Evolution of the TGF-beta Signaling Pathway and Its Potential Role in the Ctenophore, Mnemiopsis leidyi


Mnemiopsis leidyi with Jazz
1. Introduction

Warning: Before you go further, please read the overview page of M. leidyi.

The paper that will be reviewed explores the evolutionary significance and function of TGF-β pathway of M. leidyi. I know, the figures and blocks of text might bore you out, but I assure that the name of genes, what those gene does are not that relevant to what the paper has discovered. So take a deept breath, and scroll down to see what this little “Comb jelly” do with the TGF-β pathway.

See the difference?

  • M. leidyi is one of species of Ctenophore phylum. You might mistake it with jellyfish or sea anemone, but jellyfish is in phylum Cnidaria. What is the difference? Both M. leidyi and a jellyfish has a simple nerve system and a sensory organ. Sponges, which are the antecedent organisms than Ctenophore and Cnidaria, are classified as phylum Porifera. M. leidyi and sponges have two main cell layers with jelly-like layer between them while a jellyfish usually has two or three distinctive cell layers. These three phylums are closely related, suggesting the evolutionary relationship among three phylums. The chart comparing these three phylums are here. These phylums are comprises in Kingdom Animalia, the metazoans, that are multicellular organisms having eukaryotic cells with a nucleus, a cell membrane, and organelles such as mitochondria. (Virtual Fossil Museum)

So there you go. Porifera, Cnidaria, and our M. leidyi as Ctenophore. All these phylums are metazoans. Now, Why M. leidyi is used as model organism?

  • Like it was mentioned in the overview page, the entire genome of the M. leidyi has been sequenced, thus it is possible to determine its gene content, structure and a gene evolution of M. leidyi. Also, its embryos are transparent, so observing the imaging of early features of fertilization, a cell cycle, and a cell division is possible. Ctenophore has complex body structures and it lives in a hostile environment that makes it prone to injury thus it is a good candidate to study its regeneration and healing process. This could be used to understand how other species including human can be helped to regenerate themselves in the future. Ctenophores are ideal systems for studying adult wound healing and regeneration in the lab or in their natural environment.

Before I go start talking about what the paper found, I want to go over just one more, TGF-β signaling pathway.

Yes, we talked this particular pathway in the class, but there were some people who are just not good at cell biology (for example, me) and knowing this will make so much easier to understand the paper.

The TGF-β signaling pathway is a metazoan(Yep, the kingdom animalia) specific intercellular signaling pathway known to be important in many developmental and cellular processes. Detail was in the overview page, but for people who just came to my paper directly, let me copy and paste at here.

The basic components, super-family, of TGF-beta signaling pathway.

There are two receptors, TGF-β receptor I and II. There are two family who initiate(Tolloid) or inhibit(Noggin, Chordin, Follistatin, CAN family) the TGF-β ligand. There are different type of Smad proteins, which regulate the pathway inhibition or carryout inside the cell membrane.

This is what happen when the pathway is initiated: Binding of TGF ligand to TGF β receptor II  initiates signaling, and the receptor I activates Receptor-Smad. Receptor-Smad and Co-smad binds, and this complex enters the nucleus and activates the transcription of target genes. Interesting thing is, this particular pathway is highly conserved in metazoans, while non-metazoan has no TGF-β receptor or ligand. This pathway is most likely evolved in early animal evolution, and to be involved in axial patterning of cnidarians. Now, would that be true?

This paper investigates the components of TGF-β signaling pathway of M. leidyi to argue that the pathway is likely to be evolved in early metazoan evolution since none of TGF-β signaling pathway has been identified in any non-metazoans. Also, the paper suggests that TGF-β signaling pathway is involved in transducing earlier maternal signals rather than early axis specification of embryo development.

2. Results

Porifera, Cnidaria, Ctenophore are all relatively simple organism that is thought to be evolved early in animal evolution. These phylum (which are metazoans) has TGF-β receptor or ligand, and this supports TGF-β pathway most likely evolved early in animal evolution. The recent analysis of the genomic sequence of M. leidyi identified a near complete TGF-β signaling pathway component except any extracellular antagonists such as Noggin, Chordin, Follistatin, and CAN family.

Like it was mentioned at above, M. leidyi, TGF-β pathway requires many different components : Type I and II receptor, Smad, TGF-β ligand, and different signaling inhibitors. These different TGF-β superfamily(group of each components) can be divided into two major classes : TGF-β like class, BMP(bone morphogenetic protein) like class. The author identifies nine TGF-β ligands, four receptors, and five Smads from a genomic sequence anaylsis. The identified components of TGF-β pathway is classified as below.

What component? How we are classifying?
TGF-β ligands TGF-β like : MITGFbA, MITGFbBBMP-like: Mlbmp3, Mlbmp5-8
TGF-β receptors TGF-β receptor I: TgfRIa, TgfRIb, TgfRIcTGF-β receptor II: TgfRII
Receptor Smad Smad 1/5 family : MISmad1a, MlSmad1bSmad 2/3 family : MlSmad2

Table 1. Classification of TGF-β superfamily identified in the genome analysis of M. leidyi.

Let’s take a break and look at the Table 1. Among the nine TGF-β ligands, four were TGF-β like ligands. Among four TGF-β like ligand, the most TGF-β like families were MITGFbA, MITGFbB.  The BMP like ligands were identified as Mlbmp3 and Mlbmp5-8. Take a deep breath. The name of the gene, like I said, is not that important. Just know these are classified as TGF-β like, others are BMP like.

Four receptor were divided into two classes : a single Type II receptor (TgfRII) and three Type I receptor (TgfRIa, TgfRIb, TgfRIc). Also, names are not that relevant.

Among five Smad family members, there are three receptor Smads: two belonginig to Smad1/5 family(MISmad1a, MlSmad1b) and one Smad 2/3(MlSmad2). There is a single Co-Smad(Mlsmad4) and a single inhibitory.

What we have to know from Table 1 is that these components were identified as TGF-β like, and proves that M. leidyi has these components. However, there were no extracellular components such as Noggin, Chordin, or Gremlin orthologs, which are known to inhibit the TGF-β signaling.

Table 2. TGF-beta pathway members in Mnemiopsis genome. Identified TGF-beta pathway members are listed out. No need to memorize or to know all the functions. Just skim through it so you will not be confused with names later.

Table 2 shows the TGF-β pathway members in Mneomiopsis gene. No need to know functions of these genes, all it saying is these genes are classified as TGF-β pathway member gene. But do skim through the names of the genes, since author will investigate which gene is expressed at what stage of embryo development.

Next, the author uses RNA-tagging technique (specifically uses digoxigenin-labeled RNA probes. Link provided) and in situ hybridization to visualize which specific TGF-β pathway component is expressed in what stage of embryo development. The three axis of M. leidyi is explained in the overview page.

Figure. 1 Early TGF-b mRNA expression. Four of the TGF-beta genes are detected early in development prior to and during gastrulation. The schematic at the top depicts the stages of embryos during cleavage and gastrulation, at 1–2 and 3 hours post fertilization (hpf), respectively. Embryos are lateral views, otherwise oral/aboral as stated. The asterisk marks the position of the blastopore.

The TGF-β signaling genes were expressed just prior to and during gastrulation, right around along the three body axes. The earliest expression is MITgf1a and MIBmp3 at 1-2 hours post fertilization, at the onset of gastrulation.(First picture of Fig 1. B&C) MlBmp3 expression lasts only short time and does not last until the later development stage. Also, all four TGF-β signaling genes are somewhat expressed along the body axes: oral-aboral, sagittal, or tentacular.

Figure 2. Late TGF-beta mRNA expression. MlBmp5–8, MlTgf1a, MlTgf2, MlTGFbB and MlTolloid are detected during later stages of development.

Trust me, there is a reason why the figure is so big. The tentacle bud and pharynx structure location by looking at the top of the figure. These TGF-β signaling genes (MlBmp5-8, MlTgf1a, MlTgf2, MITGFb8, MITolloid) are expressed predominantly at tentacle buds(3rd and 4th column of every row) and pharynx(4th and 5th column of every row) of later embryo development.

Figure 3. TGF-beta receptor expression patterns. Expression of TGF-breceptors through development, from gastrulation (3 hpf) to cydippid (24 hpf). Views are lateral unless otherwise specified, and asterisks mark the position of the blastopore or mouth.

Unlike TGF-β type II receptor which are pervasive throughout from egg to cydippid(Link provided) stage(Figure 3A), each three Type I receptors are expressed in different area that doesn’t overlap each other.(Figure 3B,C,D) Still, all receptor are predominantly expressed around tentacle bulb and pharynx. (Again, 4th and 5th columns of every row)

From the Figure 1, except the expression is MITgf1a and MIBmp3 during 1-2hpf, most of genes were expressed from 3-5hpf to cydippid stage(24hpf). This concludes that it is hard to believe TGF-β signaling pathway is involved in establishing axis formation of M. leidyi since axis specification occurs prior to the onset of gastrulation.(R&D Systems) Even the earliest TGF-β pathway gene expression occurs at 3-5hpf, which is at the onset of gastrulation.(Figure 1B&C) Then our question is, does TGF-β signaling pathway have nothing to do with axis formation or any later embryo development?

Figure 4. SB431542 treatment during Mnemiopsis development. Effects of TGF-binhibitor, SB431542, at 12 hours post fertilization. (A,B, D– F) are treated embryos, while (C, G–I) are controls. White arrows on A-C shows each comb rows of M. leidyi. The red lines in D-I shows the pharynx(D,G) and tentacle buds(E,H).

SB431542, a protein inhibitor of TGF-β signaling by blocking Type I receptor activity, was used to interfere the pathway. The egg with a treatment of SB431542 inhibitor fails to form the normal eight rows of comb plates. Comparing Figure 5C(control) to Figure 5A and 5B (the treatment), the white arrow indicating each comb row is only two at 5A and 4 at 5B.  Instead of eight rows of comb rows, it clustered into two or four groups that does not synchronously aligned to each other. In addition, there is a thickening of the pharyngeal ectoderm and area around tentacle bulb.(Figure 5D& 5G) The tentacle bulb is smaller and does not form tentacle out from itself.(Figure 5E&5H) Overall development is delayed slightly when compared to the wild type. However, when embryos were treated with SB431542 after the gastrulation, the embryo develops normally.

3. Discussion

i) Evolution

Nearly all TGF-β pathway genes are a metazoan-specific and there are no Smad genes in any other eukaryote, except Smad-like MH2 domain in the choanoflagellate, Monosiga. Thus, the origin of this pathway may have been a key innovation in metazoan evolution. Within the metazoan, the diversity and total number of TGF-β receptors and Smads are relatively constant while TGF-β ligands are much more variable. This is consistent with hypothesis that there are more constraints on intercellular relative to the extracellular components of the signaling pathway. The Smads and intracellular regions of the TGF-β receptors can be utilized for multiple purposes and in response to various ligands and signals. On the other hand, the ligands themselves are not so highly constrained, which might explain why there are so many more lignads than receptors and why the sequences of the ligands are much less conserved than those of the receptors and Smads. It is possible that ligands diversified and were co-opted for multiple developmental processes, while the intracellular components were reused.

Figure 5. Summary of presence and absence of TGF-beta components. The rows contain the different TGF-beta components. The columns represent the four early-branching lineages of the Metazoa, plus the Bilateria. Each row represents the presence (black dot) or absence (grey dot) of a particular component in the corresponding lineage. The box shows the absences shared by Porifera and Ctenophora.

While the core components of TGF-β, the receptor and its mediators is co-evolved, the addition of antagonistic ligand regulation appear to have evolved later. The Mnemiopsis genome does not contain any of TGF-β signaling pathway inhibitors,(Figure 5) suggesting the ancestral function of BMP enhancer must have targeted proteins other than Chordin, Noggin, CAN family members.

Considering all non-bilaterians have at least one TGF-β like gene while all non-bilaterians have at least one BMP-like gene,(Figure 5) this variation must have occurred early in animal evolution. The emergence of TGF-β pathway was a key innovation in the transition to multicellularity in the metazoan ancestor

ii) Function of TGF-β pathway in embryo development

The results of TGF-β inhibitor SB431542 suggest that there is also a role of TGF-β signaling in comb row organization and morphogenesis. The SB431542 treatment has resulted the comb plate formation at the correct time and similar morphological display but in more clustered way. Recent research discovered T-box gene, branchyury, has shown when its function inhibited during the development, it blocks pharyngeal invagination.(Yamada) This is similar defect that was shown by SB431542 treament.It is possible that brachyury is a target of TGF-β signaling, similar to both frog and the chick brachyury that are direct targets of Activin-like signaling, which is one of TGF-β signaling pathway. It is yet to determine what really mediates comb row development, but inhibition supports that TGF-β signaling could be playing a role in ctenophore pharyngeal morphogenesis by activating brachyury.(Yamada)

The expression of TGF-β ligand appears right before the gastrulation, after the axes are already specified.(Figure 1,2,3) Therefore, TGF-β signaling pathway is not directly involved in early axis specification, but presumed to transfer earlier signal such as maternal protein that are localized in early embryo and egg. The paper does not talk about further into TGF-β pathway member functions in transferring earlier signal (such as maternal signals).

4. Review

A large portion of paper focuses on explaining evolution relationship among Mnemiopsis member. I did not include in the wiki page, but there are so many figures to explain the relationship among the species. However, the author does not explain how he divided the members of Mnemiopsis into TGF-like or BMP-like pathway, not he/she explains what this is significant in general evolutionary theory. Most of the discussion also briefly discusses the evolutionary significance using such data, makes me wonder why it was so important to put the data in there.

It does give supporting proof that TGF-β pathway is evolution was significant for multicellular organism, but its function in Mneomiopsis is not shown more specifically. The actual experiment that involved was only one, the treatment of inhibitor SB431542. By one experiment, it is hard to tell any specific functions of TGF-β pathway or how its components interacts. The experiment result is not fully explained why the comb rows are clustered, why the pharynx was enlarged, or why the tentacle buds are decreased in its size.

Overall, the evolution trend of Mnemiopsis is explained well, but the function of TGF-β pathway are not researched further in the paper, thus making the reader to wonder why author choose a TGF-β pathway function to prove the evolutionary trend among Porifera, Cnidaria, and Ctenophore when there is no explanation of function of TGF-β pathway.

Reference

Kevin Pang, Joseph F. Ryan, Andreas D. Baxevanis, Mark Q. MArtindale. “Evolution of the TGF-β Signaling Pathway and Its Potential Role in the Ctenophore, Mnemiopsis leidyi.” PLoS One. 2011; 6(9): e24152. Published online 2011 September 8. http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024152

The Virtual Fossil Museum. “Kingdon Animalia(Metazoa) Systematics.” The Virtual Fossil Museum. 2002-2012. http://www.fossilmuseum.net/Tree_of_Life/kingdom_animalia.htm

Yamada A, Martindale MQ, Fukui A, Tochina S. “Highly conservedfunctions of the Brachyury gene on morphogenetic movements: insights from theearly-diverging phylum Ctenophora.” Dev Biol. 2010. 339: 212–222.

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