Transgenic rats reveal functional conservation of regulatory controls between the Fugu isotocin and rat oxytocin genes

Abstract

We have asked whether comparative genome analysis and rat transgenesis can be used to identify functional regulatory domains in the gene locus encoding the hypothalamic neuropeptides oxytocin (OT) and vasopressin. Isotocin (IT) and vasotocin (VT) are the teleost homologues of these genes. A contiguous stretch of 46 kb spanning the Fugu IT-VT locus has been sequenced, and nine putative genes were found. Unlike the OT and vasopressin genes, which are closely linked in the mammalian genome in a tail-to-tail orientation, Fugu IT and VT genes are linked head to tail and are separated by five genes. When a cosmid containing the Fugu IT-VT locus was introduced into the rat genome, we found that the Fugu IT gene was specifically expressed in rat hypothalamic oxytocinergic neurons and mimicked the response of the endogenous OT gene to an osmotic stimulus. These data show that cis-acting elements and trans-acting factors mediating the cell-specific and physiological regulation of the OT and IT genes are conserved between mammals and fish. The combination of Fugu genome analysis and transgenesis in a mammal is a powerful tool for identifying and analyzing conserved vertebrate regulatory elements.

Figure 1

Schematic representation of the Fugu IT-VT locus. A 46-kb sequence (thick line) was obtained from cosmids 55F6 and 370B2. Only HindIII sites (H) are shown. Arrows represent complete genes and indicate the direction of transcription. Hatched rectangles represent the cosmid arms (not to scale). KIAA, gene with homology to a human cDNA (KIAA0264) whose function is not known; FA, fatty acid binding protein gene; SR, sepiapterin reductase gene; H1, H2, and H3, hypothetical genes.

Figure 2

(A) Expression of Fugu genes in transgenic rats. WT-Hypo, hypothalamus from wild-type rats; TG-Hypo, hypothalamus from transgenic rats; Brain (−Hypo), brain without hypothalamus. Adrenal glands and ovary from three rats were pooled to obtain adequate RNA for Northern analysis. RNA was fractionated on a 1.2% gel, transferred to nylon membrane (Amersham), and probed with various probes. Each lane contains approximately 20 μg (hypothalamus) or 50 μg (other tissues) of total RNA. IT and VT probes did not hybridize to RNA from the testis of wild-type rats (data not shown). (B) Effect of dehydration on levels of transgene expression. Each lane contains approximately 20 μg of total RNA from the hypothalamus of transgenic rat that was given tap water (control) or deprived of fluid for 3 days (dehydrated). A oligonucleotide probe recognizing rat α-tubulin (TUB) was used as internal standard for quantitation.

Figure 3

In situ hybridization of brain and testis sections from transgenic rats. Tissue sections were hybridized with ³⁵S-labeled isotocin probe. OC, optic chiasma; 3V, third ventricle; S, spermatozoa. (Left) Sections were photographed under bright-field. The same sections were photographed in the dark-field (Right) to show the distribution of silver grains. IT transcripts were found only in the SON and PVN of the hypothalamus. No VT transcripts were found in any part of the brain. The IT probe did not hybridize to brain sections from the wild-type rats. The antisense oligonucleotide for IT probe did not bind to any cells in the brain or testis from transgenic rats (not shown).

Figure 4

Double in situ hybridization analysis. Adjacent sections of brain were hybridized simultaneously with ³⁵S-labeled IT probe and digoxigenin-labeled OT probe (IT-OT) or with ³⁵S-labeled IT probe and digoxigenin-labeled VP probe (IT-VP). Silver grains represent the hybridized radioactive probe, and the gray-brown patches are the result of color reaction of the hybridized digoxigenin probe. The IT transcripts colocalize with endogenous OT transcripts in the SON and PVN, but not with the VP transcripts. OC, optic chiasma.

Figure 5

A simple model to show the rearrangement in the IT-VT locus. The open arrows represent genes (VT; IT; FA, fatty acid binding protein; SR, sepiapterin reductase; and ZF) and indicate the direction of their transcription.

Figure 6

Conserved sequences in the promoter and 3′ regions of the Fugu IT and the rat OT genes. Homologous regions from the Fugu IT-VT locus and the rat OT-VP locus (7) were compared by using Martinez/Needleman–Wunsch algorithm of the lasergene software (DNAstar, Madison, WI). Only sequences that show at least seven conserved bases at a stretch are shown. The numbers in A and B refer to the position of the bases in relation to the first base of the translation initiation codon and the termination codon, respectively. ∗ denote conserved bases.