The Rx-like homeobox gene (Rx-L) is necessary for normal photoreceptor development.
OBJECTIVE
The retinal homeobox (Rx) gene plays an essential role in retinal development. An Rx-like (Rx-L) gene from Xenopus laevis has been identified. The purpose of this study was to analyze the function of Rx-L in the developing retina.


METHODS
DNA-binding properties of Rx-L were analyzed by electrophoretic mobility shift assay (EMSA), with in vitro-translated proteins and radiolabeled oligonucleotide probe. The Rx-L expression pattern was analyzed by in situ hybridization using whole or sectioned embryos and digoxigenin-labeled antisense riboprobes. Rx-L loss of function was studied by using antisense morpholino oligonucleotides targeted to the Rx-L translation initiation site. Embryos injected with control or Rx-L morpholinos were analyzed at stage 41 or 45.


RESULTS
Rx-L shares homology with Rx at the homeo-, OAR, and Rx domains, but lacks an octapeptide motif. Rx-L is expressed in the developing retina beginning in the early tailbud stage. In the maturing retina, Rx-L expression is restricted primarily to the developing photoreceptor layer and the ciliary marginal zone. Rx-L can bind a photoreceptor conserved element-1 (PCE-1) oligonucleotide, an element conserved among all known photoreceptor gene promoters. In a promoter activity assay, Rx-L functions as a stronger transcriptional activator than Rx. Antisense morpholino-mediated knockdown of Rx-L expression resulted in a decrease in rhodopsin and red cone opsin expression levels in Xenopus retinas. Injection of the Rx-L antisense morpholino oligonucleotide also resulted in a decrease in the length of both rod and cone outer segments.


CONCLUSIONS
The results suggest that Rx-L functions to regulate rod and cone development by activating photoreceptor-specific gene expression.
Pan
Yi
Y
Center for Molecular and Human Genetics, Columbus Children's Research Institute, Ohio State University, Columbus, OH 43205, USA.
Nekkalapudi
Srivamsi
S
Kelly
Lisa E
LE
El-Hodiri
Heithem M
HM
D000595:Amino Acid Sequence; D000818:Animals; D024202:Electrophoretic Mobility Shift Assay; D005136:Eye Proteins / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000502:physiology*; D012160:Retina / Q000196:embryology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics*; D014982:Xenopus laevis
D005136:Eye Proteins; D018398:Homeodomain Proteins; D015345:Oligonucleotide Probes; D014157:Transcription Factors