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Lecture 7

November 8 Lecture 7 - (October 29, Nov 5, 8) - LIFESCI 2A03

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Life Sciences
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Joe Kim

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LECTURE 7 LIFESCI 2A03 Molecular Basis of Regeneration Planarians as a Model System October 29, Nov 5, 8 2013 Reddien, P.W. et al. (2005). SMEDWI-2 is a PIWI-Like-Protein That Regulate Planarian Stem Cells. Science. 310(5752), 1327-1330. The Sanchez lab is studying neoblasts and the role that these cells play in regeneration.  Researchers identify the neoblast cells by: 1. Morphology of the cells 2. BrdU labeling 3. Antibody to Histone H3  Explain each of these, what does the researcher see and why?  Eliminate the cells and see if the markers are still present in order to demonstrate that these markers are specific to neoblasts o Irradiated planaria are unable to regenerate because neoblastas are destroyed; observe whether markers are affected when irradiated o Before irradiation (capable of regeneration) – showing typical neoblast distribution; o After irradiation (incapable of regeneration) – no cells labeled by H3 antibody; neoblasts not dividing or functioning correlates to ability to regenerate  Neoblasts are required for regeneration o Blastema formation and regeneration of the pharynx in the irradiated posterior fragments 3 days after amputation o A-C – dorsal view of the wound region of the regeneration fragment o A – un-irradiated fragment; blastema formation  Head region – un-irradiated animal; normal typical blastema formation, anterior to eye spots; original pigmented cells and pigmented cells o B, C – irradiated fragment; no blastema  Regional Irradiation – focused irradiation at place of amputation; no normal blastema formation  Whole irradiation – irradiation all over; no normal blastema formation o SMEDWI-2 gene required for regenerative process  What genes are expressed in neoblasts?  What genes are required for neoblast function (regeneration)?  How can we answer these questions?  Determining which genes are expressed in neoblasts and which genes are required for neoblast function (regeneration) o Tracking gene expression – in situ hybridization o Manipulating gene function – RNA interference Identifying genes required for regeneration – a screen using RNAi  Identified a collection of genes needed for completing regeneration:  Effects of RNAi (phenocopies rather than phenotypes) could be placed into distinct categories, identifying genes involved each step in the regenerative process 1. Wound healing and initiation of regeneration (blastema initiation) 2. Neoblast maintenance (homeostasis) 3. Proliferation (growing the blastema) 4. Differentiation and patterning 5. Morpholaxis (integrating new tissues with old) 6. Non-regenerative homeostasis  The RNAi screen identified putative candidates in regeneration pathway including members of the piwi family of genes  Piwi – originally identified as encoding regulatory proteins responsible for maintaining incomplete differentiation in stem cells and maintaining the stability of cell division rates in germ line cells  Reddien et al decided to pursue smedwi genes due to their sequence similarity to piwi family of genes  There are two smedwi genes in the planaria genome o Expressed in neoblasts 1 LECTURE 7 LIFESCI 2A03 o When disrupted through RNAi, has an effect on regenerative process A. BrdU identifies mitotically active cells (neoblasts) B. In situ hybridization to smedwi-1 mRNAs C. In situ hybridization to smedwi-2 mRNAs o Expression pattern of the smedwi genes from both histone H2 staining and BrdU staining has similar patterns (no staining at anterior tip, pattern of neoblasts) o No staining and cannot regenerate in space near pharynx, or near anterior tip – neoblasts not present  Irradiation eliminated neoblasts and smedwi-2 expressing cells (F, G, H has irradiation) o F – gene H.1.3.B (expressed in muscle cells); gene not involved n neoblast function o G – smedwi-1 o H – smedwi-2 o Not seeing expression of smedwi genes after irradiation o Irradiation did not turn off all transcription – seen in F, in situ hybridization of H.1.3.B gene  transcription can occur after irradiation o Irradiation prevents proliferation of neoblasts and prevents function of the neoblasts – smedwi-1, smedwi-2 are not expressed after irradiation  smedwi may be expressed in neoblasts  RNAi – Decided to pursue smedwi genes due to their sequence similarity to piwi family of genes which have a known role in stem cells o Smedwi-1 RNAi did not produce robust effects; regeneration occurs o Smedwi-2 RNAi produced effects similar to irradiated animals; regeneration does not occur  pursue this gene o Figure – left no regeneration; right intact animal 1. Initiation of regeneration (blastema formation); wound healing, blastema beginning to form; is smedwi-2 required for this step? o Question: Is SMEDWI-2 required for the first step of regeneration? o Null Hypothesis  The elimination of smedwi-2 expression has no effect on the ability to form a blastema  There is no significant difference between the treatment and the control o Control – blastema formation o Negative control – unc-22 has no role in regeneration (expressed in differentiated muscle cells)  Negative controls are groups where no phenomenon is expected; they ensure that there is no effect when there should be no effect  We see normal regeneration  RNAi treatment against unc-22 – dotted lines are where cut surface; regeneration has occurred; not yet pigmented, undifferentiated cells 2 LECTURE 7 LIFESCI 2A03 o Control – eliminate blastema formation o Positive control – irradiation (eliminates neoblasts)  Positive controls – groups where a phenomenon is expected; that is, they ensure that there is an effect when there should be an effect, by using an experimental treatment that is already known to produce that effect (and then comparing this to the treatment that is being investigated in the experiment)  Healed wounds – no regeneration, no blastema  Yellow lines = cut site, no regeneration seen  Looks like no regeneration is occurring; curling o After negative and positive control – know what it looks like when it is regenerating (unc- 22) and when it is not regenerating (irradiation) o Describe the data and result  There is no regeneration in RNAi against smedwi-2; amputation surfaces (yellow arrows) have no blastema formation o Reject null hypothesis 6. Homeostasis o Question: is smedwi-2 required for homeostasis o Null Hypothesis  The elimination of smedwi-2 expression has no effect on the ability to maintain homeostasis i.e. maintain net cell number of time  there is no significant difference between the treatment and the control o Homeostasis – the ability of the animal to maintain its cells – differentiated cells are lost (due to aging, cell death etc), neoblasts o Irradiation disrupts homeostasis – see cell loss over time in the absence of functional neoblasts o Irradiation and amputation – no regeneration  6 days later – no regeneration  15 days later – whole animal morphology is changing; getting smaller; curling at edges o Irradiation, organism intact – see tissue loss in absence of amputation  4 days – indents on sides  8 days – getting smaller  11 days – regression and cell loss across anterior surface  14 days – looks similar to amputated animal; smaller curling, cell loss o Cell loss in both irradiated animals – need neoblasts to maintain homeostasis o Control – homeostasis formation o Negative control – unc-22 has no role in regeneration  Do not expect effect  See normal homeostasis o Control – eliminate homeostasis o Positive control – irradiation eliminates neoblast  Expect effect  Curling indicative of cell loss o Describe data and result  RNAi against smedwi-2  See curling, similar morphology to irradiation; looks different than negative control o Reject Null Hypothesis – smedwi seems to be required to maintain homeostasis; smedwi is expressed in neoblasts, remove smedwi2 causes loss of neoblast function 2. Neoblast Maintenance (and proliferation) o Neoblasts are maintained through cell division and are the only known mitotically dividing cells in planaria o If smedwi-2 RNA
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