Lim homeobox genes in the Ctenophore Mnemiopsis leidyi: the evolution of neural cell type specification
© Simmons et al; licensee BioMed Central Ltd. 2012
Received: 23 November 2011
Accepted: 13 January 2012
Published: 13 January 2012
Nervous systems are thought to be important to the evolutionary success and diversification of metazoans, yet little is known about the origin of simple nervous systems at the base of the animal tree. Recent data suggest that ctenophores, a group of macroscopic pelagic marine invertebrates, are the most ancient group of animals that possess a definitive nervous system consisting of a distributed nerve net and an apical statocyst. This study reports on details of the evolution of the neural cell type specifying transcription factor family of LIM homeobox containing genes (Lhx), which have highly conserved functions in neural specification in bilaterian animals.
Using next generation sequencing, the first draft of the genome of the ctenophore Mnemiopsis leidyi has been generated. The Lhx genes in all animals are represented by seven subfamilies (Lhx1/5, Lhx3/4, Lmx, Islet, Lhx2/9, Lhx6/8, and LMO) of which four were found to be represented in the ctenophore lineage (Lhx1/5, Lhx3/4, Lmx, and Islet). Interestingly, the ctenophore Lhx gene complement is more similar to the sponge complement (sponges do not possess neurons) than to either the cnidarian-bilaterian or placozoan Lhx complements. Using whole mount in situ hybridization, the Lhx gene expression patterns were examined and found to be expressed around the blastopore and in cells that give rise to the apical organ and putative neural sensory cells.
This research gives us a first look at neural cell type specification in the ctenophore M. leidyi. Within M. leidyi, Lhx genes are expressed in overlapping domains within proposed neural cellular and sensory cell territories. These data suggest that Lhx genes likely played a conserved role in the patterning of sensory cells in the ancestor of sponges and ctenophores, and may provide a link to the expression of Lhx orthologs in sponge larval photoreceptive cells. Lhx genes were later co-opted into patterning more diversified complements of neural and non-neural cell types in later evolving animals.
LIM Homeobox (Lhx) genes were first isolated from the nematode, Caenorhabditis elegans, where the Lhx homolog, MEC-3, was shown to be required for the proper differentiation of touch receptor neurons . Subsequent studies in C. elegans and rat isolated LIN-11 and Islet1, respectively, which together with MEC-3 are the founding members for the LIM family acronym [2, 3]. Phylogenetically, Lhx genes were originally subdivided into six subfamilies, Lhx1/5, Lhx2/9, Lhx3/4, Lhx6/8, Islet, and Lmx . Lhx proteins are composed of tandem zinc-finger LIM domains at the N-terminus, which function by binding specific co-factors that mediate their function, while the helix-turn-helix homeodomain (HD) interacts with DNA in a sequence-specific manner . While LIM domains and homeodomains are found in non-metazoan eukaryotes, the specific combination of LIM-LIM-HD is only found in animals .
Lhx genes have roles in cell specification, tissue differentiation and neural patterning. In both vertebrate and invertebrate taxa, Lhx genes have conserved roles in the patterning of sensory neurons, interneurons, and motor neurons (reviewed in [4, 7]). It has been suggested that cells expressing different combinations of Lhx genes form a "LIM code" that is important in specifying cell types within a tissue or organ , and motor neuron axon pathway finding .
The C. elegans Lhx1/5 genes, LIN-11 and MEC-3, are required for the terminal differentiation of non-overlapping sensory, motor neurons and interneurons [1, 9, 10]. Mouse null mutants of the Lhx1 family die during mid-gastrulation due to massive head defects , while the Lhx5 homologue is expressed in the anterior neural plate and parts of the developing diencephalon . At later stages, the expression pattern of Lhx5 extends to parts of the midbrain, hindbrain and spinal cord . A study (reviewed in  on the LIM code for axon path finding in motor neuron subtypes revealed that depending on the combination of Lhx genes expressed in motor neurons, different motor neuron subtypes are produced. Lhx1 when expressed with Islet2 produces motor neurons that project into ventral limb bud musculature, while Islet1, Islet2, and Lhx3 expressing motor neurons project into the medial motor column, and different motor neurons are produced when Isl1 and Isl2 are expressed either together or separately. In addition to neural patterning, Lhx1 is also known for its role in blastoporal organizer activity during gastrulation in Xenopus, ascidians, amphioxus, and cnidarians  as well as endoderm specification in ascidians , amphioxus  and mice .
The Lhx2/9 group, also known as the apterous group (named after the Drosophila gene), has diverse roles in patterning the nervous system, wing development, muscle development, axon guidance, and neurotransmitter choice [18, 19]. The C. elegans Lhx2/9 homologue ttx-3 is expressed in a pair of interneurons and anterior muscle cells that project into the nerve ring . The vertebrate Lhx2/9 homologues are expressed in the nerve cords, eyes, olfactory organs and limbs .
The Lhx3/4 subgroup expression patterns were found to be restricted to post mitotic neurons in Drosophila. The function of Lhx3/4 genes in Drosophila is in axon guidance of specific motor neurons when combined with the expression of Lhx gene islet . In the developing chick embryo, Lhx3/4 has been shown to specify the formation of interneurons in the presence of one of its binding partners Ldb1 (LIM domain-binding protein 1), while additional expression within the same cells with the Lhx gene Islet1 specifies the formation of motor neurons , which show that Lhx genes function in a combinatorial fashion called the 'LIM code' to specify specific neural identities [4, 8, 23].
The Drosophila Lhx6/8 homologue arrowhead is expressed in the nervous system and has yet to be functionally analyzed . The C. elegans homologue is expressed in sensory, motor and interneurons in the brain . The vertebrate members of this subfamily are expressed in the forebrain and first branchial arch .
The islet gene subfamily is expressed in mesodermal derived cells and in subsets of motor and interneurons of the central nervous system (CNS) of Drosophila . In vertebrates, Islet paralogs are expressed in many tissues, including heart, liver, pancreas, brain, and eyes [28–30].
The C. elegans homologue to the group Lmx is expressed in post-mitotic neurons controlling axon guidance and the synthesis of the neurotransmitter GABA. It is also expressed in endothelial cells of the uterus and in the excretory system . The vertebrate chick homologue Lmx-1b is involved in dorsal ventral patterning of limbs and in patterning the otic vesicle .
Cnidarians are now well accepted to be the sister group to all bilaterians [37–41]. The anthozoan cnidarian, Nematostella vectensis, possesses members of all six Lhx subfamilies, as does the placozoan, Trichoplax, suggesting that complete diversification already occurred in the ParaHoxozoan common ancestor . The sponge, Amphimedon, only has members of three subfamilies: Lhx1/5, Lhx3/4, and Islet. Amphimedon Lhx1/5 and Lhx3/4 were found to be expressed within putative photosensory cells surrounding larval pigment cells, while Islet is ubiquitously expressed . In Trichoplax, all Lhx genes were shown to be expressed in adult animals through the use of RT-PCR; however, spatio-temporal patterns were not examined . Expression patterns of NvLhx6/8, NvLhx1/5, and NvLmx in Nematostella overlap with previously described neural territories . Interestingly, while the Amphimedon and Nematostella Lhx genes are all on separate genomic scaffolds, a single Trichoplax scaffold contains a cluster of Lmx, Lhx3/4, Lhx6/8, a LIM-only gene, and Lhx2/9 further downstream, suggesting that the Lhx family arose via tandem duplications .
Ctenophores occupy a highly debated phylogenetic position, once grouped with cnidarians forming the group Coelenterata [42, 43]. Ctenophores are now thought to have diverged prior to cnidarians and may actually be the earliest extant metazoan phyla that possess definitive neurons [37, 38]. Comparisons of gene complements between the four basal metazoans will be helpful in giving support for one or more of the various competing phylogenetic hypotheses (reviewed in ). Recent gene content studies [45–47] give consistent support for Porifera and Ctenophora diverging prior to the ParaHoxozoa (Placozoa, Cnidaria, Bilateria), however identifying the earliest branching taxon remains problematic.
For this study the genomic complement of Lhx genes was examined in the lobate ctenophore M. leidyi. Using the recently sequenced genome of M. leidyi, four Lhx genes were predicted by preliminary searches . We further analyzed these genes by obtaining their full sequences through RACE PCR. Using whole mount in situ hybridization, we examined the expression patterns of the four Lhx genes throughout development. These genes are expressed in discreet sensory cell types, and in an overlapping fashion within the apical sensory organ, a highly innervated nervous structure.
Gene identification, structure and genomic organization
The genomic organization of the Mnemiopsis Lhx genes showed no apparent linkage groups and they were found to be located on separate genomic scaffolds. The size of the scaffolds is as follows: MlLhx1/5 is found on scaffold ML1325 with a length of 127,187 bases. MlLmx is found on scaffold ML0569 with a length of 734,417 bases. MlIslet is found on scaffold ML0530 with a length of 126,527 bases. MlLhx3/4 is found on scaffold ML0681 with a length of 490,590 bases.
Included in this phylogenetic study are three LIM domain-containing gene families that lack a homeodomain and are the most closely related to Lhx genes, which were used for outgrouping: nuclear LIM only (LMO), Actin binding LIM (Ablim), and Paxilin. The most closely related family to the Lhx genes are found within the nuclear LIM only (LMO) gene family . Both M. leidyi and A. queenslandica do not possess genes within this family, and neither do the unicellular non-metazoans Monosiga brevicollis and Capsaspora owczarzaki surveyed in this study. The absence of LMO genes in these taxa in addition to the current genomic and phylogenetic data available and their internal branching within the Lhx hierarchy in our phylogenetic tree (although with low support values) suggests that these genes are likely a subfamily of Lhx genes and arose through a gene duplication shortly thereafter losing their homeodomains after the split of ctenophores and sponges and before the rise of ParaHoxozoa.
Genomic complement of Lim homeobox genes
Developmental expression of M. leidyi Lhx genes
MlLhx3/4 is expressed in cells at the aboral pole, eventually being confined to the central part of the apical organ floor (Figure 5Q-X). MlLmx is expressed in two groups of cells at the aboral pole just after gastrulation, along the tentacular plane (Figure 5Y). These cells eventually form part of the tentacle bulbs (Figure 5C'-F'). At 12 to 14 hpf, there is an additional expression domain in the ectoderm of the aboral pole in four groups of cells (Figure 5C', E'), which later appear to overlap with MlLhx1/5 in the region of the putative photoreceptors in the floor of the apical organ (Figure 5O).
Lhx gene complements
By probing the full genome of M. leidyi, we find that it has a genetic complement more similar to that of the sponge lineage than to the complete repertoire of Lhx gene subfamilies found in placozoans, cnidarians, and bilaterians. These data provide further evidence of the basal position of ctenophore and sponge lineages relative to Parahoxazoans. While multiple independent gene loss in both ctenophore and sponge clades may be the cause for this apparent synapomorphy, growing arguments based on genomic content, are suggesting a close link between sponges and ctenophores, including the existence of homeodomain containing transcription factor complement , nuclear receptor repertoire , Wnt signaling pathway , and TGF beta pathway .
Mnemiopsis expression data
Lhx genes are expressed in regions which contain the highest concentration of neural elements in the cnidarian Nematostella, including the apical tuft and nerve rings around the mouth and in the pharynx . In the sponge Amphimedon, there are broader expression domains; however, there are overlapping domains associated with the photosensory pigment ring, suggesting that Lhx genes are playing a combinatorial role in neural or sensory cell fate specification across the Metazoa, including the basal metazoans . The expression data here suggest Lhx genes are deployed in a similar fashion in the ctenophore Mnemiopsis. There are both overlapping and gene-specific expression domains associated with the apical sensory organ, a highly innervated structure involved in gravity, light, and pressure detection . MlLhx1/5, MlLmx and MlIslet have overlapping expression in the putative photoreceptor cells (Figure 6), while MlLhx3/4 and MlIslet are also expressed in other parts of the apical organ. It is possible that a 'LIM code' is involved in specifying different neural territories or cell types of the apical organ. Although the molecular interactions of only Lhx3/4 and Islet have been studied in depth , it is not unlikely that other Lhx genes may be able to form complexes to differentially regulate gene expression patterns. The expression of Lhx genes also forms distinct boundaries within the apical organ and associated polar fields, which may contribute to regional specificity, axonal projection boundaries, or neural transmitter phenotypes of these neural structures as is seen in bilaterians .
Previous studies have described other genes in different regions of the apical organ of Mnemiopsis, including the T-box genes Tbx2/3 and Brachyury , the homeobox genes Prd1 and Prd3 , the Wnt genes MlWnt6 and MlWntX , as well as the zinc fingers MlGli and MlGlis . Histological and electron microscopic studies of the ctenophore apical organ have suggested it is a highly innervated sensory structure [58–71]; however, gene expression studies suggest that it is much more complex than previously thought. These data are supported by recent work in the ctenophore, Pleurobrachia pileus; immunohistochemical studies have shown that there is a highly complex nerve net underlying the apical organ, as well as distinct groups of neurons within the apical organ floor that belie its morphological simplicity . Using this information about the neural anatomy of ctenophores, we suggest that ctenophore Lhx genes are expressed in a combinatorial fashion in the developing sensory cells associated with the apical organ.
In addition to the apical organ, Lhx expression domains are also seen in putative neurosensory regions such as the polar fields expression of MlIslet. While these ciliated structures have been identified morphologically and histologically [59, 72], their function has remained largely unknown. They have been proposed to be olfactory organs [58, 73–75], however, there is little evidence to confirm this. The recent immunohistological study of P. pileus by Jager et al.  identified a special nerve net that extended throughout the polar fields, as well as extensive neuro-sensory structures, termed 'Z bodies.' The gene expression of MlIslet overlaps with the Z bodies, but is not specific to them; instead the expression domain encompasses the entirety of the polar fields.
The expression of MlLhx1/5 in the presumptive photocytes is also quite intriguing. Ctenophores are capable of bioluminescence via calcium-activated photoproteins , but the mechanism or function has yet to be elucidated. The photocytes are located around the meridional canals underlying the comb rows . In addition to photoprotein genes, these cells also express an opsin gene , which suggests that these cells may represent an ancestral neuroeffector cell type. They express a gene that senses the environment (light-sensing via opsin), and an effector (photoprotein, generating light production) preceded by a transcription factor (MlLhx1/5), such that these photocytes appear to be capable of both sensing as well as responding to stimuli.
MlLhx1/5 expression is also found around the blastopore and is regionally localized to a portion of the pharynx in later stages. Considering that Lhx1/5 is expressed around the blastopore in many bilaterians, as well as the cnidarian Nematostella, we can speculate that besides neural patterning, Lhx1/5 was an ancestral 'blastoporal' gene. However, whether it functions in organizing activity in ctenophores, as it does in bilaterians, remains to be determined.
Recent gene content studies [45–47] give support for Porifera and Ctenophora diverging prior to the ParaHoxozoa (Placozoa, Cnidaria, Bilaterian) during early animal evolution. This is further supported by the present study, in which we have further classified the genomic complement of M. leidyi Lhx genes. Comprehensive phylogenetic analyses of LIM genes  in addition to our Lhx gene phylogenetic analyses of cnidarians, ctenophores, sponges and placozoans, indicate that the ancestor of ctenophores and sponges likely had three or four Lhx gene subfamilies: Lhx1/5, Islet, Lhx 3/4, and Lmx. The common ancestor of ParaHoxozoa contained three more Lhx gene subclasses, the LIM domain binding proteins, and the gene family LMO which was formed by duplications from a pre-existing Lhx gene subclass. Mnemiopsis Lhx genes are expressed in both overlapping and non-overlapping domains within proposed neural and sensory cell territories. These data suggest that Lhx genes first likely played a role in the patterning of sensory cells, as is seen in sponge larval photoreceptive cells . In other animal lineages with more complex nervous systems, Lhx genes appear to have a conserved role in patterning neural and sensory cell types. This apparent link shows support for the hypothesis that neural cell types may have evolved from non-neural sensory cells. An alternate hypothesis may be that sponges lost components of a simple nervous system after the split with ctenophores, or the ctenophore nervous system arose independently as a case of convergent evolution. These competing hypotheses still need to be considered due to the unexpected complex repertoire of structural and patterning nervous system genes found within sponges [34, 35]. Future genomic, expression, and functional studies are needed to characterize the molecular nature of the ctenophore nervous system.
Animal collection, RNA extraction and cDNA synthesis
M. leidyi adults were collected from Eel Pond or the NOAA Rock Jetty in Woods Hole, MA, during the months of June and July and spawned as previously described . RNA was extracted from embryos at regular intervals from fertilization to 36 hpf using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) . RNA was reverse transcribed to generate cDNA using the SMARTer RACE cDNA Amplification Kit (BD Biosciences, San Jose, CA, USA).
Identification of LIM homeobox genes in ctenophores
The Mnemiopsis genome was scanned in silico for genes of interest using a reciprocal BLAST approach. Human, Drosophila, and Nematostella orthologs were used as queries for TBLASTN searches. Candidate matches were then used in BLASTP searches of the human genome to find the closest hit. If the closest match was not the original ortholog or the E-value was greater than 0.001, then it was coded as being absent from the genome. For all genes of interest, gene-specific primers were designed for RACE PCR (MacVector, Cary, NC, USA). RACE PCR fragments were then conceptually spliced and aligned back to genomic contigs for comparison of intron-exon boundaries using Sequencher (Gene Codes, Ann Arbor, MI, USA). The following genes were isolated and fully sequenced: MlLhx1/5 [Genbank: JF912807], MlLhx3/4 [Genbank: JF912808], MlIslet [Genbank: JF912806], and MlLmx [Genbank: JF912809].
Whole mount in-situ hybridization
Embryos were fixed at various stages from freshly collected uncleaved embryos (0 hpf) to cydippids (24 to 36 hpf) . They were stored in methanol at -20°C until ready to use. Digoxygenin-labeled M. leidyi Lhx gene riboprobes (Ambion/Applied Biosystems, Inc., Foster City, CA, USA of the following sizes: MlIslet 1200 bp, MlLmx 1100 bp, MlLhx1/5 850 bp, and MlLhx3/4 1500 bp, were hybridized for 48 hours at 60°C at 0.1 ng/ul and detected using an alkaline phosphatase conjugated antibody (Roche Applied Science Inc, Indianapolis, IN, USA) and the colorimetric substrate NBT/BCIP (Roche Applied Science Inc, Indianapolis, IN, USA) Following detection, specimens were washed with phosphate-buffered saline and transferred through a glycerol series up to 70% glycerol. They were then mounted on glass slides, viewed under an Axioskop 2 compound microscope, and imaged using an AxioCam HRc with Axiovision software (Zeiss Inc, Jena, Germany). Color balance and brightness were adjusted using Adobe Photoshop CS3. The only modification to the in situ protocol is a change in acetic anhydride treatment (treated in 0.1 M triethanolamine rather than 1% w/v). For the most recently updated protocols, contact the authors. All in situ images presented here, as well as additional developmental stages and/or views, are available online via the comparative gene expression database, Kahikai http://www.kahikai.com.
M. leidyi Lhx protein sequences were aligned to the complements of Lhx proteins of the various species: H. sapiens, Drosophila melanogaster, N. vectensis, T. adhaerens, and A. queenslandica, and outgroup sequences of LIM-only domain containing gene 4 (LMO4) in addition to the above species when present, G. gallus, D. rerio, and the related unicellular choanoflagellates M. brevicollis, and C. owczarzaki LIM domain-containing genes paxilin and ablim (full sequence data provided in Additional file 2, using MUSCLE (default parameters) and trimmed by eye in Jalview  to include the conserved LIM domains and Homeodomains. Construction of the Lhx gene orthology was conducted in RaxML v7.0.0 , using maximum likelihood analysis under the JTT I+G model of evolution determined by ProtTest v1.4 analysis . A total of 1,000 searches was performed and 500 bootstrap replicates were applied to the tree with the best likelihood score used to generate branch support values. Bayesian phylogenetic analyses were also performed with MrBayes 3.1  using the JTT I+G model with two runs of 2,000,000 generations sampled every 100 generations. The first 5,000 trees were disregarded as burn-in.
actin binding LIM
Basic Local Alignment Search Tool
central nervous system
hours post fertilization
- Ldb :
LIM domain-binding protein
LIM homeobox containing genes
LIM-only domain containing gene
LIM-only domain containing gene 4
Placozoa: Cnidaria: Bilateria
polymerase chain reaction
randomized accelerated ML.
This work was supported by NASA and NSF grants to MQM, and the Intramural Research Program of the National Human Genome Research Institute (National Institutes of Health). We would like to thank Bernard J Koch, Joseph F Ryan and Andreas D Baxevanis for continued motivation and inspiration. In addition we would like to thank Alice Young, Brian Schmidt, Natalie Gurson, Richelle Legaspi, and Betsy Novotny, who were involved with the Mnemiopsis genomic sequencing at NISC.
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