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TGF-B signaling pathway overview
- TGF-beta superfamily ligands (including BMP ligands) bind to both Receptor II and I.
- These ligands cause receptor dimerization and then phosphorylation of both receptor II and I, with Ser/thr kinase domain and GS box, respectively.
- This leads to the activation of Smads, both 2 and 3, and 1 and 5, from Receptors II and I respectively.
- These smads then dimerize with Smad4, where they then translocate into the nucleus to aid in transcription.
DEVELOPMENTAL EVENT THAT UTLIZES TGF-BETA PATWHAY
- TGF-beta pathway is important for tissue patterning, organo morphogenesis, cell proliferation, and in this paper for neural crest and plate formation.
- Bone morphogenic protein (BMP) is a subfamily of TGF-beta pathway. In Xenopus and zebrafish embryos a low dorsal to high ventral gradient of BMP activity is thought to contribute to embryonic patterning. High BMP activity is required for epidermal fate, a moderate level of activity is necessary for neural plate border fates , including competency to become neural crest , and BMP inhibition is a prerequisite for neural fate. Neural and neural crest fates require BMP signaling at temporally distinct periods in addition to the requirement for different levels of BMP activity.
1st EXPERIMENTAL APPROACHES FROM THE PAPER THAT SUPPORTS A ROLE FOR TGF-B/BMP signaling IN THIS DEVELOPMENTAL EVENT:
- First, semi quantitative reverse transcription PCR (qPCR): used to look for a regulator of SNW1 and BMP. Results in Xenopus embryos show SNW1 transcripts present in both dorsal and ventral sides, but at Neurula stages is present more DORSALLY.
- Western blot: showed SNW1 protein levels lowest at earlier stages and continued to increase through the developmental stages.
- Knocking down SNW1: show reduced SNW1 protein levels starting at the level where it was seen to be lowest to start, but NOT before, indicating that the protein prior to this stage is maternally deposited, and the rest is zygotically produced.
- Experiments were repeated in zebrafish, and similar patterns observed.
- SNW1 knockdown: INHIBITS neural crest induction as measured by loss of neural crest markers Snail, Slug and Sox 9 at the injected side at stage 14.
2nd EXPERIMENTAL APPROACHES FROM THE PAPER THAT SUPPORTS A ROLE FOR TGF-B/BMP signaling IN THIS DEVELOPMENTAL EVENT:
- To investigate if SNW1 might elicit its effects on neural crest fate by functioning intracellularly in the TGF beta superfamily signal trunsduction pathways, authors used cell lines in tissue culture for these assays
- siRNA depletion of SNW1 to see if SNW1 may act intracellularly to function in the TGF-B signal transduction pathway: Results show no effect on the levels of phsoprylated Smad1/5 or smad 2 in response to purified TGF-B or BMP4 ligands, arguing against SNW1 functioning in either the core BMP or TGF-beta pathways. SNW1 depletion resulted in a reduction of pSmad1 levels equivalent to that caused by overexpression of the BMP antagonist noggin.
- SNW1 affects BMP signaling only in the context of the whole embryo and not in an isolated tissue or in cell culture, further indicating that SNW1 is not a core component of the intracellular BMP pathway, although SNW1 is required for BMP activity in vivo.
- SNW1 role, however, is by acting at the level or upstream of the BMP receptors, and not in the pathway, where it plays a role in the boundary of the BMP activity in the neural plate border