The transforming growth factor-β (TGF-β) signaling pathway was first discovered about 30 years ago, a pathway in which certain secreted proteins had the capability of transforming cells and tissues. The first TGF-β gene was cloned in 1985 
. Since then, similar proteins were discovered in animals as diverse as flies, nematodes, and vertebrates, all of which had similar functions in tissue morphogenesis (reviewed in 
). Through the use of cloning and sequencing technologies, it was soon discovered that the genes encoding for these proteins were all related and diversified from a common ancestral gene. There are roughly a dozen families belonging to the TGF-β superfamily, and these can be divided into two major classes: the TGF-β-like class and the bone morphogenetic protein-like (BMP) class. The TGF-β-like class includes TGF-β sensu stricto
, Lefty, Activin/Inhibin, and Myostatin/Gdf8. The BMP class includes Bmp2/4/Dpp, Bmp5–8, Bmp3, Gdf2, Gdf5–7, Vg1/Univin, ADMP, and Nodal. Besides being known for its roles in morphogenesis, TGF-β signaling, especially via Bmp2/4/Dpp, is also known for its role in dorsal-ventral patterning in both protostomes and deuterostomes (reviewed in 
The TGF-β precursor protein has three distinct regions: (1) the signal peptide, which targets it to the endoplasmic reticulum and secretion; (2) the propeptide, or the latency associated peptide; and (3) the mature peptide, which is cleaved from the precursor protein and is actively involved in signaling 
. Whereas the mature peptide is highly conserved across different families, the propeptide is not. The mature peptide is cleaved by Furin, a convertase, at a dibasic arginine-X-X-arginine (RXXR) site 
. The active peptide forms a hetero- or homodimer, which binds to a specific TGF-β Type II receptor () 
. The Type II receptor then recruits a TGF-β Type I receptor and phosphorylates it via its serine/threonine kinase domain. Phosphorylated Type I receptors then phosphorylate (and thereby activate) receptor-associated Smad proteins (R-Smads), including Smad1/5 and Smad2/3 (For reviews, 
). R-Smad proteins are composed of two main functional domains, the Mad-homology domains 1 and 2 (MH1 and MH2). Smad1/5 is associated with BMP-like signaling, while Smad2/3 is associated with TGF-β-like signaling. Inactive R-Smads are associated with the membrane via the Smad anchor for receptor activation (SARA) protein, which contains a zinc finger FYVE domain 
. Activated R-Smads are released into the cytosol where they interact with the common-mediator Smad (Co-Smad, aka Smad4), and then become translocated to the nucleus. This heteromeric complex then regulates TGF-β target genes by interacting with transcription factors, including Fos/Jun and Myc, or co-activators, such as the Creb-binding protein (CBP) 
. The MH1 domain is capable of interacting with DNA, while the MH2 domain interacts with Type I receptors and is involved with protein-protein interactions, such as R-Smad/Co-Smad binding.
Basic overview of TGF-β signaling pathway.
Inhibition of TGF-β signaling can occur at multiple levels: extracellularly, cytoplasmically, and in the nucleus. Extracellularly, diffusible antagonists such as Chordin, Noggin, Follistatin and the CAN family (Cerberus/DAN/Gremlin) act as ligand traps, interfering with ligand binding to receptors 
. In turn, the zinc metalloprotease Tolloid is capable of cleaving Chordin, thereby releasing BMPs to become active, showing that there are many levels of regulation involved with TGF-β signaling 
. Besides cleaving Chordin, Tolloid also functions to cleave pro-collagens of the extracellular matrix 
, as well as other proteoglycans, some of which also are known to bind TGF-β ligands 
Intracellularly, the pathway can be inhibited at many levels. At the level of the receptors, FKBP12 can block Type I receptor phosphorylation by binding to the GS domain 
. BAMBI, a pseudoreceptor, can prevent the Type I and Type II receptors from forming a receptor complex 
. Pathway modulation can also occur via inhibitor-Smads (I-Smad, Smad6/7), which have an MH2 domain (like other Smads) and can bind to Type I receptors, interfering with R-Smad binding and phosphorylation 
. I-Smads can also compete with R-Smad in binding with Co-Smads. Another intracellular regulator of TGF-β signaling is the Smad ubiquitin regulatory factor (SMURF), an E3 ubiquitin ligase that targets R-Smads for degradation 
. SMURF can also be recruited by I-Smads to degrade Type I receptors at the membrane. TGF-β signaling is also regulated within the nucleus by the binding of co-repressors Ski/Sno 
. These proteins recruit other repressors to block the activation of TGF-β target genes.
All levels of the TGF-β signaling pathway are highly conserved in metazoans, with pathway members present in all animals studied to date 
. Outside the metazoa, no TGF-β receptor or ligand has been discovered, so this pathway most likely evolved early in animal evolution. In the choanoflagellate, Monosiga brevicollis
, an MH2 domain is present; however, it is unlike all known Smad proteins in that it is accompanied by a zinc finger domain 
. Amongst the non-bilaterians (cnidarians, poriferans, the placozoan, and ctenophores), most of our knowledge regarding this pathway is gleaned from cnidarians 
. Interestingly, this pathway has been implicated in axial patterning in cnidarians, similar to its role in dorsal-ventral patterning in bilaterians. Work in the sponge, Amphimedon queenslandica
, has also shown that TGF-β signaling may be involved in axial patterning 
. To date, there is nothing known about this pathway in the final group of non-bilaterians, the ctenophores. To better understand the evolution of this pathway, we need to be able to compare all the non-bilaterian taxa.
The ctenophore body plan and body axes are specified early in development. Developmental potential is segregated to different lineages; however, the exact molecules involved are unknown. Analysis of the genomic sequence of the lobate ctenophore, Mnemiopsis leidyi, allowed us to identify a near-complete TGF-β signaling pathway composed of nine ligands, four receptors, and five Smads, revealing that the core components are present in all metazoans studied to date. Notably absent are extracellular diffusible antagonists, including Chordin, Follistatin, Noggin, and CAN family members. We looked at the expression of these genes during ctenophore development and found expression of ligands to be differentially expressed along all three body axes (oral-aboral, tentacular, and sagittal). While we do not believe this pathway is necessarily specifying these axes, since they are expressed after the axes are already specified, we do believe they are involved with transducing earlier signals.