Trichoplax adhaerens and the development of eyes

On the development of eyes

Several types of eyes exist and these include the camera-type eye, the compound eye, and the mirror eye (Figure 1). Ernst Mayr proposed that eyes evolved in all animal phyla 40 to 60 times independently.
A monophyletic program governing the development of the different eye types is proposed and the Pax6 gene is posited to be the master control gene. The Pax6 gene also plays a part in controlling the development of the nose, ears and parts of the brain.

What is needed for the developmental program of eyes?
A few core genes include:
Pax6 (eyeless [eye]) in Drosophila)
Six-type genes (E.g. Six3)
Sox-type genes (E.g. Sox2)
atonal ( E.g. Atoh7)
Retinoid receptors
Fox transcription factors (E.g. FoxN4)
Pitx

Fascinating experiments have been conducted by shuffling around the genetic program architecture of genes associated with eye development in various animals.
For example in Drosophila:
Ectopic eye structures are able to be induced on the antennae, legs, and wings of fruit flies. This is done by targeted expression of the eyeless gene (Pax6 Drosophila homologue) (Figure 2). The Pax6 gene from the mouse is able to do the same job as the Drosophila version (Figure 3). And in Xenopus embryos, ectopic eye structures in can also be induced by the Drosophila eyeless (Pax6) version (Figure 4).

What about the Trichoplax adhaerens genome? Any genes for eye development?
It seems quite a chunk of the circuitry needed for eye development is present. (From table 1)
PaxB (eyeless?)
Six genes
Sox gene
Atonal gene
Retinoid X Receptor
Fox transcription factors
Pitx

All that is missing seems to be crystalins (plays a part in lens formation). However, Darwin posited that "The simplest organ which can be called an eye consists of an optic nerve, surrounded by pigment-cells and covered by translucent skin, but without any lens or other refractive body." Thus large chunks of the circuitry for eye development in Trichoplax is present but no eyes!

Now compare the developmental program to evolution.
Here is an interesting article that shows the parallels between evolution and development.

For development:
Primordial germ cells (PGC) are prevented from entering the somatic program and are demethylated (genome-wide erasure of existing epigenetic modifications). Then the gametes are imprinted (targeted DNA methylation) during gametogenesis, only to be demethylated again after fertilization. Then during development, DNA is methylated again, causing totipotential cells to become pluripotent. X-inactivation and reactivation (of the paternal gamete I think) also occurs. The whole process is governed by the genetic (and epigenetic?) program. During the unfolding of this somatic program, random variation and selection occur, ultimately leading to just a few endpoints, every time it is successful. The process is constrained (few end points) as a result of pre-existing information that is set up during the inititiation of the process. All this is controlled by information in the genome.

For evolution:
There also seems to be only a few endpoints (small subset, limited variation) out of all the possible endpoints.
In the article:
An End to Endless Forms: Epistasis, Phenotype Distribution Bias, and Nonuniform Evolution
It is argued to be as a result of genetic instructions dating earlier in evolutionary time. Preadaptations...

As already seen in the evolution of eyes, as soon as these sets of genes were formed (E.g. Pax genes), through whatever mechanism), evolution seemed to have been biased to a few end points, and these few endpoints arose 40-60 times, independently, as a result of pre-existing (preadaptations) information in the case of eyes.

What other "biased" end points can there be? Nervous systems, smell, hearing? And why would evolution be biased, as in development, to only reach a few end points over and over?

Trichoplax adhaerens: The Otp gene

Otp: The trend of neurolically associated developmental genes in the genome of the Trichoplax continues...

The various versions:
Trichoplax: Function unknown at present. Interested in its function in a basal eumetazoan.
Human otp. Its function:
The role of Otx and Otp genes in brain development.

Quote:

Over the last ten years, many genes involved in the induction, specification and regionalization of the brain have been identified and characterized at the functional level through a series of animal models. Among these genes, both Otx1 and Otx2, two murine homologues of the Drosophila orthodenticle (otd) gene which encode transcription factors, play a pivotal role in the morphogenesis of the rostral brain. Classical knock-out studies have revealed that Otx2 is fundamental for the early specification and subsequent maintenance of the anterior neural plate, whereas Otx1 is mainly necessary for both normal corticogenesis and sense organ development. A minimal threshold of both gene products is required for correct patterning of the fore-midbrain and positioning of the isthmic organizer. A third gene, Orthopedia (Otp) is a key element of the genetic pathway controlling development of the neuroendocrine hypothalamus. This review deals with a comprehensive analysis of the Otx1, Otx2 and Otp functions, and with the possible evolutionary implications suggested by the models in which the Otx genes are reciprocally replaced or substituted by the Drosophila homologue, otd.

The same for mice:
The murine Otp homeobox gene plays an essential role in the specification of neuronal cell lineages in the developing hypothalamus.

Quote:

Hypothalamic nuclei, including the anterior periventricular (aPV), paraventricular (PVN), and supraoptic (SON) nuclei strongly express the homeobox gene Orthopedia (Otp) during embryogenesis. Targeted inactivation of Otp in the mouse results in the loss of these nuclei in the homozygous null neonates. The Otp null hypothalamus fails to secrete neuropeptides somatostatin, arginine vasopressin, oxytocin, corticotropin-releasing hormone, and thyrotropin-releasing hormone in an appropriate spatial and temporal fashion, and leads to the death of Otp null pups shortly after birth. Failure to produce these neuropeptide hormones is evident prior to E15.5, indicating a failure in terminal differentiation of the aPV/PVN/SON neurons. Absence of elevated apoptotic activity, but reduced cell proliferation together with the ectopic activation of Six3 expression in the presumptive PVN, indicates a critical role for Otp in terminal differentiation and maturation of these neuroendocrine cell lineages. Otp employs distinct regulatory mechanisms to modulate the expression of specific molecular markers in the developing hypothalamus. At early embryonic stages, expression of Sim2 is immediately downregulated as a result of the absence of Otp, indicating a potential role for Otp as an upstream regulator of Sim2. In contrast, the regulation of Brn4 which is also expressed in the SON and PVN is independent of Otp function. Hence no strong evidence links Otp and Brn4 in the same regulatory pathway. The involvement of Otp and Sim1 in specifying specific hypothalamic neurosecretory cell lineages is shown to operate via distinct signaling pathways that partially overlap with Brn2.

The Zebrafish version. Its function: More of the same.
Differential regulation of the zebrafish orthopedia1 gene during fate determination of diencephalic neurons

Quote:

The homeodomain transcription factor Orthopedia (Otp) is essential in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates. However, there is little information on the intercellular factors that regulate Otp expression during development

In the sea urchin.
Evolution of OTP-independent larval skeleton patterning in the direct-developing sea urchin, Heliocidaris erythrogramma.

Quote:

The Orthopedia gene (Otp) encodes a homeodomain transcription factor crucial in patterning the larval skeleton of indirect-developing sea urchins.

A clear example of a cooption, whereby the same gene plays a role in neurological development in vertebrates and skeletal development in the sea urchin. Recycling of pre-existing genes for various, distinct, developmental processes.

Trichoplax adhaerens: The Pitx gene

Pitx: Another neurologically associated Hox gene present in the Trichplax genome.

The various versions:
Trichoplax. Function unknown at present. Would be interesting to find out what it is.
Human Pitx1 Its function:

Quote:

This gene encodes a member of the RIEG/PITX homeobox family, which is in the bicoid class of homeodomain proteins. Members of this family are involved in organ development and left-right asymmetry. This protein acts as a transcriptional regulator involved in basal and hormone-regulated activity of prolactin.

Human Pitx2 Its function:

Quote:

This gene encodes a member of the RIEG/PITX homeobox family, which is in the bicoid class of homeodomain proteins. The encoded protein acts as a transcription factor and regulates procollagen lysyl hydroxylase gene expression. This protein plays a role in the terminal differentiation of somatotroph and lactotroph cell phenotypes, is involved in the development of the eye, tooth and abdominal organs, and acts as a transcriptional regulator involved in basal and hormone-regulated activity of prolactin. Mutations in this gene are associated with Axenfeld-Rieger syndrome, iridogoniodysgenesis syndrome, and sporadic cases of Peters anomaly. A similar protein in other vertebrates is involved in the determination of left-right asymmetry during development. Alternatively spliced transcript variants encoding distinct isoforms have been described

Human Pitx3 Its function:

Quote:

This gene encodes a member of the RIEG/PITX homeobox family, which is in the bicoid class of homeodomain proteins. Members of this family act as transcription factors. This protein is involved in lens formation during eye development. Mutations of this gene have been associated with anterior segment mesenchymal dysgenesis and congenital cataracts.

Zebrafish Pitx1
Zebrafish Pitx2
Zebrafish Pitx3
Drosophila Ptx1 (fruitfly)

More interesting facts about Pitx:
The Pitx homeobox gene in Bombyx mori: Regulation of DH-PBAN europeptide hormone gene expression

Quote:

The diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is expressed exclusively in seven pairs of DH-PBAN-producing neurosecretory cells (DHPCs) on the terminally differentiated processes of the subesophageal ganglion (SG). To help reveal the regulatory mechanisms of cell-specific DH-PBAN expression, we identified a cis-regulatory element that regulates expression in DHPCs using the recombinant AcNPV-mediated gene transfer system and a gel-mobility shift assay. Bombyx mori Pitx (BmPitx), a bicoid-like homeobox transcription factor, binds this element and activates DH-PBAN expression. The BmPitx was expressed in various tissues, including DHPCs in the SG. Suppression of DH-PBAN expression by silencing of the BmPitx successfully induced non-diapaused eggs from a diapause egg producer. To the best of our knowledge, this report is the first to identify a neuropeptide-encoding gene as a target of the Pitx transcriptional regulator in invertebrates. Thus, it is tempting to speculate that functional conservation of Pitx family members on neuropeptide gene expression occurs through a "combinational code mechanism" in both vertebrate and invertebrate in neuroendocrine systems.

PITX genes are required for cell survival and Lhx3 activation
Zebrafish pitx3 is necessary for normal lens and retinal development.

And the trend of neurologically associated genes present in this basal eumetazoan continues

Trichoplax adhaerens: Hex and Dbx genes

More preadaptations from Trichoplax:

The Hex Homeobox gene

Its function?
Critical element in the development of the liver:
The role of Hex in hemangioblast and hematopoietic development.
The homeoprotein Hex is required for hemangioblast differentiation.
The homeobox gene HEX regulates proliferation and differentiation of hemangioblasts and endothelial cells during ES cell differentiation.

Detoxification of free radicals and damaging molecules play a crucial part in cellular homeostasis as well as systems homeostasis. The liver is mainly responsible for system homeostasis as it contains the highest concentration cells (hepatocytes do the heavy lifting) capable of detoxification, modification and excretion of hazardous molecules. It would be interesting to see what a gene that is associated with the development of the liver is doing in this simple organism at the base of the eumetazoan tree.

And the Dbx gene?

The trend of neurologically associated homeobox genes continues.
Regulation and function of Dbx genes in the zebrafish spinal cord.

Quote:

Dbx homeodomain proteins are important for spinal cord dorsal/ventral patterning and the production of multiple spinal cord cell types. We have examined the regulation and function of Dbx genes in the zebrafish. We report that Hedgehog signaling is not required for the induction or maintenance of these genes; in the absence of Hedgehog signaling, dbx1a/1b/2 are expanded ventrally with concomitant increases in postmitotic neurons that differentiate from this domain. Also, we find that retinoic acid signaling is not required for the induction of Dbx expression. Furthermore, we are the first to report that knockdown of Dbx1 function causes a dorsal expansion of nkx6.2, which is thought to be the cross-repressive partner of Dbx1 in mouse. Our data confirm that the dbx1a/1b/2 domain in zebrafish spinal cord development behaves similarly to amniotes, while extending knowledge of Dbx1 function in spinal cord patterning. 2007 Wiley-Liss, Inc

Trichoplax adhaerens: The Hmx gene

Another Hox gene in the Trichoplax genome involved in central nervous system development.

Hmx
Its function:
Hmx homeobox gene function in inner ear and nervous system cell-type specification and development.

Quote:

The Hmx homeobox gene family is comprised of three members in mammals, Hmx1, Hmx2, and Hmx3, which are conserved across the animal kingdom and are part of the larger NKL clustered family of homeobox genes. Expression domains of Hmx genes in distantly related species such as Drosophila and mouse suggest an ancestral function in rostral central nervous system development. During vertebrate evolution, the Hmx genes appear to have been recruited into additional roles in inner ear morphogenesis and specification of vestibular inner ear sensory and supporting cell types. Being derived from a common ancestor, the vertebrate Hmx gene family is thus a strong candidate to investigate functional overlap versus the unique roles played by multiple genes belonging to the same family. The functions of Hmx2 and Hmx3 were investigated via directed gene mutagenesis and the primary regions where Hmx2 and Hmx3 exert their individual functions are consistent with their expression domains, such as the vestibule and uterus. Meanwhile, it is notable that some tissues where both Hmx2 and Hmx3 are extensively expressed were not severely affected in either of the Hmx2 or Hmx3 single mutant mice, suggesting a possible functional overlap existing between these two genes. Compound Hmx2 and Hmx3 double mutant mice showed more severe defects in the inner ear than those displayed by either single knockout. Furthermore, novel abnormalities in the hypothalamic-neuroendocrine system, which were never observed in either of the single mutant mice, confirmed a hypothesis that Hmx2 and Hmx3 also function redundantly to control embryonic development of the central nervous system.

The Trichoplax Hmx sequence. BLAST it.

Where is this critter's brain? Did its mind get lost somewhere along the line, or is it just ready for a bit of cooption here and there?

Trichoplax adhaerens: The Mnx (aka HB9) gene

The Mnx (aka HB9) gene

The Trichoplax Mnx sequence: ABC86118
Comparison of this sequence with a few others: Cladogram

The human Mnx1 gene.
The fly Mnx gene (exex)
The Zebrafish Mnx gene

What does it do?
It is involved in the development of the pancreas and motor neurons.
1) Zebrafish mnx genes in endocrine and exocrine pancreas formation.
2) The Mnx homeobox gene class defined by HB9, MNR2 and amphioxus AmphiMnx.

Quote:

The HB9 homeobox gene has been cloned from several vertebrates and is implicated in motor neuron differentiation. In the chick, a related gene, MNR2, acts upstream of HB9 in this process. Here we report an amphioxus homologue of these genes and show that it diverged before the gene duplication yielding HB9 and MNR2. AmphiMnx RNA is detected in two irregular punctate stripes along the developing neural tube, comparable to the distribution of 'dorsal compartment' motor neurons, and also in dorsal endoderm and posterior mesoderm. We propose a new homeobox class, Mnx, to include AmphiMnx, HB9, MNR2 and their Drosophila and echinoderm orthologues; we suggest that vertebrate HB9 is renamed Mnx1 and MNR2 be renamed Mnx2.

Interesting research:

Directed Evolution of Motor Neurons from Genetically Engineered Neural Precursors.

Quote:

Stem cell-based therapies hold therapeutic promise for degenerative motor neuron diseases such as amyotrophic lateral sclerosis and for spinal cord injury. Fetal neural progenitors present less risk of tumor formation than embryonic stem (ES) cells but inefficiently differentiate into motor neurons, in line with their low expression of motor neuron-specific transcription factors and poor response to soluble external factors. To overcome this limitation, we genetically engineered fetal rat spinal cord neurospheres to express the transcription factors HB9, Nkx6.1 and Ngn2. Enforced expression of the three factors rendered neural precursors responsive to sonic hedgehog and retinoic acid and directed their differentiation into cholinergic motor neurons that projected axons and formed contacts with co-cultured myotubes. When transplanted in the injured adult rat spinal cord, a model of acute motor neuron degeneration, the engineered precursors transiently proliferated, colonized the ventral horn, expressed motor neuron-specific differentiation markers and projected cholinergic axons in the ventral root. We conclude that genetic engineering can drive the differentiation of fetal neural precursors into motor neurons which efficiently engraft in the spinal cord. The strategy thus holds promise for cell replacement in motor neuron and related diseases.

What did these guys do? They enforced the expression of 3 genes associated with neuronal development in order to direct the development of motor neurons. Sonic hedgehog also played a role.
So four genes played a role:

1. HB9
2. Nkx6.1
3. Ngn2
4. Sonic hedgehog

Are similar genes present in the Trichoplax genome?
1. HB9 (mnx)
Yes (see above).

2. Nkx6.1
Here is the human Nk6 gene
And here is the Trichoplax version

3. Ngn2
Here is the human neurogenin 2 (ngn2) gene
And here is the Trichoplax version.
A quick BLAST (blastp) the human genome shows this sequence to be closely related to ngn2 (E-value = 3^-8).

4. Sonic hedgehog (shh)
Here is the human shh gene
This gene seems to absent in from the Trichoplax genome, however, the presence of shh in Monosiga brevicollis (unicellular eukaryote that diverged before Trichoplax) suggest the possibility of gene loss in this lineage.

Wonder what will happen if shh is co-expressed and together with mnx, Nk6 and ngn2 in Trichoplax, or whether these genes will function like their counterparts in higher animals.

A complex array of neurologically associated developmental pathways present in this eumetazoan that has no nerves, sensory cells and muscle cells, and there is more

Trichoplax adhaerens: The Dlx gene

More Trichoplax preadaptations:

The Dlx gene

What does it do (wiki)?

Quote:

Dlx genes are required for the tangential migration of interneurons from the subpallium to the pallium during vertebrate brain development [3]. It has been suggested that Dlx promotes the migration of interneurons by repressing a set of proteins that are normally expressed in terminally differentiated neurons and act to promote the outgrowth of dendrites and axons [4]. Mice lacking Dlx1 exhibit electrophysiological and histological evidence consistent with delayed-onset epilepsy [5]. Dlx2 has been associated with a number of areas including development of the zona limitans intrathalamica and the prethalamus. Dlx5/6 expression is necessary for normal lower jaw patterning in vertebrates [6]. Dlx7 is expressed in bone marrow

A quick BLAST of the sequence reveals it is closely related to human Dlx1, as well as Dlx1 in other vertebrates (including Zebrafish, the mouse, rat opossum, dog etc.)

More specifically, what does Dlx1 do?
Pubmed

Quote:

This gene encodes a member of a homeobox transcription factor gene family similiar to the Drosophila distal-less gene. The encoded protein is localized to the nucleus where it may function as a transcriptional regulator of signals from multiple TGF-{beta} superfamily members. The encoded protein may play a role in the control of craniofacial patterning and the differentiation and survival of inhibitory neurons in the forebrain. This gene is located in a tail-to-tail configuration with another member of the family on the long arm of chromosome 2. Alternatively spliced transcript variants encoding different isoforms have been described.

It is possible to create a homology of this protein to look at its possible structure. The closest match is the human Dlx 5 protein structure. Sequence alignment places the Dlx sequence of Trichoplax closer to human Dlx5 than to human Dlx1.

What does Dlx 5 do?
Pubmed:

Quote:

This gene encodes a member of a homeobox transcription factor gene family similar to the Drosophila distal-less gene. The encoded protein may play a role in bone development and fracture healing. Mutation in this gene, which is located in a tail-to-tail configuration with another member of the family on the long arm of chromosome 7, may be associated with split-hand/split-foot malformation.

The homology model of the protein:
A good quality protein was generated

So, a Hox gene responsible for a sundry of neurologically associated developmental processes present in an organism with no nerve, sensory or bone cells at the base of the evolutionary tree.
Awesome

Trichoplax adhaerens.

The intriguing genome of the Trichoplax (Placozoa meaning flat animals):
Trichoplax Genome Sequenced: 'Rosetta Stone' For Understanding Evolution

Nice Pic


Place on the evolutionary tree

Currently there is only one named species in the phylum: Trichoplax adhaerens.

Quote:

It's a flattened blob, a few millimeters across and made up of a few thousand cells. It's main claim to fame is its remarkable simplicity: it is a multicellular animal that consists of only four apparent cell types, and the only obvious organization is into an upper and lower surface. The upper surface consists of a sheet of covering cells, while the lower surface contains two cell types: the gland cells that secrete digestive enzymes onto whatever the animal is sitting on, and the cylinder cells that absorb whatever nutrients are released. In between is a loose network of fiber cells that are responsible for the animal's movement.

Link
Nerves, sensory cells and muscle cells are absent.
Interestingly (from the link):

Quote:

One other strange thing: in culture, Trichoplax is consistently asexual and reproduces by fission, but older cultures at high density begin to produce small motile presumptive sperm cells, and as individual animals desintegrate, they spew out ova. The two have never been observed to come together, though, so there is no fertilization, and while the ova may divide a half dozen times, they all eventually die. It is possible that there is another stage in the life cycle that is not viable under laboratory conditions and has never been observed.

The genome of this critter is even more fascinating.
From the nature article:
The Trichoplax genome and the nature of placozoans

Table 1 | Developmental transcription factors in the Trichoplax genome
Homeobox (Hox genes)

• A) ANTP-class: Trox-2 (Hox/ParaHox-like), Not, Dlx, Mnx, Hmx, Hex, Dbx and seven others.
• B) PRD-class (paired box and homeobox): PaxB, Pitx, Otp, Gsc and five others
• C) POU-class(POU domain and homeobox): POU class 4 (Brn-3), one other
• D) LIM-class (LIM domain and homeobox): islet, apterous, Lhx1/5 and one other
• E) SIX-class (sine oculis homeobox): Six3/6 and one other
• F) TALE-class: Pbx/Exd, Irx, Meis
• G) HNF-class: Hnf

Going down the list, what are the functions of these Hox genes?
1) Trox-2 (Hox/ParaHox-like)
Hox/paraHox-like genes are involved in axial patterning in bilaterarian organisms. Basically, they control the formation of the anterior–posterior (AP) axis. Function of Trox-2?

Quote:

We speculate that Trox-2 functions within a hitherto unrecognized population of possibly multipotential peripheral stem cells that contribute to differentiated cells at the epithelial boundary of Trichoplax.

2) Not
In mice, Not controls the development of the caudal notochord. What is the notochord?
Wiki

Quote:

The notochord is a flexible, rod-shaped body found in embryos of all chordates. It is composed of cells derived from the mesoderm and defines the primitive axis of the embryo. In lower vertebrates, it persists throughout life as the main axial support of the body, while in higher vertebrates it is replaced by the vertebral column. The notochord is found on the ventral surface of the neural tube.

What does it do in this flat, simple organism?

Quote:

The homeobox gene Not is highly conserved in Xenopus, chicken and zebrafish with an apparent role in notochord formation, which inspired the name of this distinct subfamily. Interestingly, Not genes are also well conserved in animals without notochord such as sea urchins, Drosophila or even Hydra, but appear to be highly derived in mammals. A search for homeobox genes in the placozoan Trichoplax adhaerens, one of the simplest organisms available today, revealed only two homeobox genes: a Not homologue and the previously described gene Trox-2, which is most similar to the Gsx subfamily of the Hox/ParaHox cluster genes. Not has a unique expression profile in Trichoplax. It is highly expressed in folds of intact animals and in the wounds of regenerating animals. The dynamic expression pattern of Trichoplax Not is discussed in comparison with the invariable expression pattern of Trox-2 and the putative secreted protein Secp1. The high sequence conservation of Not from Trichoplax to lower vertebrates, but not to mammals, represents a rare example of an apparent gene decay in the lineage leading to humans.

Interesting preadaptations. It gets better.

Next, a look at the other Hox genes in this organism and their functions in higher animals.
Dlx, Mnx, Hmx,Hex, Dbx etc...

Hox genes video
Nice overview of Hox genes.