Failed blastema formation in nbl. (A) Whole-mount in situ hybridization and longitudinal sections of msxb in WT and nbl fin regenerates at 24 hpa. msxb (red arrowhead) marks early blastema cells. Note the absence of msxb expression in nbl, indicating failure of early blastema formation. (B) Sections from WT and nbl regenerates immunostained for BrdUrd (green) and DAPI (blue). Fish were treated with BrdUrd for 6 h before harvesting at 24 hpa. No blastema is observed in nbl regenerates at 33°C. However, mesenchymal cell proliferation in nbl is slightly increased. Epidermal cell proliferation in nbl is comparable to WT at 33°C. Data in graph are the mean ± SEM; n = 7; *, P < 0.01. (C) Sections from WT and nbl regenerates immunostained for active Caspase-3 (red) and DAPI (blue). WT and nbl fish regenerated for 16 h at 25°C followed by a 4-h incubation at 33°C. Note there are Caspase-3-positive cells proximal to the amputation plane in nbl. Normally, this is where mesenchymal tissue disorganization occurs, a process that immediately precedes early blastema formation.

hsp60 is the nbl gene. (A) Genetic and physical map of nbl. Linkage map (Top), transcripts (Middle), and physical map (Bottom) places nbl between z54324 and z7144. Numbers in parentheses show recombination events from 2,302 meioses between nbl phenotype and linked genetic markers. (B) DNA sequence analysis from nbl and WT strains revealed a unique thymidine to adenosine mutation in nbl, causing valine-324 to glutamic acid missense mutation in Hsp60 (red arrows). (C) Multiple species alignment of the hsp60 region containing the V324E mutation. Note complete conservation of valine-324 among species, including E. coli. (D) Ribbon drawing structure of one subunit in the cis GroEL (Hsp60) ring. Figures were generated by using molmol (32) and coordinates from Protein Data Bank file 1AON (33). The location of the mutation site is shown in red space-filling form in the apical domain (light blue). Intermediate (green) and equatorial domains (blue) are also shown. (E) Northern blot analysis of hsp60 expression in regenerating caudal fin. hsp60 levels are up-regulated during blastema formation at both 25°C and 33°C. β-actin expression shown as a control. (F) Whole-mount in situ hybridization of hsp60 during regeneration. Fish were incubated at 25°C to ensure expression is not induced by heat-shock treatment. hsp60 expression is first induced beneath the wound epidermis, where mesenchymal tissue disorganization occurs at 24 hpa. At 48 hpa, hsp60 is expressed in the newly formed blastema. At 120 hpa, hsp60 expression is up-regulated in the distal blastema where msxb is expressed.

GroEL V300E causes reduced chaperone function. (A) The E. coli strain KY1156 (26) requires functional GroEL for growth. GroEL V300E is analogous to Hsp60 V324E. KY1156 transfected with GroEL V300E decreased growth at 25°C, suggesting this mutation reduces GroEL function. (B) GroEL V300E shows decreased affinity for nonnative form of LDH. LDH activity was measured after addition of nonpermissive solution (without ATP at 42°C) at time 0 in the presence of mutant (blue) or WT GroEL (red). Negative control was without GroEL (orange).

nbl, a temperature-sensitive regeneration mutant. (A) Whole-mount WT and nbl caudal fin regenerates at 0 and 7 dpa. nbl shows a clear block in fin regeneration. Red dashed lines demarcate amputation plane. Red arrows show clotted blood in nbl.(B) Sections of regenerating fins stained with hematoxylin at 3, 6, 16, and 24 hpa (33°C). A fluid- or bloodfilled space separates the wound epidermis from mesenchyme in nbl (black arrow). nbl fins fail to form blastema (red arrow; 24 hpa). (C) Heart sections 7 dpa stained with acid fuchsin-orange G to stain fibrin (orange) and collagen (blue) deposits. nbl ventricle did not initiate cardiac regeneration. Yellow dashed line demarcates the amputation plane. (D) Heart regeneration at 12 dpa. Myocyte enhancer factor 2 staining marks cardiomyocytes (red), and BrdUrd (green) identifies cycling cells. A low percentage of cardiomyocytes incoporate BrdUrd (orange) in WT. No evidence of myocyte or nonmyocyte proliferation was observed in nbl hearts.

Failed blastema maintenance in nbl.(A) Whole-mount in situ hybridization of msxb in WT and nbl fin regenerates. Fish were incubated for 5 days at 25°C before an 8-h incubation at 33°C. msxb expresson is absent in the nbl distal blastema (red arrowhead). (B) Sections of WT and nbl regenerates immunostained for BrdUrd (green) and DAPI (blue). Fish were incubated with BrdUrd during the final 2 h at 33°C. Note that an 8-h incubation at 33°C does not affect mesenchymal and epidermal cell proliferation levels during regenerative outgrowth but distal blastemal cells disappear in nbl (red arrowhead). (C) Apoptosis of blastema cells in nbl. WT and nbl fish regenerated for 5 days at 25°C followed by 4 h at 33°C. Fins were immunostained with active Caspase-3 (red) and DAPI (blue). Note that distal blastema cells undergo apoptosis in nbl. (D) Electron micrographs of distal blastema in WT and nbl fish. Fins regenerated for 5 days at 25°C followed by 8-h incubations at 33°C. Note the larger mitochondria with empty matrix (red arrowhead) in nbl mutants. (Bars, 2 μm.)

hsp60 rescues nbl.(A) nbl embryos raised at 33°C develop pericardial effusion and die at 5 dpf. Embryos from homozygous nbl crosses were injected with 1 nl of 200 ng/μl mRNA (hsp60WT, hsp60V324E, or enhanced GFP) and then transferred to 33°C. Blue arrows mark pericardial effusion; red arrow shows normal cardiac anatomy. Note that hsp60WT mRNA rescues the pericardial effusion phenotype. (B) nbl embryos were scored for the presence of pericardial effusion and ability to swim at 5 dpf. Hsp60WT mRNA rescues the nbl phenotype. (C) WT embryos injected with morpholino against hsp60 show pericardial effusion at 3 dpf at 28.5°C. Blue arrows mark pericardial effusion. These results indicate that hsp60 dysfunction, and not heat shock, caused the nbl phenotype.