Zebrafish Hsp70 is required for embryonic lens formation.
Heat shock proteins (Hsps) were originally identified as proteins expressed after exposure of cells to environmental stress. Several Hsps were subsequently shown to play roles as molecular chaperones in normal intracellular protein folding and targeting events and to be expressed during discrete periods in the development of several embryonic tissues. However, only recently have studies begun to address the specific developmental consequences of inhibiting Hsp expression to determine whether these molecular chaperones are required for specific developmental events. We have previously shown that the heat-inducible zebrafish hsp70 gene is expressed during a distinct temporal window of embryonic lens formation at normal growth temperatures. In addition, a 1.5-kb fragment of the zebrafish hsp70 gene promoter is sufficient to direct expression of a gfp reporter gene to the lens, suggesting that the hsp70 gene is expressed as part of the normal lens development program. Here, we used microinjection of morpholino-modified antisense oligonucleotides (MOs) to reduce Hsp70 levels during zebrafish development and to show that Hsp70 is required for normal lens formation. Hsp70-MO-injected embryos exhibited a small-eye phenotype relative to wild-type and control-injected animals, with the phenotype discernable during the second day of development. Histological and immunological analysis revealed a small, underdeveloped lens. Numerous terminal deoxynucleotidyl transferase-mediated dUTP-fluoroscein nick-end labeling (TUNEL)-positive nuclei appeared in the lens of small-eye embryos after 48 hours postfertilization (hpf), whereas they were no longer apparent in untreated embryos by this age. Lenses transplanted from hsp70-MO-injected embryos into wild-type hosts failed to recover and retained the immature morphology characteristic of the small-eye phenotype, indicating that the lens phenotype is lens autonomous. Our data suggest that the lens defect in hsp70-MO-injected embryos is predominantly at the level of postmitotic lens fiber differentiation, a result supported by the appearance of mature lens organization in these embryos by 5 days postfertilization, once morpholino degradation or dilution has occurred.
Evans
Tyler G
TG
Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5.
Yamamoto
Yoshiyuki
Y
Jeffery
William R
WR
Krone
Patrick H
PH
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism / Q000473:pathology; D004625:Embryo, Nonmammalian; D047108:Embryonic Development; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism*; D007150:Immunohistochemistry; D007908:Lens, Crystalline / Q000196:embryology* / Q000637:transplantation; D008845:Microinjections; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014184:Transplantation, Homologous; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
D018840:HSP70 Heat-Shock Proteins; D016376:Oligonucleotides, Antisense