QUESTION 1. (17 points) In the Drosophila female germline, germline stem cells (

Question

QUESTION 1. (17 points) In the Drosophila female germline, germline stem cells (GSCs, red) divide asymmetrically away from a population of cells called cap cells (green) that are thought to be the stem cell niche (similar to the hub of the male germline). GSCs.are surrounded by somatic.escort.cells (blue). Cap cells secrete BMP. Ectopic overexpression of BMP in the whole germarium causes overproliferation of GSCs and inhibits their differentiation. You propose two hypotheses for how BMP affects GSCs: 1. BMP acts directly on the GSCs, or 2. N BMP acts indirectly via signaling first to the early escort cells that then signal to GSCs (assume the escort cells that surround the differentiating cystoblasts have differentiated themselves and are different from the escort cells that surround the GSCs) Cap cell Regions: me Escort usome I a) 5 points) You decide to test these hypotheses by performing an immunostain for Phospho-SMAD in the germarium. Describe a result that would support hypothesis 1 and rule out hypothesis 2. Also describe a result that would rule out 1 and support 2. In each case, explain your interpretation, Figure. Schematic of the Drosophila ovary b) (12 points) Consider that your Phospho-SMAD immunostain supports hypothesis 2, which states that the early escort cells respond to BMP and not the GSCs themselves. For the 4 experimental conditions listed below, describe how GSC proliferation and differentiation would be changed if this hypothesis were true 1. (3 points) Overexpress BMPR-dominant negative in GSCs 2. 3. 4. (3 points) Overexpress BMP in early escort cells (3 points) Overexpress a constitutively activated BMPR in early escort cells (3 points) Overexpress a SMAD dominant negative in all cells

Explanation / Answer

a. Phospho SMAD stain on GSC alone would show that BMO signalling is active at that location and there is a direct signalling/crosstalk between germarium and GSCs whereas differential phospho smad stain in GSC along with escort cells would tell us that the signalling/crosstalk is not direct, rather indirect.

b.

1. Overexpressing BMPR dominant negative in GSC's would lead to abrogation/reduction in function of BMP signalling. Thus early escort cells would receive less/no BMP signal , causing differentiation of GSC rather than proliferation and maintenance of stem cell nature.

2. If one overexpresses BMP in early escort cells, it would lead to more BMP signaling leading to an increased GSC's proliferation and less differentiation.

3.Constitutively active BMPR iin early escort cells would lead to more signalling that is passed downstream to GSC's leading to an increased GSC proliferation.

4. SMAD is downstream in BMP signaling and interact with more smads . Upon phosphorylation, phospho smad would translocate to the nucleus where it would transcribe genes like RUNX2/BMP related genes. If one overexpresses a dominant negative form of smad, the translocation of smad would be affected and hence BMP signaling is affected leading to abrogation of GSC proliferation but it will lead to an increased differentiation along with several signaling being affected as BMP crosstalks with TGFbeta as well.

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