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IGP Genetics and Developmental Biology

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IGP Genetics and Developmental Biology

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    3. You have seen this “worm life cycle” before. Most of the cycle is devoted to larval growth. The larval stages are demarcated by a molt in which the outer cuticle is shed. The underlying skin or “hypodermis” secretes a new extracellular cuticle for the next period of larval growth. These processes are tightly coordinated and reproducible from animal to animal. This is actually quite remarkable when you think about it…because it involves the coordinated activities of multiple cell types throughout the animal that execute their developmental program in concert with each other…and at the right time. The question that interested Victor Ambros and Bob Horvitz was,”How are these events timed? Is there a genetic program devoted to controlling the timing of larval development? You might agree that this is an interesting problem but what does the nematode molting cycle have to do with human biology? you might ask. Afterall, we don’t have an exoskeleton. The cuticle is a specialized structure unique to nematodes (as evidenced by the interesting fact that nematodes actually have many more collagen genes (~50) than humans)You have seen this “worm life cycle” before. Most of the cycle is devoted to larval growth. The larval stages are demarcated by a molt in which the outer cuticle is shed. The underlying skin or “hypodermis” secretes a new extracellular cuticle for the next period of larval growth. These processes are tightly coordinated and reproducible from animal to animal. This is actually quite remarkable when you think about it…because it involves the coordinated activities of multiple cell types throughout the animal that execute their developmental program in concert with each other…and at the right time. The question that interested Victor Ambros and Bob Horvitz was,”How are these events timed? Is there a genetic program devoted to controlling the timing of larval development? You might agree that this is an interesting problem but what does the nematode molting cycle have to do with human biology? you might ask. Afterall, we don’t have an exoskeleton. The cuticle is a specialized structure unique to nematodes (as evidenced by the interesting fact that nematodes actually have many more collagen genes (~50) than humans)

    4. Wormatlas. Hypodermis…most of the animal is covered with a single multinucleated cell, Hyp 7 (>100 nuclei?)Wormatlas. Hypodermis…most of the animal is covered with a single multinucleated cell, Hyp 7 (>100 nuclei?)

    5. Seam cell image (psuedo colored GFP) from Rougvie 2005Seam cell image (psuedo colored GFP) from Rougvie 2005

    6. Images of cuticle, hypodermal cells that give rise to it….seam cells that produce alae…THEN talk about how remarkable this is…etc..why care? DIC image of adult alae from Rougvie 2005. EM of seam cell and alae from Worm atlasImages of cuticle, hypodermal cells that give rise to it….seam cells that produce alae…THEN talk about how remarkable this is…etc..why care? DIC image of adult alae from Rougvie 2005. EM of seam cell and alae from Worm atlas

    8. Rougvie 2001Rougvie 2001

    9. Rougvie 2001. Rougvie 2001.

    10. Rougvie 2001. Rougvie 2001.

    11. Prediction: LIN-14 activity is high in L1 and then declines. Draw out this model on the boardPrediction: LIN-14 activity is high in L1 and then declines. Draw out this model on the board

    12. Ruvkun and Giusto 1989 Control?Ruvkun and Giusto 1989 Control?

    13. Ruvkun and Giusto 1989 HYPOTHESIS: LIN-14 protein maintains L1 lineage/inhibits execution of L2, etc. lineage….Ruvkun and Giusto 1989 HYPOTHESIS: LIN-14 protein maintains L1 lineage/inhibits execution of L2, etc. lineage….

    15. Fewer seam cells in lin-4…dueto reiteration of L1 division pattern.GFP expression in seam cells pseudo colored for emphasis from Rougvie 2005Fewer seam cells in lin-4…dueto reiteration of L1 division pattern.GFP expression in seam cells pseudo colored for emphasis from Rougvie 2005

    16. Rougvie 2001. Rougvie 2001.

    17. Slack & Ruvkun 1997 No lin-4 = too much lin-14Slack & Ruvkun 1997 No lin-4 = too much lin-14

    18. Board, lin-4 ----l lin-14Board, lin-4 ----l lin-14

    19. Lin-14(gf) affects 3’ UTR Model: lin-14 3’ UTR restricts lin-14 expression Hypothesis: LIN-4 protein binds to lin-14 3’ UTRLin-14(gf) affects 3’ UTR Model: lin-14 3’ UTR restricts lin-14 expression Hypothesis: LIN-4 protein binds to lin-14 3’ UTR

    21. Genetic linkage Lin-4 deletion removes 5 kb fragment Complemenation ---> 700bp fragment No hits in cDNA library (actually did get cDNAs but none include sequence from 700bp lin-4 rescuing fragment) Disrupted orf with insertion = frame shift…still rescuesGenetic linkage Lin-4 deletion removes 5 kb fragment Complemenation ---> 700bp fragment No hits in cDNA library (actually did get cDNAs but none include sequence from 700bp lin-4 rescuing fragment) Disrupted orf with insertion = frame shift…still rescues

    22. Show approximate locations of lin-4L and lin-4S in 693bp lin-4 rescuing fragmentShow approximate locations of lin-4L and lin-4S in 693bp lin-4 rescuing fragment

    23. Scheme for isolating lin-4 allele by noncomplementation screen Optional…show them how mnc1 works…. Put lin-4(ma161) inScheme for isolating lin-4 allele by noncomplementation screen Optional…show them how mnc1 works…. Put lin-4(ma161) in

    24. Lin-14 miRNA is complementary to specific sites in lin-14 3’ UTR…note bubble Red arrow points to lin-4(ma161) point mutationLin-14 miRNA is complementary to specific sites in lin-14 3’ UTR…note bubble Red arrow points to lin-4(ma161) point mutation

    26. 10 years to molecularly identify lin-4 and lin-14 Lin-4 and lin-14 = no obvious homologs outside nematoda Who cares about worm heterchronic genes/hypodermis…etc.10 years to molecularly identify lin-4 and lin-14 Lin-4 and lin-14 = no obvious homologs outside nematoda Who cares about worm heterchronic genes/hypodermis…etc.

    27. Heterochronic genes regulating timing of larval development. Lin-4 is negative regulator of lin-14 and lin-28 via interaction with 3’ UTR Let-7 is second miRNA discovered also negative regulator of gene expression through interaction with 3’UTR of lin-41Heterochronic genes regulating timing of larval development. Lin-4 is negative regulator of lin-14 and lin-28 via interaction with 3’ UTR Let-7 is second miRNA discovered also negative regulator of gene expression through interaction with 3’UTR of lin-41

    32. Bartel Cell 2004Bartel Cell 2004

    36. Precocious expression of miR-1 leads to heart defect…premature termination of cellular proliferation “Tiny brakes for a growing heart”Precocious expression of miR-1 leads to heart defect…premature termination of cellular proliferation “Tiny brakes for a growing heart”

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