Misexpression of Xsiah-2 induces a small eye phenotype in Xenopus.

Recent data demonstrate a structural and functional conservation of factors crucial for the development of the insect and the vertebrate eye. We isolated Xenopus siah-2, a protein with 67% identity to Drosophila sina (seven in absentia) and 85% identity to the mouse and human siah-2 proteins. Sina is required in Drosophila for the R7 photoreceptor cell formation during eye development, because it down regulates proteins that inhibit R7 differentiation via the ubiquitin/proteasome pathway. Nothing is known about the developmental function of the siah protein in vertebrates. We show that in Xenopus siah-2 is expressed maternally and is later restricted to the brain, spinal cord and the developing and mature eye. To demonstrate that the vertebrate factor participates in the process of eye formation we over expressed Xsiah-2 during Xenopus development and observed the formation of a small eye phenotype. The vertebrate counterpart of a C-terminal loss of function sina mutant, that causes a deficiency of the R7 photoreceptor cells in Drosophila, induces in Xenopus also smaller eyes. The small eyes are characterized by a reduced size of the lens, the retina and the pigmented epithelium. As this phenotype has been also described for flies expressing sina ectopically, the data demonstrate the functional and structural conservation of Xsiah-2 and sina in metazoan eye development.

Ectopic pigmentation in Xenopus in response to DCoH/PCD, the cofactor of HNF1 transcription factor/pterin-4alpha-carbinolamine dehydratase.

DCoH, the dimerization cofactor of the HNF-1 homeodomain proteins (hepatocyte nuclear factor-1alpha and beta), is involved in gene expression by associating with these transcription factors. The protein also called PCD for pterin-4alpha-carbinolamine dehydratase is a bifunctional factor as it catalyzes also the regeneration of tetrahydrobiopterin. This coenzyme is used by the enzyme phenylalanine hydroxylase, which generates tyrosine, the precursor of catecholamines and melanin. DCoH/PCD presumably cooperates with other partners, because it is expressed earlier than HNF1 and phenylalanine hydroxylase (PAH) in early vertebrate development. It is also found in cells lacking HNF1 and PAH like skin, brain and the pigmented epithelium of the eye suggesting a yet unknown function. We show that the overexpression of DCoH/PCD in Xenopus induces the formation of ectopic pigment cells in the epidermis, that are visible earlier than the endogenous pigmentation and broader distributed. This ectopic pigmentation is accompanied by an increase in tyrosinase activity and the amount of melanin. Overexpression of DCoH/PCD induces the appearance of pigment cells also in animal cap explants, that normally differentiate into atypical epidermis. DCoH/PCD mutants with impaired carbinolamine dehydratase activity retain the potential to induce pigmentation and we propose therefore that DCoH/PCD is not simply an essential enzyme for melanin biosynthesis, but also a regulator for the differentiation of pigment producing cells.

Loss of HNF1alpha function in human renal cell carcinoma: frequent mutations in the VHL gene but not the HNF1alpha gene.

Human renal cell carcinoma (RCC) is a common malignant disease of the kidney characterized by dedifferentiation of renal epithelial cells. Our previous experiments showed that most RCCs have a loss of function of the tissue-specific transcription factor hepatocyte nuclear factor (HNF) 1alpha. Detailed analyses of the 10 exons encoding HNF1alpha in 32 human RCCs by single-strand conformation polymorphism analysis and direct DNA sequencing revealed no tumor-associated mutation, whereas with the same probes we frequently found mutations in the von Hippel-Lindau tumor suppressor gene. No mutation leading to loss of HNF1alpha function was detected by analyzing the integrity of the HNF1alpha transcripts in the RNA derived from RCCs by the protein truncation test. Investigating human RCC cell lines by western blotting and gel retardation assays showed a dramatic loss in the expression of the tissue-specific transcription factor HNF1alpha in eight of 10 cell lines. As the HNF1alpha-related transcription factor HNF1beta was expressed in all these tumor cell lines, the loss of HNF1alpha expression was a specific event and was maintained in RCC cell lines. The loss of HNF1alpha expression in RCC cell lines on the RNA level was confirmed by reverse transcription polymerase chain reaction. We propose that tumor-associated mutations in the HNF1alpha gene do not occur in human RCC and that the loss of function is partially due to a transcriptional inactivation of the HNF1alpha gene.