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Lecture 5

Lecture 5 - Stem Cell Niche - October 1 - LIFESCI 2A03

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Life Sciences
Joe Kim

LECTURE 5 LIFESCI 2A03 Lecture 5: Stem Cell Niche October 1, 2013  Preformationist theory of development – semen contains an embryo, a preformed miniature infant, or homunculus, that becomes larger during development th o View persisted into the 18 century o Problems  Genetics from both parents not possible  No evidence (microscopy at the time not sufficient)  Sperm is single cell; suggests many tiny cells within sperm forming homunculus  Generational problem – inside homunculus there are millions of sperm cells with homunculus, inside whith there are millions of sperm etc – too microscopic  Other theory was that animal emerges gradually from a relatively formless egg  During the 19 century, improvements in microscopy allowed biologists to see that embryos took shape in a series of progressive steps after the fusion of egg and sperm (germline cells) Adult Stem Cells  Adult Stem ells – cells that can divide to create two cells; another stem cell or a cell more differentiate than itself o Cells:  A (purple) = stem cell  B (blue) = progenitor cell  Terms stem cell and progenitor cell are often used interchangeably; stem cell pluripotent; progenitor cell more differentiated (multipotent, unipotent)  C (yellow) = differentiated or specialized cell o Processes  1 = symmetric cell division  2 = asymmetric cell division  3 = progenitor division  4 = differentiation  Symmetric Cell Division – two identical daughter cells are created (both stem cells)  Asymmetric Cell Division – the two daughter cells are different from one another (one stem cell and a progenitor cell with limited self-renewal potential or a differentiated cell) o Maintains homeostasis of a system that tends to maintain a stable, constant condition o One stem cell and one differentiating cell – maintains a balance between stem cells and undifferentiated cells o Difference between two cells after asymmetric division 1. Differential segregation of  mRNAs  cystolic proteins  cell membrane proteins 2. Differences in surrounding environments o Two mechanism of asymmetric cell division A. Extrinsic fate determinants = the daughter cells are placed in different microenvironments  One cell stays in stem cell niche (maintains contacts and signals with niche); one cell pushed out 1 LECTURE 5 LIFESCI 2A03 B. Intrinsic fate determinants = asymmetric localization of components within the dividing cell that results in their differential segregation  Cell begin asymmetric; one cell has determinants, other does not  Stem Cell Niche – microenvironment that maintains stem cell identity o The niche has been studied in greatest detail for the germ line stem cells, but niches are being defined for many somatic adult stem cells  Adult stem cells fall into two categories o Somatic stem cells (that we have been discussing) – different somatic stem cell lineages go on to create or replenish body cells o Adult stem cells – create germ line cells (sperm and eggs)  Eg/ half the body of a female fruit fly is for developing eggs  Drosophila ovaries – model system for studying the stem cell niche o Ovaries divides into ovariole (string of maturing eggs; bottom mature egg, follicles along ovariole are developing eggs) o Far right = ovaduct o Drosophila ovaries contain germ line cells that will go on to form the egg and somatic cells that support egg development o We will focus on the anterior end of the ovariole where the germ line stem cells reside in their niche o Developmental life span (egg to adult) = 12 days o Grow and breed quickly; do not require a lot of maintenance; cheap medium to grow them on o Protein coding sequences are similar between drosophila and humans – can identify important gene in humans and observe what happens in drosophila  Germ Line Stem Cells (GSCs) – undergo asymmetric cell division’ o One cell retains stem cell identity, one cell (cystoblast, CB) eventually becomes the egg o Cyst – group of germ line cells that result from mitotic division of the cystoblast – one of the cells will become the egg, other cells become supporting cells  Drosophila ovary or germarium o Stem Cell Niche (red) – terminal filament, cap and inner sheath cells express molecules important for the maintenance and self-renewal of female GSCs o GSCs (light green) – undergo asymmetric cell division, giving rise to one daughter cell that will retain stem cell identity and one daughter cell, a cystoblast (CB), which will initiate differentiation (dark green)  While the germ line cells go on to form the egg, somatic cells form the niche  To identify the germ line cells, use a cellular marker 2 LECTURE 5 LIFESCI 2A03 o The fusome (or spectrosome) is found only in germ line cells – use antibody to proteins within this structure to identify the germline cells o Germ cells identified to follow through development; green stains the fusome of germline cells o Germline cells:  GSCs – germline stem cells (red); red when toching niche  CB – cystoblast; no longer red when out of niche  CS - cysts o Somatic Cells of the Niche (blue)  TF – terminal filament  TGS – inner sheath cells  Cpc – cap cells o Germline markers (green)  FS – fusome; grows as cystoblast divides  SS – spectrosome; in cells at end of germarium touching niche o Two or three GSCs are located at the tip of the ovariole (germarium) and are surrounded by the niche which is made up of: terminal filament cells, cap cells and inner sheath cells  GSCs undergo asymmetric cell division o GSC stays in the niche and retains stem cell identity o Cystoblast (CB) leaves the niche; undergoes mitotic divisions to form the cyst (in which, one cell will become the egg)  The GSC and the cystoblast differ in the intrinsic factors and extrinsic factors that determine their identity o Intrinsic Factors  Cyst oblasts require the expression of two genes  Bag of marbles (bam) and benign gonial cell neoplasm (bgcn) – code for products expressed in germline stem cell  While germline stem cells express a different set of intrinsic factors to maintain their identity o Extrinsic Factors  Germline stem cell niche  Germline Stem Cell Niche o Papers regarding the GSC niche in Drosophila ovary – trying to identify signaling roles in stem cell niche  Song et al. 2003. Bmp signals from niche cells directly repress transcription of a differentiation-promoting gene, bag of marbles, in germline stem cells in the Drosophila ovary. Development. 131: 1353-1364.  Chen, D. and D. McKearin. 2003. Dpp signalling silences bam transcription directly to establish assymetirc divisions of germline stem cells. Current Biology. 13(20): 1786-1791.  Review Article – looks at the whole field; not in depth  Primary Article o Germline stem cell niche in Drosophila ovary o Different between stem cells and differentiated cells  GSC – needs to remain undifferentiated, needs to undergo cell division (self-renewal of stem cells)  CB – needs to differentiate 3 LECTURE 5 LIFESCI 2A03  Intrinsic differences between gene expression  GSC does not transcribe the gene bad of marbles (bam)  The gene bam is transcribed in the CB and in developing cysts  Can visualize bam gene through activity of promoter and green fluorescent protein (GFP  [bamP-GFP] o Promoter – specific DNA sequences required for binding of RNA polymerase and transcription factors that recruit RNA polymerase; upstream of DNA sequence o Fusion between bam gene promoter and the coding sequence for the GFP o Figure: Left = original bam gene; right = bam promoter sequence fused to GFP gene o Results in GFP production when bam promoter is bound to by RNA polymerase o Evidence for transcription of bam absent in GSCs but present in CB’s o Notation – P [bamP-GFP]  P {bamP-GFP} – a P-element is a piece of mobile DNA used to introduce exogenous sequences into the genome of a fly; [bamP-GFP is carried by the P-element  P {bamP-GFP } – P represents promoter sequences that control (turn on) gene expression; here bamP means the promoter is normally associated with the bam gene  P {bamP-GFP} – on the right of the promoter is the gene that is being controlled by the promoter; here it is the gene coding for the green fluorescent protein  Question: What happens if bam gene is not transcribed? o Hypothesis – the bam gene is required for germline cell development o Null Hypothesis – there is no significant difference in the development of the germline cells in the presence or absence of bam gene expression o Mutation that removes a functional bam gene results in the following effect: A) Experimental – disrupted appearance (mutant)  Germ cells fail to differentiate remains as CB  No developing cysts  All cells are single cells with round fusomes  Mutant bam gene = bam[86] o  = deletion; no functional gene present o Lost bam activity  bam gene promoter (P{bamP-GFP} is activated o See green in undifferentiated CBs  These CBs are not GSCs  Not dividing asymmetrically (to develop GSCs), dividing symmetrically (only CBs) B) Control – normal appearance (wildtype)  CBs are formed 4 LECTURE 5 LIFESCI 2A03  Differentiated and developing cysts (groups of cells derived from CB)  Wild type bam gene is present o Can see bam product because of GFP  Bam gene promoter (P{bamP-GFP} is activated o See green in only CB and cysts, not in GSCs  Bam protein – required for germ cell development; activity is unknown; located in fusome  Cells in the niche (GSC) do not turn on the bam promoter, do not transcribe bam  Cells outside of the niche (CB) do turn on the bam promoter, do transcribe bam  Many proteins are expressed by the niche cells o Eg/ protein Dpp – a signal or a ligand for a receptor  Protein Dpp o Produced by stem cells, then secreted into extracellular space o Bone morphogenic proteins (BMPs) – known to induce bone formation; similar to Dpp o Dpp receptor is found on the stem cells o Hypothesis – if Dpp is required for maintaining stem cell, then too much Dpp should increase the number of stem cells o Null Hypothesis – there is no significant difference in the number of s
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