Analysis of cell lineage stability during X.laevis tadpoles tail regeneration. Dr. Cesare Gargioli Centre of Regenerative Medicine University of Bath

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1 Analysis of cell lineage stability during X.laevis tadpoles tail regeneration Dr. Cesare Gargioli Centre of Regenerative Medicine University of Bath

2 Lentivirus - virus animali a ssrna+ diploide (HIV-1, HIV-2, FIV, SIV) - ciclo replica?vo molto lento - il genoma virale a RNA viene retrotrascriao in DNA ed integrato nel genoma della cellula ospite da enzimi del virione COSTRUZIONE DI SISTEMI DI ESPRESSIONE GENICA STABILI VANTAGGI DEI VETTORI LENTIVIRALI: - Infezione di cellule quiescien? e in divisione - Elevata capacità di inserimento del transgene e sua espressione stabile Spirin et al., 2008

3 Vettori lentivirali di terza generazione VETTORI LENTIVIRALI DIFETTIVI PER LA REPLICAZIONE BASATI SUL GENOMA DI HIV-1: separazione dei faaori cisagen? (sequenze di integrazione, promotori, ecc.) da quelli trans-agen? p l a s m i d i d i impaccheaamento e dell envelope dele? delle sequenze di impaccheaamento ed integrazione eliminazione della regione enhancer-promotore al 3 -LTR eliminazione di tuu i geni Spirin et al., 2008

4 Produzione di vettori lentivirali ricombinanti VETTORI DI ESPRESSIONE UTILIZZATI: CMV-GFP ptween Dpβcatenina

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6 Regenerating Processes Preblastematic: wound healing from epidermis Blastematic: cellular dedifferentiation and proliferation Morphogenetic: cellular redifferentiation or transdifferentiation. Is Metaplasia occurring? Metaplasia: the conversion of one differentiated cell type into another

7 Tail Regeneration Wt tail transverse section 3dd reg. longitudinal section 3dd reg. notochord 3dd reg. Neural Ampulla

8 Research Aim Does metaplasia occur during Xenopus tail regeneration? Specific tail core tissue labeling by grafting tissue segment from CMV-GFP transgenic donor into Wt hosts Analysis of the GFP expression stability in the regenerating labeled tissues Investigation of the cell populations directing the tail regeneration process

9 X.laevis Transgenesis Cit. egg extract Transgene sperm nuclei Non-transgenic nuclei Transgenic nuclei 50-70% Gastrul. Eggs Embryos 10-20% 10-40% TG

10 CMV-GFP TG St. 13 CMV-GFP TG St. 27 CMV-GFP TG St. 48

11 CMV-GFP TG 5 d. post-amputation CMV-GFP TG 10 d. post-amputation CMV-GFP TG 15 d. post-amputation

12 Grafts from transgenic embryos (CMV-GFP) Neural-Plate Notochord Somites

13 Neural-Plate CMV-GFP NP St. 32 CMV-GFP NP St. 49

14 Notochord CMV-GFP Notochord St. 32 CMV-GFP Notochord St. 49

15 Somites CMV-GFP Somites St. 32 CMV-GFP Somites St. 49

16 Anti-GFP Immunostaining NP-GFP st.28 Not-GFP st.28 Som-GFP st.28 NP-GFP st.49 Not-GFP st.49 NP-GFP st.49

17 CMV-GFP NT amp. 2G9 mabαnt CMV-GFP Not amp. αcol II mabαnot CMV-GFP Som amp. 12/101 mabαsom

18 CMV-GFP NT 5 d. post-amputation CMV-GFP NT 10 d. post-amputation CMV-GFP NT 20 d. post-amputation

19 CMV-GFP Not 5 d. post-amputation CMV-GFP Not 10 d. post-amputation CMV-GFP Not 20 d. post-amputation

20 Tissue specific Promoter NβTub Col II αcaract

21 αcardact driving GFP and CRE CarAct GFP CarAct CRE CMV loxp stop loxp GFP inactive Cre recombinase CRE CMV loxp GFP X loxp active eliminated stop

22 αcardact-gfp αcardact-cre + CMV-loxSTOPlox-GFP St. 28 St. 49

23 αcardact-gfp αcardact-cre + CMV-loxSTOPlox-GFP 3 d. p.amp. 5 d. p.amp.

24 Mesodermal tissues

25 Somites Formation

26 Skin Graft (CMV-GFP) 24 hr post-transplants 3 d. p.amp. 5 d. p.amp. 7 d. p.amp.

27 LDL-diI heart injection injection 24 hr post inj. 3 d. post amp. 5 d. post amp.

28 Presomitic Mesoderm (PSM) Graft Dors Med PSM st. 13 Dors Med PSM st. 17 Dors Lat PSM st. 13 St. 28 St. 49

29 Dors Med PSM st d. post-amputation 5 d. post-amputation 10 d. post-amputation 20 d. post-amputation

30 Dors Med PSM st d. post-amputation 5 d. post-amputation 10 d. post-amputation 20 d. post-amputation

31 Dors Lat PSM st d. post-amputation 5 d. post-amputation 10 d. post-amputation 20 d. post-amputation

32 Muscle Development Myf5 + Pax3 MyoD myogenin/mrf4 Muscles Pax7 Muscle Satellite Cells

33 Muscle transversal sections GFP Imm. Med PSM st. 13 GFP Imm. Med PSM st. 17 GFP Imm. Lat PSM st. 13 Pax7 Imm. Satellite cells

34 BrdU Immunostaining Muscles trasversal sections Muscles longitudinal sections

35 Transversal muscle Immunofluorescence Pax7 Imm. GFP Imm. Pax7+GFP Imm. Pax7+GFP Imm.

36 3 d. Pax7 positive blastemal cells

37 Immunostaining anti PCNA 2 d. p.amp. Longit.sect. 5 d. p.amp. Longit.sect. Transverse section

38 Summary Host tadpoles are able to regenerate a complete and functional tail like the original Each tissue behaves independently: Notochord and Spinal Cord regenerate from the same original tissue During normal tail regeneration there is no Metaplasia The regenerating muscle fibers do not come from the stump muscle but from muscle satellite cells as the mammals. The early dorsal PSM does not have the competence to give rise to satellite cells as lateral and late PSM.

39 Xenopus regeneration bud Gargioli and Slack, Development 2004