life sci 2a03

LS2A03: Research Methodologies in Life Sciences.��

" "The Stem Cell Niche�

Preformationist theory of development: ��This is the idea that semen contains an embryo — a preformed, miniature infant, or "homunculus" — that simply becomes larger during development. ��This view persisted into the 18th century.��The other theory was that an animal �emerges gradually from a relatively �formless egg. �

Homunculi in sperm as drawn by N. Hartsoecker in 1695�

During the 19th 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: ��These cells can divide to create two cells: another stem cell or a cell more differentiated than itself.�

Cells: �A = stem cell�B = progenitor cell�C = differentiated or specialized cell�

Processes: �1 = symmetric cell division �2 = asymmetric cell division �(3 = progenitor division)�4 = differentiation �

Image from Wikipedia

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)�

mitosis�

mitosis�

This maintains homeostasis: of a system that tends to maintain a stable, constant condition. �

Asymmetric cell division producing one stem cell and one differentiating cell maintains a balance between stem cells and differentiated cells.�

http://www.bioscience.org/2009/v14/af/3430/figures.htm

What might be different between the two cells after an asymmetric division? �

mitosis�

What might be different between the two cells after an asymmetric division? �

1) Differential segregation of: � - mRNAs� - cytosolic proteins� - cell membrane proteins.��2) Differences in the surrounding environment.��Some combination of these.�

mitosis�

Two mechanisms of asymmetric cell division: ��A) Extrinsic fate determinants = the daughter cells are placed in different microenvironments ��B) Intrinsic fate determinants = asymmetric localization of components within the dividing cell that results in their differential segregation �

http://www.bioscience.org/2009/v14/af/3430/figures.htm

Stem cell niche: microenvironment that maintains stem cell identity.��The niche has been studied in greatest detail for the germline stem cells, but niches are being defined for many somatic adult stem cells�

What factors define the a stem cell niche?�

Adult stem cells fall into two classes: ��1) The somatic stem cells that we have been discussing. These different somatic stem cell lineages go on to create or replenish body cells��2) Adult stem cells that create germ line cells : sperm and eggs.�

Drosophila ovaries: A model system for studying the stem cell niche�

www.cgm.cnrs-gif.fr/pret/images/fig1.jpg

drjes3.com/~JES3/projects.html

biology.mcgill.ca/faculty/nilson/research.html

Drosophila ovaries contain germline cells that will go on to form the egg and somatic cells that support egg development.�

We will focus on the anterior end of the ovariole where the germ line stem cells reside in their niche.�

Germ line stem cells (GSCs) undergo asymmetric cell division.��One cell retains stem cell identity�One cell, the cystoblast (CB), will eventually become the egg.��

Cyst: group of germline cells that result from mitotic division of the cystoblast – one of the cells will become the egg.�

Drosophila ovary or germarium: �

http://jcs.biologists.org/cgi/content-nw/full/118/4/665/FIG1

Stem cell niche: The terminal filament, cap and inner sheath cells (red) express molecules important for the maintenance and self-renewal of female GSCs.�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 germline cells go on to form the egg, somatic cells form the niche.��Which cells are somatic and which cells germline?�

In order to identify the germline cells use a cellular marker: �A structure called the fusome (also known as the spectrosome) is found only in germline cells.��You can use an antibody to proteins within this structure to identify the germline cells.��

�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 �

Drosophila ovaries: A model system for studying the stem cell niche�

Germline cells: �GSCs: germline stem cells �CB, cystoblast �(CS, cysts) ��Somatic cells of the niche: �TF: terminal filament �IGS, inner sheath cells�Cpc, cap cells��Germline markers: (green)�FS: fusome�SS: spectrosome�

GSCs undergo asymmetric cell division.��The GSC stays in the niche and retains stem cell identity�The cystoblast (CB) leaves the niche. This cell undergoes mitotic divisions to form the cyst and one of these cells will become the egg.��

The GSC and the cystoblast differ in the intrinsic factors and extrinsic factors that determine their identity.�

Intrinsic factors: ��Cystoblasts require the expression of two genes: ��bag of marbles (bam) and benign gonial cell neoplasm(bgcn)��While germline stem cells express a different set of intrinsic factors to maintain their identity. �

Extrinsic factors: ��The niche. What factors and mechanisms define the germline stem cell niche?�

We will look at two papers that help to define the GSC niche in the Drosophila ovary.�

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.�

Extrinsic factors: ��The niche. What factors and mechanisms define the germline stem cell niche?�

We will look at two papers that help to define the GSC niche in the Drosophila ovary.�

What is the difference between a: ��Review article and a primary literature article?�

What are the other components of a niche?��The topic of your independent essay will be a component of the stem cell niche.��You will choose a type of somatic adult stem cell niche and one protein to study.��Your written analysis will be similar to this – analysis of a primary research paper that describes the role of the protein in stem cell niche function.�

�A list of topics will be provided Friday and you will choose a topic on A2L.��

Niche: blue cells (somatic cells)�

Germline stem cell (GSC); or simply stem cell�

Cystoblast (CB); differentiating germline cell that will form an egg �

Spectrosome = germ cell specific marker�

Our model system for studying stem cell niches: �"The germline stem cell niche in the Drosophila ovary.�

How are the stem cells and the differentiated cells different from one another?��

GSC: needs to remain undifferentiated, needs to undergo cell division (self-renewal of stem cells)��CB: needs to differentiate�

How are the stem cells and the differentiated cells different from one another?�� bam

Intrinsic differences between " " " " " " " " " " "the cells:

gene expression.��GSC does not transcribe the gene bag of marbles (bam) ��The gene bam is transcribed in the CB and in developing cyst cells.�

How can we visualize bam gene transcription ... �" " " " " " " " "through activity

of its promoter�Promoter: specific DNA sequences required for binding of RNA polymerase and transcription factors that recruit RNA polymerase.�

TOOL: bam gene promoter and green fluorescent protein= {bamP-GFP} � �Fusion between bam gene promoter and the coding sequence for the green fluorescent protein (GFP). See green wherever bam is normally transcribed.�

Green fluorescent protein coding sequence " "

"= GFP protein �

Promoter sequences control transcription �

bam coding sequence = codes for protein �

bam promoter sequences control transcription �

No green in GSC �

Green in CB and cyst cells = differentiated germ cells�

Figure 1B: Chen and McKearin, 2003

Fusome can be seen in red; (also cytoskeleton of egg chambers�

P {bamP - GFP} �

1) P represents promoter – sequences that control (turn on) gene expression.��Here bam P means the promoter normally associated with the bam gene.�

2) 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 (GFP).�

Notation:�

3) This P is different. 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} �

So, what happens if bam gene is not transcribed?��

Hypothesis: The bam gene is required for germline cell development. ��Null hypothesis: There is no significant difference in the developent of the germline cells in the presence or absence of bam gene expression �

So, what happens if bam gene is not transcribed?��A mutation that removes a functional bam gene results in the following effect: �

Control: normal appearance �" " "(wildtype) �

Experimental: disrupted appearance (mutant)�

CBs are formed, how do we know? We see differentiated and developing cysts (groups of cells derived from CB). �

Germ cells fail to differentiate – remain as CB. How do we know? There are no developing cysts. All cells are single cells with round fusomes.�How do we know these are not GSCs?�

Figure 1: Chen and McKearin, 2003

Control: normal appearance �" " "(wildtype) �

Experimental: disrupted appearance (mutant)�

Wildtype bam gene is present ��bam gene promoter

"(P{bamP-GFP} is activated = �(see green in only in CB and cysts, not in GSCs)��

Mutant bam gene = bam[∆86] (∆ = deletion – no functional gene present)��bam gene promoter " "

"(P{bamP-GFP} is activated = �(see green in undifferentiated CBs)�

What is the Bam protein? While it is required for germ cell development, its activity is unknown – located in the fusome.�

So, these are CBs, not GSCs�

bam

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�

Is there a signal from the niche to the stem cell that controls bam transcription?�

?�

?�

Many proteins are expressed by the niche cells: ��One example is the protein Dpp which is signal or a ligand for a receptor.��Dpp is similar to other proteins called bone morphogenic proteins (BMPs) that are known to induce bone formation ��A signal needs a receptor, a Dpp receptor is found on the stem cells�

Dpp protein = signal or ligand; made by niche cells�

Dpp receptor = binds to signal; found on GSC �

Hypothesis: If Dpp is required for maintaining stem cell, then too much Dpp should increase the number of stem cells. ��Null hypothesis: There is no significant difference in the number of stem cells in the presence of normal levels of Dpp and increased levels of Dpp.�

Experiment: Induce over-expression of Dpp outside of niche.�

How can we control Dpp expression – that is transcription of the dpp gene and synthesis of Dpp protein.�

How to make too much Dpp?�

Two added DNA sequences: ��{hs-Gal4} = Gal4 is a DNA binding protein that can activate gene expression. Gal4 gene expression is controlled by a promoter called a heat-shock promoter.� �The gal4 gene is transcribed only at elevated temperatures.�

{UAS-dpp} = the dpp gene codes for the functional Dpp protein. The promoter controlling expression of the gene is called UAS (upstream activating sequence). The UAS can turn on gene expression only when bound to the Gal4 protein.��So, in turn, dpp is only transcribed at elevated temperatures.�

Induced expression of �GAL4 when temperature is

increased�

GAL4 gene�Heat-shock promoter

dpp gene�UAS�

Leads to induced expression of �target gene such as dpp �

Gal4-UAS expression system: �

Result: 6 Days of elevated temperature applied and the following was seen: �

Looks the same as a lack of bam expression, BUT, notice that (P{bamP-GFP} is not expressed, no green).��All germ cells are stem cells (GSCs).��Conclusion: Dpp can maintain stem cell identity.�


Fusome can be seen in red�

The bam promoter contains a silencer element (SE).��A transcriptional regulator protein called Mad binds to the SE and inhibits bam gene expression.�

Hypothesis: Regulation of bam expression by Dpp occurs through the silencer element with the bam promoter. ��Null hypothesis: ?�

Experiment: Similar setup as previously, but now: �� P {bamP∆SE - GFP} �

Bam promoter lacks the silencer element = deletion of SE.�

Result: 6 Days of elevated temperature applied and the following was seen: �

Still see overproliferation of stem cells due to increased dpp expression (this is still controlling the normal bam gene), but now the bam promoter is active (bam-GFP is transcribed if SE is removed)�

Conclusion: Dpp controls bam expression through the silencer element (SE) in the bam promoter.�


How do we get from an extracellular signalling molecule (Dpp) to regulation of gene expression?��Mad is a DNA binding protein that regulates gene expression.�

" "It is also a target of the signalling pathway initiated by Dpp.��Hypothesis: The SE of the bam promoter contains DNA sequences that are recognized by Mad. ��Null hypothesis: ?�

Experiment: sequence analysis�

+1 transcriptional start site�

Transcriptional regulatory sequences. While this could be activating (enhancers) or inhibitory (silencers), this is an example of a silencer element.�

Conclusion: bam regulatory sequences may bind to Mad.�

Result: �

" " "�Dpp over-expression results in an increase in stem cells due to the inhibition of bam expression. ��Hypothesis: This defect can be reversed (rescued) by increasing Bam expression��Null hypothesis: ?��

Can you reverse a mutant phenotype (defect) by compensating for the error?�

Dpp over-expression : increase in stem ��Can this effect be reversed (rescued) by expressing Bam from the same heat-shock promoter. �

" " "Essentially replacing the lost bam expression.�

Too much Dpp inhibits bam expression outside of the niche: too many stem cells.�



Simultaneous misexpression of bam and dpp looks normal: cysts form�


Song et al, 2003. Use similar tools to monitor gene expression: ��Dad is a target of Dpp. �Expression of Dad indicates that the Dpp signal is active, so we can use this as a marker for Dpp signalling. �Detecting Dad expression (transcription): �

" "Dad promoter – lacZ gene�LacZ gene : an enzyme that cleaves B-galactosidase. Here we can monitor enzyme activity by cleavage of a substrate. The product is seen in pink. �

Dad promoter sequences control transcription �

Bam is expressed in CB and cyst cells. We can visualize Bam expression using the BamP-GFP from the McKearin lab.�

Green fluorescent protein coding sequence = GFP protein �

bam promoter sequences control transcription �

Hypothesis: dpp signalling activity is restricted to GSCs and some cystoblasts where bam transcription is actively repressed �

Cell types� Expression of the gene Dad marks cells with active dpp signalling: GSC and one cystoblast; �dad-lacZ = pink�Fusome = green �DNA = blue�

Expression of the gene bam in one cystoblast and cyst; �bam-GFP = green �Fusome = red�DNA = blue�

Figure 1: Song et al, 2003

bam-GFP = green �dad-lacZ = pink �DNA = blue�1) GSCs and two cystoblasts (arrows) express high Dad, but no bam, �2) a cystoblast (arrowhead) has low Dad and begins to �express bam.�

Conclusion: If there is dpp signalling there is no bam; as dpp signalling is reduced, bam expression increases.�


Hypothesis: If the phosphorylation of the transcription factor Mad is an end result of the dpp signalling pathway, then we should see pMad in cells that are not expressing bam and no pMad in cells expressing bam. �

low pMad (pink), �increased bam expression (green)�

High pMad (pink), �no bam expression (green)�

b/w is DNA (Dapi) of same germarium�


p. 1356, Song et al, 2003. “These results further support the idea that the dpp signaling pathway is activated in GSCs at high levels, whereas bam transcription is actively repressed.”�

But, does dpp signaling repress bam transcription in GSCs? �

Hypothesis: dpp is essential for repressing bam transcription in the GSCs.�

Experiment: �Identify germ cells by labelling the fusome – a fluorescent antibody to a protein within the fusome called Hts (RED)�Identify all cells by labelling DNA (with a stain called DAPI) (BLUE)�Expression of GFP is under the control of different promoters (GREEN)��

Compare two different genetic backgrounds: ��Normal or wildtype�

" " "vs �dpphr56 = a mutation in the dpp gene that prevents dpp expression, but only at high temperatures (29oC). Effect? Gradual loss of stem cells.�

Normal: �29oC, 7 days�No bam-GFP in stem cells�

Dpphr56�

29oC, 2 days�See some bam-GFP in stem cells�

Dpphr56�

29oC, 4 days�Reduced stem cell number.�See bam-GFP in stem cells�

Dpphr56�

29oC, 7 days�Reduced stem cell number.�See bam-GFP in stem cells�

Conclusion: �dpp signal is required to repress bam transcription in GSCs��(Table 1: higher levels of bam transcripts in dpphr56)�


Results: �

It is known that the Dpp signal leads to phosphorylation of the transcription factor, Mad (pMad, where 'p' is phosphorylation)��What is the relationship between the presence of pMad and bam-GFP expression? Hypothesis?�

Normal (wildtype) at 29oC, 4 days��pMad (pink) is present in GSC (*), �no bam-GFP expression (green) is seen.�

Mutant (dpphr56) at 29oC, 4 days��pMad (pink) low in GSC (*),�bam-GFP expression (green) is seen.�


Can you make the niche larger?��Extend dpp expression using heatshock Gal4 ; UAS-dpp system�

With heat, more single germ cells containing round fusome = pink �These cells do not express bam-GFP�

No heat, normal, GSC with round fusome (pink) at tip; these do not express GFP. CB and cyst cells do express bam-GFP (green)� Conclusion: dpp overexpression is sufficient for

repressing bam transcription in the cystoblast �


In addition, both studies demonstrated that Mad protein was able to physically bind to a silencer element within the transcriptional regulatory sequences of the bam gene��



P X

P = Phosphorylated form of Mad protein �

SE

In Niche: �Dpp signalling leads to the production of phosphorylated Mad transcription factor in the stem cells. Phosphorylated Mad represses bam transcription.�

Outside of the niche: �No Dpp signalling, No phosphorylated Mad and therefore no repression of bam transcription.�

Dpp � Mad�

phosphorylated �Mad�


bam coding sequence = codes for protein �P

P X

Dpp � Mad�



X

So, the model is that an asymmetric division of the GSC leaves one cell in the niche (no bam) and pushes the other cell out of the niche (bam transcribed). Bam is required for cystoblast differentiation.��The niche represses differentiation and maintains the stem cell identity.�

Dpp and Mad are just two proteins in this regulatory system.�

What are the other components?��The topic of your independent essay will be a conponent of the stem cell niche.��You will choose a type of somatic adult stem cell niche and one protein to study.��Your written analysis will be similar to this – analysis of a primary research paper that describes the role of the protein in stem cell niche function.�

How stable is the germline stem cell niche?��This question was asked by Tai and Spradling in 2003.�

What happens if it is “empty”?��What might be your hypothesis?��Design an experiment that might address your hypothesis.�

Toshie Kai and Allan Spradling. 2003. An empty Drosophila stem cell niche reactivates the proliferation of ectopic cells. PNAS. 100(8): 4633-4638.�

This has implications for disease states. What are the implications?

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