 (1986), Matsui et al. Cell 70:841 (1992)).
This differentiation is similar to the "reprograming" of somatic nuclei seen in Oocytes and ES cells.
EGCs are one of only a few pluripotent stem cells currently known. Can contribute to all cell types in embryos.

17. Human Embryonal Germ Cells
Human EGCs
Mouse ES cells
Shamblott et al. PNAS 95:13726 (1998)
Cells were grown in DMEM supplemented with 15% fetal bovine serum, 0.1mM nonessential amino acids), 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 1 mM sodium pyruvate, 100 units/ml of penicillin, 100 ug/ml of streptomycin, 1,000 units/ml of human recombinant leukemia inhibitory factor (hrLIF), 1 ng/ml of human recombinant basic fibroblast growth factor (hrbFGF), and 10 mM forskolin.