Convergent evolution of two mammalian neuronal enhancers by sequential exaptation of unrelated retroposons

Abstract

The proopiomelanocortin gene (POMC) is expressed in a group of neurons present in the arcuate nucleus of the hypothalamus. Neuron-specific POMC expression in mammals is conveyed by two distal enhancers, named nPE1 and nPE2. Previous transgenic mouse studies showed that nPE1 and nPE2 independently drive reporter gene expression to POMC neurons. Here, we investigated the evolutionary mechanisms that shaped not one but two neuron-specific POMC enhancers and tested whether nPE1 and nPE2 drive identical or complementary spatiotemporal expression patterns. Sequence comparison among representative genomes of most vertebrate classes and mammalian orders showed that nPE1 is a placental novelty. Using in silico paleogenomics we found that nPE1 originated from the exaptation of a mammalian-apparent LTR retrotransposon sometime between the metatherian/eutherian split (147 Mya) and the placental mammal radiation (≈ 90 Mya). Thus, the evolutionary origin of nPE1 differs, in kind and time, from that previously demonstrated for nPE2, which was exapted from a CORE-short interspersed nucleotide element (SINE) retroposon before the origin of prototherians, 166 Mya. Transgenic mice expressing the fluorescent markers tomato and EGFP driven by nPE1 or nPE2, respectively, demonstrated coexpression of both reporter genes along the entire arcuate nucleus. The onset of reporter gene expression guided by nPE1 and nPE2 was also identical and coincidental with the onset of Pomc expression in the presumptive mouse diencephalon. Thus, the independent exaptation of two unrelated retroposons into functional analogs regulating neuronal POMC expression constitutes an authentic example of convergent molecular evolution of cell-specific enhancers.

Fig. 1.

nPE1 evolutionary divergence in placental mammals. (A) The tree represents phylogenetic relationships among the orthologous nPE1 enhancers of mammals. (B) Evolutionary divergence sliding-window plot of nPE1. The histogram shows the number of substitutions per site along each branch of the phylogeny displayed in A, estimated every 25 bases in 50-base intervals. Each layer of the plot represents a branch or node of the tree (numbers 1–13), and colors are maintained with respect to the tree. Substitutions were estimated from the best-fit maximum-likelihood model, which incorporates unequal equilibrium nucleotide frequencies and unequal rates of transitional and transversional substitutions (HKY85). The histograms are stacked so that the total height represents the density of substitutions over the entire phylogeny. (C) Schematic of nPE1 showing the most conserved region nPE1core in yellow. (D) Sequence alignment of nPE1core enhancer, among representative placental mammals. The shading of the alignment is based on the identity of residues and shows percentage of conservation within each column. One hundred percent identical aligned nucleotides are shaded in black. More than 80% conservation is depicted in dark gray. Columns with less than 80% and more than 60% conservation are shaded in light gray, whereas nucleotides with less than 60% are not shaded.

Fig. 2.

Functional dissection of the neuronal POMC enhancer nPE1 in transgenic mice. (A) Two structurally similar transgenes were constructed to express EGFP (green box) under the transcriptional control of the 144-bp core region of nPE1 (yellow box) or a deleted core version of nPE1 (blue boxes). The deletion of nPE2 is indicated by an asterisk, and the three mouse Pomc exons are boxed. (B) Expression analysis in the anterior (AL) and intermediate (IL) lobes of the pituitary (Pit) and the hypothalamic arcuate nucleus (Arc) in coronal sections of P1 founder transgenic mice carrying nPE1corePomc-EGFP (Left) or nPE1ΔcorePomc-EGFP (Right). (C) A coronal hypothalamic section of an nPE1corePomc-EGFP transgenic mouse shows native EGFP expression (Left) and immunolabeled ACTH neurons (red, Center). A superimposed image (Right) shows that the large majority of EGFP signal coexpresses within POMC neurons.

Fig. 3.

nPE1 is an exapted retroposon of the MaLR family. (A) Sequence alignment of the human, chimpanzee, and macaque nPE1 enhancer sequences, three representative human instances of the LTR-like retrotransposon MaLR, and the THE1B internal consensus sequence from RepBase (THE1B-int). Green filled vertical bars and white spaces represent conserved and nonconserved residues, respectively, in each column of the alignment. (B) Diagram shows nPE1 and nPE1core. (C) nPE1core region of the alignment is shown in detail. (D) Diagram shows MaLR functional regions according to ref. .

Fig. 4.

Spatiotemporal reporter gene expression driven by nPE1 and nPE2 in compound transgenic mice. (A) Two structurally similar transgenes were constructed to express either the red fluorescent protein tomato under the transcriptional control of nPE1 or EGFP by nPE2. (B) Immunofluorescence using an antibody against ACTH (red, Left) and EGFP natural fluorescence (green, Center) detected in coronal sections of nPE2Pomc-EGFP adult transgenic mice at the level of the medial basal hypothalamus at a lower (Upper) and higher (Lower) magnification. Right: Superimposed images demonstrate an almost complete penetrance of eutopic expression of the transgene in POMC (ACTH immunolabeled) neurons. White arrows denote rare ACTH immunolabeled neurons with no detectable EGFP expression. (C) Immunohistochemistry using an antibody against tomato in coronal brain slices of knockin mutant mice expressing lacZ from the Pomc locus reveals high penetrance of eutopic expression of nPE1Pomc-tomato (brown reaction product) in POMC neurons visualized by X-gal staining (blue). (D) Expression of the fluorescent proteins tomato (Left) or EGFP (Center) in coronal brain sections of adult (Top and Middle) and sagittal sections of e10.5 (Bottom) compound transgenic mice carrying nPE1Pomc-tomato and nPE2Pomc-EGFP. Right: Superimposed images demonstrate an almost complete level of cellular coexpression of both fluorescent reporter proteins. White arrows indicate rare neurons expressing tomato but not EGFP, whereas a gray arrow points to a neuron expressing EGFP but not tomato.

Fig. 5.

Evolutionary history of POMC, nPE1, and nPE2. The drawing represents the phylogeny of all mammalian orders (16, 35), vertebrate classes, and tunicates as an outgroup. POMC (black arrow) appeared before the radiation of all fishes including Agnathans, Chondrichthians, and Osteichthians (10). nPE2 (blue arrow) appeared in the lineage leading to mammals, and nPE1 (purple arrow) is found only in placental mammals.