Early evolution of the LIM homeobox gene family

Abstract

Background: LIM homeobox (Lhx) transcription factors are unique to the animal lineage and have patterning roles during embryonic development in flies, nematodes and vertebrates, with a conserved role in specifying neuronal identity. Though genes of this family have been reported in a sponge and a cnidarian, the expression patterns and functions of the Lhx family during development in non-bilaterian phyla are not known.

Results: We identified Lhx genes in two cnidarians and a placozoan and report the expression of Lhx genes during embryonic development in Nematostella and the demosponge Amphimedon. Members of the six major LIM homeobox subfamilies are represented in the genomes of the starlet sea anemone, Nematostella vectensis, and the placozoan Trichoplax adhaerens. The hydrozoan cnidarian, Hydra magnipapillata, has retained four of the six Lhx subfamilies, but apparently lost two others. Only three subfamilies are represented in the haplosclerid demosponge Amphimedon queenslandica. A tandem cluster of three Lhx genes of different subfamilies and a gene containing two LIM domains in the genome of T. adhaerens (an animal without any neurons) indicates that Lhx subfamilies were generated by tandem duplication. This tandem cluster in Trichoplax is likely a remnant of the original chromosomal context in which Lhx subfamilies first appeared. Three of the six Trichoplax Lhx genes are expressed in animals in laboratory culture, as are all Lhx genes in Hydra. Expression patterns of Nematostella Lhx genes correlate with neural territories in larval and juvenile polyp stages. In the aneural demosponge, A. queenslandica, the three Lhx genes are expressed widely during development, including in cells that are associated with the larval photosensory ring.

Conclusions: The Lhx family expanded and diversified early in animal evolution, with all six subfamilies already diverged prior to the cnidarian-placozoan-bilaterian last common ancestor. In Nematostella, Lhx gene expression is correlated with neural territories in larval and juvenile polyp stages. This pattern is consistent with a possible role in patterning the Nematostella nervous system. We propose a scenario in which Lhx genes play a homologous role in neural patterning across eumetazoans.

Figure 1

Phylogeny of LIM homeobox genes. The maximum likelihood tree based on an alignment of two LIM domains and the homeodomain is shown here with support values from Neighbor-joining/Likelihood/Bayesian analyses shown for the major nodes (relationships within the major classes were well supported only for vertebrate sequences). Neighbor-joining and Likelihood bootstrap values above 50% are shown, as are Bayesian posterior probabilities above 0.95. Full trees from each analysis are shown in Additional file 1. Aq = Amphimedon queenslandica(blue); Ce = Caenorhabditis elegans; Dm = Drosophila melanogaster; Dr = Danio rerio; Hm = Hydra magnipapillata (orange); Hs = Homo sapiens; Nv = Nematostella vectensis (green); Rn = Rattus norvegicus; Sp = Strongylocentrotus purpuratus; Ta = Trichoplax adhaerens (red); Xt = Xenopus tropicalis.

Figure 2

Synteny and intron conservation of LIM homeobox genes. (a) Four of the six Trichoplax LIM homeobox genes are present on one scaffold, three of these are present in tandem. This tandem cluster also contains a gene coding for a protein of the LIM only (Lmo) class. This scaffold is in the same putative ancestral linkage group as human chromosome segments that contain 6 of the 12 human LIM homeobox genes. (b) Two introns that interrupt the homeodomain in the Lmx class proteins are well conserved across animals, but one has been lost in both Nematostella and C. elegans. Introns are represented with square brackets with the enclosed number indicating the phase of the intron.

Figure 3

LIM homeobox gene expression during Nematostella development. (a-d) The arrowhead (Lhx6/8) ortholog is first expressed in the apical tuft of the late planula (c) but disappears in the juvenile polyp (d). (e-h) The lin-11 (Lhx1/5) ortholog is first expressed in the putative pharyngeal endoderm in the early planula and later resolves into an endodermal ring around the pharynx (g' = oral view of g; h' = cross-section through h). (i-l) The Lmx ortholog is first transcribed in the oral ectoderm of the early planula and then spreads into the pharynx and directive mesenteries (l' = oral view of l). (m-p) The apterous(Lhx2/9) ortholog is expressed in the planula endoderm in a speckled pattern and later its expression spreads to the end of the pharynx and throughout the directive mesenteries (p' = lateral view of p). (q-t) The islet ortholog starts out in the putative pharyngeal endoderm and over time spreads into the directive mesenteries. This gene is transcribed in cells of the planula body wall endoderm and in the polyp stage there it shows restricted expression in the aboral endoderm (t' = lateral view of t).

Figure 4

LIM homeobox gene expression during Amphimedon development. (a, c, f, h) Whole-mount micrographs; (b, d, e, g, i, j) micrographs of sectioned embryos. (a-e) The Lhx3/4 ortholog is expressed in the inner cell mass during late gastrulation, when pigment cells form a spot (a,b) and then a ring (c,d). A stronger expression domain appears transiently under the photoreceptor ring when it is forming (arrowheads in d). Expression is ubiquitous in the prehatched larva, with higher expression levels in the subepithelial layer (e). (f-j) The Lhx1/5 ortholog appears to be expressed in the outer layer at the pigment spot stage, especially around the spot (f,g). When the pigment ring forms (h,i), the gene is highly expressed in the inner cell mass, especially inside the developing ring and at the anterior end. A strong expression domain also appears in the micromeres surrounding the developing pigment ring (arrowheads in i). Expression seems to be ubiquitous in the larva before it hatches (j).

Figure 5

Schematic diagrams of Nematostella developmental stages showing combinatorial expression of LIM homeobox genes and overlap with known functionally different neural types. Neurons with putatively different functions emerge over the course of embryonic development (as assayed by neurotransmitter antibodies and in situ hybridization to detect neuropeptide or neurotransmitter synthesis enzyme mRNA) [20]. LIM homeobox genes have dynamic expression patterns that overlap with each other, as well as with territories of different neural types. The oral nerve ring (marked by 3,4-dihydroxyphenylalanine (DOPA- β-monoxygenase and RFamide), the pharyngeal nerve ring (marked by ©-aminobutyric acid (GABA)) and the apical tuft (marked by GABA) correspond to Lmx, Lhx1/5 and arrowhead expression respectively. DOPA-β-monoxygenase expression over developmental time is mirrored by Lmx expression. Two-color stripes show expression of two neural markers in the same region.