Identification and expression of Hop, an atypical homeobox gene expressed late in lens fiber cell terminal differentiation.
OBJECTIVE
To identify transcripts expressed late in lens fiber cell maturation that might regulate fiber cell fusion, organelle degradation, or other events associated with the maturation of lens fiber cells.


METHODS
cDNA libraries were prepared from microdissected regions of chicken embryo lenses using a PCR-based method. Subtractive hybridization was used to identify transcripts expressed exclusively in fiber cells that had detached from the lens capsule. Database searches and PCR amplification with degenerate primers were used to identify human, mouse, rat, rabbit, and bovine orthologs of one such sequence and to confirm its expression in the lenses of these animals. The ability of in vitro-transcribed and translated protein to bind DNA was assessed by mobility shift assays. The locus encoding this transcript and an area about 6 kb upstream of the translation start site were sequenced. The microscopic morphology of lenses from mice in which the locus encoding this protein had been disrupted by the insertion of a nuclear-targeted bacterial lacZ sequence were analyzed. Gene expression was analyzed by PCR, in situ hybridization, and by staining for beta-galactosidase activity in lenses expressing lacZ in place of the coding sequence. Knockout lenses expressing green fluorescent protein in a mosaic pattern were sectioned in the equatorial plane and viewed with a confocal microscope to assess the presence of cell-cell fusions during fiber cell maturation.


RESULTS
Subtractive hybridization identified transcripts encoding Hop, a short, atypical homeodomain-containing protein that had previously been shown to be an important regulator of gene expression in the heart and lung. Chicken Hop did not bind to known homeodomain-binding sequences in DNA. In chicken embryos, Hop transcripts were first detected at E6. At all stages analyzed, Hop mRNA was only detected in cells that had detached from the lens capsule. Mice in which the Hop coding sequence was replaced with nuclear-targeted beta-galactosidase showed that Hop was expressed in the mouse lens in a similar pattern to the chicken lens. Characterization of lenses from mice lacking Hop revealed no morphological phenotype and no apparent defects in the degradation of nuclei or fiber cell fusion during fiber cell maturation.


CONCLUSIONS
The expression pattern of Hop provides the first evidence that new transcription is initiated in lens fiber cells after they detach from the capsule. Hop may be the first of a class of genes with this pattern of expression. Although lens abnormalities have yet to be identified in mice lacking Hop, the genomic sequences that regulate Hop expression in the lens may be useful for expressing exogenous transcripts selectively in fiber cells just before they fuse with their neighbors and degrade their organelles.
Vasiliev
Oleg
O
Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, MO 63110, USA. moisad@yandex.ru <moisad@yandex.ru>
Rhodes
Simon J
SJ
Beebe
David C
DC
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002459:Cell Fusion; D016922:Cellular Senescence; D002642:Chick Embryo; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D047108:Embryonic Development; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology / Q000196:embryology* / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D013997:Time Factors; D014158:Transcription, Genetic
D018398:Homeodomain Proteins; C466740:Hop protein, mouse