Identification of the endostyle as a stem cell niche in a colonial chordate.

Animals

Colonies of Botryllus schlosseri were maintained as described ( Boyd et al., 1986 ). Colonies were chosen for the experiments as described in supplementary . In vivo fluorescent cell labeling assay Cell drawing, labeling and transplanting were performed under a microscope (Diaphot 200, Nikon, NY, USA). We used an air compressed microinjector (PLI-188, Nikon, NY, USA) and a glass needle micropipette (50–60μm diameter sharp tip) to draw cells from one of the tested sites. Cells were drawn into a glass micropipette which contained 1μl Vybrant DiD dye solution (emission-665nm; Molecular Probes, Eugene OR USA) diluted in a tunicate saline buffer (TS; Negm et al., 1991 ; details in supplemental ). The cells in the micropipette were counted under a microscope, incubated for 5 minutes (room temperature), and then 10–40 cells (~0.5μl), were injected back into the relevant tested sites ( STable 1 ). As a control, 0.5μl of the dilution solution (TS) was injected. In addition we performed heterotypic transplantation experiments where ~40 cells taken from the vasculature or zooid lateral wall, were labeled and injected into the EN ( STable 1 ). In several colonies, including the control groups, the vasculature (marginal vessel and ampullae) was dissected away from the colonies following cell labeling (n=13, STable 1 ).

Imaging

Time lapse imaging was performed by an automated microscopy (ImageXpress, Molecular devices Corp., Palo Alto, CA) as described ( Voskoboynik et al., 2007 ). Phase contrast images and fluorescent images (cy5, maximum emission at 670nm; Fig 3 ) at varying magnifications were performed every 30–90 minutes during the first 5 days following labeling and twice a day thereafter (days 6–8). Time-lapse sequences were generated from consecutive images of same location. Proliferation/migration and fluorescent intensity measurements Labeled cells in organs (zooid, bud, vasculature) were counted using Image J software, version 1.32j (NIH, USA). Proliferation migration ratios and fluorescent intensity were calculated on fluorescent images that were taken from the labeling sites, 2, 10 and 30 hours following labeling ( Fig 3 ; detailed description in supplemental ). Transplantation experiments Cells and hemolymph from each of the tested organs (EN, stomach and ampullae; STable 2 ) were drawn into a glass micropipette as described above. 5–20 cells were microinjected into the EN or the bud of the recipient colony ( STable 2 ). Cells were counted under the microscope while being transplanted into the colonies semitransparent body (Diaphot 200, Nikon, NY, USA).

Genotyping

Somatic and germ tissues were collected every month, 1–6 months following transplantation. Samples were dissected and flash frozen in liquid nitrogen. 259 DNA samples were extracted using a modified version of the Hoss and Pabbo protocol (1993) as described ( De Tomaso et al., 1998 ). The samples were screened for polymorphism using amplified fragment length polymorphism (AFLP’s). AFLP was preformed as described ( Vos et al., 1995 ; Rinkevich et al., 1998 ; details in supplementary ).

Histology

Colonies were fixed and sections from paraplast embedded tissues were made, cross and longitudinal sections (5μm) were stained with Azan Heidenhain or Hematoxyline Eosin as described ( Moiseeva et al., 2004 ). PCNA immunohistochemistry staining and Raldh in situ hybridization were preformed as described ( Rinkevich et al., 2007 ).

Confocal analysis Vybrant

DiD labeled colonies (n=7) were fixed in 4% paraformaldehyde at 4°C (6–8 hours). Colonies were washed with PBS, cryoprotected overnight in 30% sucrose, and quick-frozen in optimum cutting temperature (OCT) compound. Frozen sections (5–7um) were cut at −20°C from OCT-embeded tissues using a microtome (Bright Instruments, Huntington, UK). Nuclei were stained with Hoechst 33342 (Molecular Probes; 1uM for 2 min) and washed with PBS. Samples were analyzed by laser scanning confocal microscopy (Lecia SP2 AOBS confocal laser scanning microscope with 5 lasers lines).

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