Figure 1.
Botryllus schlosseri Morphology.
(A) Dorsal and (A′) ventral view of adult Botryllus schlosseri colony showing individual adult animals known as zooids (z), asexual propagating buds (b) terminal projections of the extracorporeal vasculature known as ampullae (a) all encompassed in a cellulose based extracellular matrix known as the tunic (t). (B) Dorsal and (B′) ventral view of the inset showing detail of Botryllus colony including the gut (g) located in the individual zooids (z). (C) Dorsal and (C′) ventral view of GFP injected colony showing the peripheral ampullae (a) and marginal vessel (mv) which are part of the extracorporeal vasculature that connects individual zooids and buds through a common blood supply, distinguished from the auto-fluorescent pattern of pigmented cells in the zooid (asterisk). Scale bars: A = 1 mm, B–C = 500 µm.
Figure 2.
Cells within vascular tissue exhibit stochastic regenerative proliferation.
(A) Dorsal view of adult Botryllus schlosseri colony prior to ampullaectomy surgery. (B) Dorsal view of the same colony immediately following ampullaectomy surgery (dashes) to remove peripheral ampullae and portions of the marginal vessel. (C) Ventral view of the same colony 24 hours post-ampullaectomy surgery with areas of regenerated ampullae (arrows). (D–E) Phospho-Histone H3 (pHH3) immunohistochemistry staining of peripheral ampullae (a) and marginal vessel (mv) in a control colony (D) and AMPX colony showing proliferation in the ampullae (a), marginal vessel (mv), and tissues of the zooid (z) (E). (F) pHH3 positive cell counts in gut and peripheral ampullae tissue in control and AMPX colonies (n = 6, * = p<0.05). Scale bars: A–C = 1 mm, D–E = 100 µm.
Figure 3.
Vascular cell labeling is achieved though injection of mCherry protein but not GFP.
(A) Ventral view of extracorporeal vasculature of Botryllus schlosseri immediately following injection of 1 µl of 0.1 µg/ml recombinant GFP protein into the circulation and (A′) 24 hours post injection. (B) Ventral view of extracorporeal vasculature of Botryllus schlosseri immediately following injection of 1 µl of 0.1 µg/ml recombinant mCherry protein into the circulation and (B′) 24 hours post injection. (C) Ventral view of mCherry labeled vasculature within a single system of Botryllus schlosseri and (C′) vascular labeling during a takeover event with regressing zooids (rz) and the appearance of mCherry labeling in ventral island phagocytes (arrows). (D) Ventral view of Botryllus schlosseri colony co-injected with Alexa Fluor 488 conjugated BSA and Alexa Fluor 594 conjugated Bio-Particles showing labeled vessels and phagocytic island populations (arrows). (D′) Same co-injected Botryllus schlosseri colony following apoptotic clearance of regressing zooids showing double labeling of phagocytic island populations (arrows). Scale bars: A–B = 200 µm, C = 800 µm, C′ = 1 mm, D = 800 µm.
Figure 4.
pH-stable macromolecule fluorophores are maintained in vascular lysosomes.
pH-stable macromolecules fluorophores such as mCherry (A) and FITC-conjugated dextran (B) are able to be taken up by vascular tissue and label the same internal cellular structures (arrows) as seen in animals that are co-injected with FITC-conjugated dextran and mCherry (C). mCherry fluorescence is co-localized with lysosomes in Botryllus schlosseri vasculature cells as indicated by (D) Hoechst 33342 staining of marginal vessels and peripheral ampullae, (E) LysoTracker Green stain of these vessels, (F) uptake of mCherry in these vasculature tissues, and (G) the co-localization of mCherry and LysoTracker fluorescence. Scale bars: A–B = 200 µm, C = 75 µm, D–G = 200 µm.
Figure 5.
Resident vascular cells exhibit bi-potentiality during vascular regeneration.
(A) Ventral view of a mCherry labeled Botryllus schlosseri system with labeled vasculature tissue, regressing zooids (rz) and labeled ventral phagocytic islands (arrows). (B) Ventral view of mCherry labeled system immediately flowing AMPX surgery (dots). (C) Ventral view of system 24 hours post AMPX showing regenerated labeled ampullae (arrows). (D) Ventral view of system showing complete regeneration of extracorporeal vasculature at 72 hours post AMPX. (E) Regenerated blood vessels (bv) and peripheral ampullae (am) showing mCherry labeling 72 hours post AMPX. (F) Live cell image of mCherry labeling inside cells of regenerated ampullae (am) and blood vessels (bv) counterstained with Hoechst 33342. Scale bars: A–D = 1 mm, E = 200 µm, F = 50 µm.
Figure 6.
Resident vascular cells differentially express progenitor and differentiated markers during regenerating.
(A) In situ hybridization of CD133 showing expression in developing ampullae and a small subset up blood cells (arrows). (B) In situ hybridization showing VEGFR-2 expression in both ampullae (a) and marginal vessels (mv). (C) Immunohistochemistry of Cadherin showing expression in developing ampullae (a), marginal vessels (mv), and blood cells (arrows). (D) FITC-Dextran fluorescence vs. Side Scatter FACS analysis showing the auto-fluorescence of unlabeled control animals of a Botryllus schlosseri whole cell isolation following mechanical cell dissociation (n = 12). (E) FITC-Dextran fluorescence vs. Side Scatter FACS analysis of FITC-Dextran vascular labeled animals (n = 12). (F) QRT-PCR analysis of FACS isolated FITC-dextran labeled vascular cells showing 11.0 (+/− s.e.m. = 0.26) fold up regulation of CD133, 2.2 (+/− s.e.m. = 0.35) up regulation of VEGFR-2 and 0.74 (+/− s.e.m. = 0.88) regulation of Cadherin in AMPX animals (n = 12) compared to stage matched controls (n = 12). * = p<0.01, ** = p<0.001. Scale bars: A = 50 µm, B = 70 µm, C = 50 µm.
Figure 7.
Resident vascular cells maintain individual identity in regenerating chimeras.
(A) Ventral view of two Botryllus schlosseri colonies injected with Alexa Fluor 488 conjugated BSA (left) also showing auto-fluorescence in the gut (g) and a colony injected with Alexa Fluor 594 conjugated BSA (right) prior to vascular fusion of ampullae. (B) Ventral view of the chimeric colony vasculature following fusion (arrows) 48 hours after initial interaction of ampullae. (C) Ventral view of fused vasculature indicating surgical separation (dashes) of parabiosed vasculature to generate chimeric vessels with either Alexa Fluor 488 conjugated BSA (green arrow) or Alexa Fluor 594 conjugated BSA (red arrow) labeled distal tips. (D) Ventral view of colony injected with Alexa Fluor 488 conjugated BSA labeled vasculature showing blood chimerism in ventral islands (yellow arrows) and regenerating ampullae (green arrows) 24 hours post AMPX (E) Ventral view of colony injected with Alexa Fluor 594 conjugated BSA labeled vasculature showing blood chimerism in ampullae (yellow arrows) and regenerating ampullae (red arrows) 24 hours post AMPX. (F) Ventral view of surgically separated chimeric colonies 24 hours post AMPX showing chimeric ampullae regeneration and remodeling of chimeric vessels with Alexa Fluor 488 conjugated BSA (green arrow) and Alexa Fluor 594 conjugated BSA (red arrow) distal tips. Scale bars: A = 500 µm, B = 200 µm, C = 50 µm, D–F = 250 µm.