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The human insulin-family genes regulate cell growth, metabolism, and tissue-specific functions. Among these different members, only INSL4 gene shows a predominant placenta-specific expression. Here, we show that the human INSL4 gene is tightly clustered with three other members of the human insulin superfamily (RLN1, RLN2, and INSL6) within a 176-kilobase genomic segment on chromosome region 9p23.3‐p24.1. We also report evidence that INSL4 is probably the only insulin-like growth factor gene to be primate-specific. We identified an unexpected human endogenous retrovirus (HERV) element inserted into the human INSL4 promoter with a sequence similar to that of env gene, flanked by two long terminal repeats(LTRs). The emergence of INSL4 gene and genomic insertion of HERV appear to have occurred after the divergence of New World and Old World monkeys (;45 million years ago). Transient transfection experiments showed that the placenta-specific expression of INSL4 is mediated by the 39 LTR of the HERV element, and that the latter may have a major role in INSL4 up-regulation during human cytotrophoblast differentiation into syncytiotrophoblast. Finally, we identified an INSL4 alternatively spliced mRNA species that encodes putative novel INSL4-like peptides. These data support the view that ancient retroviral infection may have been a major event in primate evolution, especially in the functional evolution of the human placenta. gene regulation, insulin, placenta, pregnancy, relaxin

Polypeptide growth factors play an important role in the regulation of human embryonic development. Insulin-like 4 gene (INSL4) is a member of the insulin family, which includes insulin, IGF-I, IGF-II, relaxin, and INSL3. Using RT-PCR, we previously found abundant INSL4 mRNA in the human placenta. In this study, we examined the chronology and spatial expression of this gene in sections of human placenta and conceptus by means of in situ hybridization. Expression of the IGF-II gene was studied as a positive control. INSL4 distribution was tissue- and cell-specific. Indeed, INSL4 mRNA was most abundant in syncytiotrophoblast cells. In fetal tissues, INSL4 mRNA was identified in the perichondrium of all four limbs, vertebrae, and ribs. Moreover, INSL4 mRNA was abundant in interbone ligaments. These findings indicate that the INSL4 gene may play an important role in trophoblast development and regulation of bone formation. IGF-II mRNA, in agreement with the literature, are mainly located in the mesodermal core in the villous trophoblast and in most embryonic tissues. Mol. Reprod. Dev. 51:123-129, 1998. © 1998 Wiley-Liss, Inc.

The relaxin/insulin-like (RLN/INSL) gene family comprises a group of signaling molecules that perform physiological roles related mostly to reproduction and neuroendocrine regulation. They are found on three different locations in the mammalian genome, which have been called relaxin family locus (RFL) A, B, and C. Early in placental mammalian evolution, the ancestral proto-RLN gene at the RFLB locus underwent successive rounds of small-scale duplications resulting in variable number of paralogous genes in different placental lineages. Most placental mammals harbor copies of the RLN2 and INSL6 paralogs in the RFLB. However, the origin of an additional paralog, INSL4 (also known as placentin), has been controversial as its phyletic distribution does not converge with its phylogenetic position. In principle, by searching for INSL4 genes in representative species of all major groups of mammals we can gain insights into when the gene originated and better reconstruct its evolutionary history. Here we identified INSL4 pseudogenes in two laurasiatherian, (alpaca and dolphin) and one xenarthran (armadillo) species. Phylogenetic and synteny analyses confirmed that the identified pseudogenes are orthologs of INSL4. According to these results, the proto-RLN gene in the RFLB underwent two successive tandem duplications which gave rise the INSL6 and INSL4 paralogs in the last common ancestor of placental mammals. The INSL4 gene was subsequently inactivated or lost from the genome in all placentals other than catarrhine primates, where its product became functionally relevant. Our results highlight the contribution of relatively old gene duplicates to the gene complement of extant species.

The relaxin/insulin-like gene family is related to the insulin gene family, and includes two separate types of peptides: relaxins (RLNs) and insulin-like peptides (INSLs) that perform a variety of physiological roles including testicular descent, growth and differentiation of the mammary glands, trophoblast development, and cell differentiation. In vertebrates, these genes are found on three separate genomic loci, and in mammals, variation in the number and nature of genes in this family is mostly restricted to the Relaxin Family Locus B. For example, this locus contains a single copy of RLN in platypus and opossum, whereas it contains copies of the INSL6, INSL4, RLN2 and RLN1 genes in human and chimp. The main objective of this research is to characterize changes in the size and membership composition of the RLN/INSL gene family in primates, reconstruct the history of the RLN/INSL genes of primates, and test competing evolutionary scenarios regarding the origin of INSL4 and of the duplicated copies of the RLN gene of apes. Our results show that the relaxin/INSL-like gene family of primates has had a more dynamic evolutionary history than previously thought, including several examples of gene duplications and losses which are consistent with the predictions of the birth-and-death model of gene family evolution. In particular, we found that the differential retention of relatively old paralogs played a key role in shaping the gene complement of this family in primates. Two examples of this phenomenon are the origin of the INSL4 gene of catarrhines (the group that includes Old World monkeys and apes), and of the duplicate RLN1 and RLN2 paralogs of apes. In the case of INSL4, comparative genomics and phylogenetic analyses indicate that the origin of this gene, which was thought to represent a catarrhine-specific evolutionary innovation, is as old as the split between carnivores and primates, which took place approximately 97 million years ago. In addition, in the case of the RLN1 and RLN2 genes of apes our phylogenetic trees and topology tests indicate that the duplication that gave rise to these two genes maps to the last common ancestor of anthropoid primates. All these genomic changes in gene complement, which are particularly prevalent among anthropoid primates, might be linked to the many physiological and anatomical changes found in this group. Given the various roles of members of the RLN/INSL-like gene family in reproductive biology, it might be that changes in this gene family are associated to changes in reproductive traits.

The insulin like growth factor 4 (INSL4) gene belongs to the insulin gene superfamily and has been mapped by fluorescent in situ hybridization to 9p24. Expression of INSL4, of unknown function, has been recently detected in the perichondrium and ligaments of the human embryo. Here we have mapped INSL4 within the framework of a partial YAC contig covering the distal part of 9p to find out whether this gene lies within the critical region defined for the monosomy 9p syndrome. The gene was also located using a human-rodent radiation hybrid panel. INSL4 was found to be distal to the del(9p) critical region and excluded as a candidate for the syndrome. In addition, the positions of two relaxin genes (RLN1, RLN2) that belong to the same superfamily as INSL4 and which have also been mapped to chromosome 9, were refined. We have shown that the three genes are clustered in the same region.

A new member of the insulin gene superfamily was identified by screening a subtracted cDNA library of first-trimester human placenta and, hence, was tentatively named early placenta insulin-like peptide (EPIL). In this paper, we report the cloning and sequencing of the EPIL cDNA and the EPIL gene (INSL4). Comparison of the deduced amino acid sequence of the early placenta insulin-like peptide revealed significant overall and structural homologies with members of the insulin-like hormone superfamily. Moreover, the organization of the early placenta insulin-like gene, which is composed of two exons and one intron, is similar to that of insulin and relaxin. By in situ hybridization, the INSL4 gene was assigned to band p24 of the short arm of chromosome 9. RT-PCR analysis of EPIL tissue distribution revealed that its transcripts are expressed in the placenta and uterus.