Psf2 plays important roles in normal eye development in Xenopus laevis.
OBJECTIVE
Psf2 (partner of Sld5 2) represents a member of the GINS (go, ichi, ni, san) heterotetramer [1] and functions in DNA replication as a "sliding clamp." Previous in situ hybridization analyses revealed that Psf2 is expressed during embryonic development in a tissue-specific manner, including the optic cup (retina) and the lens [2]. This article provides an analysis of Psf2 function during eye development in Xenopus laevis.


METHODS
A morpholino targeted to Psf2 mRNA was designed to knockdown Psf2 translation and was injected into specific embryonic cells during early cleavage stages in the frog, Xenopus laevis. Injected embryos were assayed for specific defects in morphology, cell proliferation, and apoptosis. Synthetic Psf2 RNA was also co-injected with the morpholino to rescue morpholino-mediated developmental defects. It is well known that reciprocal inductive interactions control the development of the optic cup and lens. Therefore, control- and morpholino-injected embryos were used for reciprocal transplantation experiments to distinguish the intrinsic role of Psf2 in the development of the optic cup (retina) versus the lens.


RESULTS
Morpholino-mediated knockdown of Psf2 expression resulted in dosage-dependent phenotypes, which included microphthalmia, incomplete closure of the ventral retinal fissure, and retinal and lens dysgenesis. Defects were also observed in other embryonic tissues that normally express Psf2 including the pharyngeal arches and the otic vesicle, although other tissues that express Psf2 were not found to be grossly defective. Eye defects could be rescued by co-injection of synthetic Psf2 RNA. Examination of cell proliferation via an antibody against phospho-histone H3 S10P revealed no significant differences in the retina and lens following Psf2 knockdown. However, there was a significant increase in the level of apoptosis in retinal as well as forebrain tissues, as revealed by TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay.


CONCLUSIONS
The results demonstrate intrinsic roles for Psf2 in both retinal and to a lesser extent, lens tissues. Observed lens defects can mainly be attributed to deficiencies in retinal development and consequently the late phase of lens induction, which involves instructive cues from the optic cup. Developmental defects were not observed in all tissues that express Psf2, which could be related to differences in the translation of Psf2 or redundant effects of related factors such as proliferating cell nuclear antigen (PCNA).
Walter
Brian E
BE
Biology Department, Illinois Wesleyan University, Bloomington, IL, USA.
Perry
Kimberly J
KJ
Fukui
Lisa
L
Malloch
Erica L
EL
Wever
Jason
J
Henry
Jonathan J
JJ
D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004475:Ectoderm / Q000187:drug effects / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000002:abnormalities / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D005123:Eye / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism / Q000473:pathology; D005124:Eye Abnormalities / Q000196:embryology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D007908:Lens, Crystalline / Q000002:abnormalities / Q000187:drug effects / Q000637:transplantation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D012313:RNA; D012160:Retina / Q000187:drug effects / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*
D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D016376:Oligonucleotides, Antisense; C489515:TAP2 protein, Xenopus; D029867:Xenopus Proteins; D012313:RNA