Genes regulated by the transcription factor Xrx1: a microarray analysis

Eye development is a multistep process that requires specific inductive signals and precise morphogenetic movements, starting in a well-definite region of the anterior neural plate, the eye field. Recently, it was demonstrated that an elaborate gene network of eye field transcription factors (EFTFs) contributes to specify the neural and retinal fate of the eye field. Among these EFTFs, Xrx1 is involved in proliferation and neurogenesis in the eye field and is necessary for the correct development of the retina . By means of Affymetrix microarrays, a high throughput screening was performed, looking for genes that can mediate the function of Xrx1, by comparison of the expression profiles of whole embryos in which the Xrx1 function was either overexpressed or down-regulated. A suitable experimental design has been designed, and a PCR-screening procedure was developed to correctly select embryos microinjected either with Xrx1 mRNA (gain of function) or Morpholino antisense oligonucleotides (loss of function). The selected injected embryos were used in the microarray experiments in five biological replicates for each condition for higher statistical consistency. The intensity data were resumed, normalized and analyzed via the GC-RMA algorithm: a row by row T-test between control and injected groups revealed 793 differentially expressed transcripts for the gain of function experiment and 1893 differentially expressed transcripts for the loss of function experiment. 122 transcripts were present in both gene lists. A complete reannotation, based on the Xenopus laevis Unigene #73 was performed for each transcript via newly developed algorithm in a fraction of the time needed by other applications. The analysis of the resulting datalists showed that more than 30% of the identified transcripts was poorly or not annotated. An initial exploration of annotated transcripts in the in the gain of function gene list showed several transcripts involved in patterning, cell proliferation and cell signaling. Attention was focused as well on genes whose expression was coherently affected in the experiments (i.e. genes activated in gain of function and repressed in loss of function experiments, and vice-versa). An initial pool of candidate targets was selected among the common list of 122 genes : the relative EST clones were transcribed into antisense probes and used for whole mount in situ experiments on embryos at different developmental stages (13, 16, 19, 27, 33/34 and fully developed sectioned eyes) for a first validation of the microarray data. Whole mount hybridizations of non annotated ESTs provided as well confirmations for the consistency of the microarray data: many ESTs resulted actually transcribed within the Xrx1 expression domain, including the eye, eye-field or diencephalic structures. Selected candidate transcripts from the microarray experiments, showing an overlapping expression domain with Xrx1 in the developing retina and/or in the ciliary marginal zone will be tested for the ability to respond to Xrx1 overexpression by in situ hybridization. Their identity and predicted molecular function will be defined based on BLAST analysis, Gene Ontology and NCBI Unigene annotation. An inducible GR-Xrx1 construct will be used to assess if the candidate transcripts are direct targets of Xrx1.