Mnemiopsis leidyi: How nervous system developed in the Tree of life.

The human nervous system is one of the most incredible structures that has ever evolved within the history of living organisms. Millions of neurons are firing constantly, even when someone is not doing much of anything. However nervous systems are not only a uniquely human thing. Almost every organism in the Kingdom Animalia has some sort of nervous system from the small 302 neuronal cells in Caenorhabditis elegans to 10^11 (that is a hundred billion) neurons in the human body. So it is no surprise that many scientists want to figure out when this incredible network of neurons became a staple for animals. It is not that easy though, and it has stirred up some recent debate as to what animals are truly at the bottom of the tree of life. One of these organisms at the bottom is the ctenophore. Ctenophores are pelagic marine organisms that looks similar to jelly fishes, but they do not have stingers. Other comparisons between cnidarians(the jelly fish) and the ctenophores can be found here.
A video of ctentphores can be found here.

Typical examples of species in Ctenophora. Images curtesy of Wikimedia Commons:

Ctenophores do have a defined nervous system complete with a nerve net and an apical organ which help these creatures determine the water pressure. The apical organ also helps to sense how much light is in the environment and which way is up. Ctenophores are one of the four groups of organisms at the bottom of the animal tree of life. The other three are sponges, placozoa, and cnidarians. These four groups have evolved from single-celled organisms but never became bilateral. Only two of those four groups have defined neurons. These two groups are the cnidarians and the ctenophores.

There have been recent studies suggesting that ctenophores may be the earliest animals to have defined neurons. The ctenophore Mnemiopsis leidyi is the most study species in the entire group. It has been in many genomic studies since they are easy to raise in a lab environment, and their entire genome is sequenced. The sequenced data can be found here. If these organisms were the first to develop neurons, what genes were necessary to make its neurons? Are these neurons similar to the genes that have been already found in other animals? Do they have the same functions? Did M. leidyi use the genes in a different way and they just evolved different functions in other animals? To find these answers out, the genome of M. leidyi was probed for LIM homeobox(Lhx) genes. These genes are known to be pivotal in the neuronal development of many bilaterians.

What are Lhx genes?

Lhx genes encode for proteins that work as transcription factors. All Lhx proteins contain two zinc-finger LIM domains and a homeodomain, hence the name LIM homeobox genes. The two LIM domains can bind to many specific co-factors and the co-factors they bind to depend on the type of LIM domain. Having two LIM domains is common in Animalia. There are other eukaryotes that have LIM domains but their proteins only have one LIM domain while animals have two LIM domains. The homeodomain has a helix-turn-helix structure which helps it to bind to specific regions of DNA and make the regions more viable for transcription.

A cartoon of a typical Lhx gene complete with two LIM domains and a homeodomain. From Hobert 2000:

The homeodomain of the different types of Lhx gene is more similar to each other than to the rest of the homeobox genes. This means that all Lhx genes can trace their distinctive DNA-binding specificity to a common ancestor. Lhx genes have been highly conserved in the evolution of animals. Lhx genes were first isolated from Caenorhabditis elegans. The gene that was found in C. elegans is called MEC-3 and is required for the differentiation of touch receptor neurons. Several other studies afterwards found the genes LIN-11 and Islet1 in C. elegans and rat genomes which also dealt with neuronal specification. LIN-11, Islet1, and MEC-3 are the founding members for the LIM family acronym.

What do Lhx genes do?

There are six classes of Lhx genes. Each Lhx gene is divided into the classes based on the specific functions of the genes. Genes from each class do not function independently of each other. During neural development, cells express different combinations of Lhx genes which form a “LIM code” to determine the fate of the neuron. For example, when Lhx 3 is expressed with Islet in the same cell, that cell will develop into a motor neuron.


  • also known as LIN-11 group in invertebrates such as C. elegans
  • MEC-3 gene (in C. elegans) and LIN-11 gene are both needed for terminal differentiation of non overlapping sensory, motor and interneurons
  • Lhx1 functions in neural induction
  • in mice, Lhx1 is needed at mid-gestation or the mice die
  • Lhx5 is needed at gastulation or organism dies
  • Lhx5 is found in the hypothalamus
  • Lhx5 helps neural-precursor cells to poliferate amd move during hippocampus formation
  • LIN-11 gene and MEC-3 gene also affect neural migration


  • also known as apterous group
  • Lhx2 functions in brain and eye development and in hematopoiesis in vertebrates
  • in vertebrates, Lhx9 is expressed in interneurons of the dorsal side of the spinal cord
  • Lhx2 and Lhx9 have overlapping expression


  • Lhx3 and Lhx4 are both necessary during pitutary organogenisis and motor neuron development
  • Lhx3/4 are expressed in motor neuron that have their axons projecting ventually from the neural tube
  • Lhx 3/4 is needed for diversification of motor neuron axonal projections


  • Lhx 6/8 is found in Drosophila but not fully funtionally analyzed
  • in C. elegans, Lhx6/8 is expressed in sensory, motor and interneurons of the brain
  • in vertebrates, Lhx6/8 is expressed in the forebrain and first branchial arch.


  • Rat Isl-1 was the first Islet gene found
  • Islet is important for motor neuron and panceas development in mice
  • in Drosophila, Islet is important in axonal targeting


  • In C. elegans, Lmx functions in axon guidance and GABA synthesis and excatory system
  • In vertebrates, L,x functions in dorsal ventral patterning of the limbs and patterning of the otic vesicle

How does Mnemiopsis leidyi relate to any of this?

The ctenophores are one of the four basal groups of multicellular metazoans stuck between single-celled organisms and organisms that are bilateral. The other three groups are Porifera, Cnidaria, and Placozoa. Many studies say that Porifera and ctenophores diverged before the ParaHoxoazans (cnidaria, placozoa, and bilateria), but there are still debates about this positioning.

A basic diagram of the relationships of between ctenophores and other basal organisms. From Simmons 2012 :

As animals grew more complex in their body plans, the need for more complex nervous systems arose. However, when nervous systems arose in the evolution of animals and how they became so complex has been the topic of some debates. Ctenophores used to be grouped with cnidarians. However, ctenophore do not contain cnidocytes, one of the defining characteristics of cnidarians. Several people believe that the ctenophore evolved before the cnidarians. Since only cnidarians and ctenophore have neurons while porifera and placozoa does not, ctenophores may be the first animals to develop and process neurons. It is a problem though to place the earliest branches in order of evolutionary developments since very little fossil records exist. Genomic data is only just beginning to be synthesized for these four basal groups. To determine where M. leidyi belongs in the neuronal development phylogeny, a genomic study was preformed closely looking at the Lhx genes of M. leidyi and comparing them to other sequences of other species. Also to determine the roles of the Lhx genes during development, in situ hybridization(ISH) was used to observe the organism during different stages of development with the Lhx genes tagged.

Finding Lhx Genes in Mnemiopsis leidyi

Before the Lhx genes in Mneomiopsis leidyi can be compared to Lhx genes of different species, it must be determined if the Lhx genes are even present in the M. leidyi genome. The researchers in “Lim homeobox genes in the Ctenophore Mnemiopsis leidyi: the evolution of neural cell type specification” took RNA from embryos of M. leidyi at different intervals of development, from fertilization to 36 hpf (hours post-fertilization). This RNA was reverse transcribed into cDNA. The Lhx genes were isolated from all of the cDNA by using 5′ and 3′ RACE RT-PCR.

After this step had been preformed, four classes of Lhx genes were identified. They are MlLhx1/5,MlLhx3/4, MlIslet, and MlLmx, with the Ml prefix standing for M. leidyi. All of the four of these genes encode for proteins that have two LIM domains followed by a homeodomain, which is the main characteristic of Lhx proteins. These genes are not linked and found on different places on the genome. The exact sizes and locations are listed on the table below.

Lhx gene class Genomic Scaffold Size(in bases) Genbank code
MlLhx1/5 ML1325 127,187 JF912807
MlLhx3/4 ML0681 490,590 JF912808
MlIslet ML0530 126,527 JF912806
MlLmx ML0596 734,417 JF912809

Comparing M. leidyi genes to other species

Since Lhx genes were found in Mnemiopsis leidyi genome, it can now compare to Lhx genes from other species. The species that the researchers compared M. leidyi to include the cnidarian Nematostella vectensis, the placozoan Trichoplax adherens, the sponge Amphimedon queenslandica, Homo sapiens, Danio rerio, Gallus gallus and Drosophila melanogaster. Each species Lhx genes were compared to the other by looking at similarities of the LIM domains and homeodomains. These similarities were processed by using maximum likelihood and bayseain analysis. From this data, a tree comparing each of the Lhx gene classes against one another was made. The genes were organized based on introns between the LIM domains and the homeodomain binding sites on the genes.

A tree made by maximum likelihood and bayseian analysis of Lhx genes. The outgroup used are actin binding LIM(Ablim) and Paxalin. Ml is M. leidyi, Aq. is A. queenslandica, Ca is Capsaspora owczaraki, Dan is Danio rerio, Dm is Drosophila melanogaster, Gal is Gallus gallus, Hs is Homo sapiens, Mon is Monosiga brevicollis, Nm is Nematostella vectensis, Ta is Trichoplax adhaerens. From Simmons 2012 :

A. queenslandica does not have any homologue to Lmx. It was noted in the paper that the researchers are not certain why A. queenslandica does not have Lmx. They suggest it could be due to either the sponge losing the gene or the sponge arose before Lmx became a staple in the animal lineages.

The outgroups used in the tree were genes that have one or more LIM domains but no homeodomain. One of the groups, known as LIM only(LMO) is the most similar to the Lhx genes. LMO was not found in A. queenslandica and M. leidyi but it was found in the other species Trichoplax adhaerens, Nematostella vectensis, Gallusgallus, Danio rerio, Drosophila melanogaster, and Homo sapiens. It is interesting to note that the cnidarian and the placozoan both have the gene. This suggested that the gene LMO, which is a possible class of Lhx genes, arose after the split of sponges and ctenophores but before ParaHoxozoa.

Both A. queenslandica and M. leidyi are missing Lbd or the LIM-domain binding factor. Lbd is found in all other animals genomes including the Parahoxoans. Also, the Lhx genes of the sponge and M.ledyi are very similar to each other. Both only have three to four of the six classes of Lhx genes while the rest of the animals including cnidrains and placozoa have all seven Lhx genes (the six classes and LMO).

What these genes do in Mnemiopsis leidyi

The four Lhx genes were found in embryos during different stages of development.

This is the whole mount of M. leidyi during different stages of development starting at gastrulation up till the cyyippid stage. The purple staining on the organism represnts the localization of detected mRNA transcripts of the various Lhx genes. A-H is MlIslet, I-P is MlLhx1/5, Q-X is MlLhx3/4 and Y-F' MlLmx. From Simmons 2012 :


Of the four Lhx genes expressed in the embryo during development, MlIslet is the first to be found during gastrulation which is 4 hpf. MlIslet appears in cells at the aboral pole of the embryo along the sagittal axis. MlIset is continually expressed from gastrulation onwards, forming the polar fields and the apical organ floor. The part of the apical organ floor with MlIslet is the most sagittal part of the animal.


MlLhx1/5 is expressed after gastrulation or about 5 hpf. It is initially expressed in cells around the blastopore and in mesodermal cells at the oral pole that invaginated during gastrulation. The cells around the blastopore later on become the pharnyx while the mesodermal cells become photocytes. These photocytes reside under four of the comb rows. MlLhx1/5 is also expressed in late stage of development in the apical organ floor and become the photosensory cells.


MlLhx is expressed after gastrulation at the aboral pole and is expressed in the ectoderm. Just like MlIslet, it becomes apart of the apical organ floor.


MlLmx is expressed after gastrulation in two groups of cells along the aboral pole. This group of cells is formed along the tentacular plane, which form the tentacular blub apparatus. Later on, MlLmx is also expressed in the apical organ, overlapping expression with MlLhx1/5 in the apical organ and gives rise to the photoreceptors.

The researchers found MlIslet, MlLhx1/5, MlLhx3/4, and MlLmx are all expressed during the cydippid stage in the apical organ. Within the apical organ, MlIslet, MlLhx1/5, and MlLmx are all expressed in four groups of cell. These groups give rise to the photosensory cells. MlLhx3/4 does not overlap with any of the other genes in the apical organ and is expressed somewhere else in the apical organ. The non-overlapping regions become different types of nervous tissue.

This diagram shows the various locations of M. leidyi genes in the cydippid stage of development. From Simmons 2012 :

Overall, it seems that the Lhx genes in sponges and M. leidyi are more similar to each other than to the other classes of organism. This suggest that M. leidyi and sponges are very closely linked to one another. The Lhx genes in other animals, including sponges seem to have two types of expression, overlapping expression and gene-specific expression areas. The highest concentrations of Lhx genes are almost always found in regions of neural elements. M. leidyi has most of its expression in the apical organ which is the organ used for most of the sensing of the organism. MlIslet, MlLhx1/5, and MlLmx have overlapping expression in the photoreceptor cells. MlLhx3/4 and MlIslet also work together in a different part of the apical organ. The different concentrations, combinations, and locations of Lhx genes forms a “LIM code’ which helps specify different neuronal regions and cells within the apical organ.

Lhx genes can combine with other Lhx genes and can regulate expression patterns. In areas which Lhx genes do not overlap, they can confine themselves to different regions. This confinement of genes can lead to regions that perform specific tasks, can control the boundaries of axons from neurons and/or have different neural transmitter phenotypes. These features found in M. leiydi are common in bilaterians. This study and other have shown that the simplicity of the morphology of the apical organ is not as simple as it seems. There is a highly complex nerve net that makes up the apical organ. This nerve network is made up of many different groups of neurons are found in the apical organ floor. The ctenophore Lhx genes need to be expressed in a combination of genes during the development of the apical organ to account for this complexity.

M. leidyi can bioluminesce, but the process of how it lights up is not particularly well understood. Photoproteins and opsin are proteins that are both found in photoreceptor cells of M. leidyi. The transcription of theses two proteins are controlled by MlLhx1/5. Photoproteins generate light and opsin senses light in the environment. Using these two genes, M. leidyi can sense and react to stimuli in the environment. All animals can sense and respond to stimuli. While it was found that MlLhx1/5 was important in blastoceol neural development, how it works in ctenophore blastoceol development is not well known. How ctenophore blatoceol formation is similar to bilaterans is still unknown.

The Lhx genes in M. leidyi does have similar functions to Lhx genes in bilaterans. Unlike the ParaHoxozoans, M. leidyi only has four of the six classes of Lhx. Therefore, this study confirms that the ctenophores are at the bottom of the tree of life with the sponge. It is still a mystery as to how the ParaHoxozoans gained the two extra genes. Also, it is hard to tell whether sponges or ctenophores are truly at the bottom of the tree. As always, more studies are needed to find the answers to these questions.


Hobert, O., and H. Westphal. “Functions of Lim-Homeobox Genes.” Trends in Genetics 16.2 (2000): 75-83. Print.

Ryan, Joseph F., et al. “The Homeodomain Complement of the Ctenophore Mnemiopsis Leidyi Suggests That Ctenophora and Porifera Diverged Prior to the Parahoxozoa.” Evodevo 1 (2010). Print.

Simmons, David K., Kevin Pang, and Mark Q. Martindale. “Lim Homeobox Genes in the Ctenophore Mnemiopsis Leidyi: The Evolution of Neural Cell Type Specification.” Evodevo 3 (2012). Print.

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