Skapek

Skapek- Muscle cell differentiation

Talking about general concepts in muscle that can be extended in principle to other types of differentiation.
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important concepts.
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Skeletal muscle develops from the mesoderm layer of the embryo. Mesodermal cells are pluripotent progenitors. Some stage determines cell to skeletal muscle lineage. Happens in somites (clusters of cells along developing notochord and neural axis in segments). Initially all look the same. Cells in dorsoventral aspect are dermal myotome. Get cues from epithelial cells and from medial notochord which set up gradients of diffusible proteins. Determined at that stage, but must migrate. Myogenic cells migrate to limbs or body wall. Differentiate after migration. While or after migration start undegoing morphological and biochem chnges. Become myotube or myofiber of myotubes. Can get multiple nuclei by failure of cytokinesis or fusion of cells. The latter causes multinucleation in skeletal muscle cells.
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Some epigenetic events allow expression of transcription factors. Cell undergoes some changes due to autonomous factors (programming) or signalling from outside of cell.
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Expression of a few key genes is necessary. MyoD was the first transcription factor discovered. Found exclusively in myoblasts. Myf5 has overlapping function. Expression MyoD enough to make cell muscle. Works with other transcription factors. Others are not muscle-specific. Environmental cues from notochord and ectoderm important as shown in mouse and chick models.
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MyoD and Myf5 may guide lineage commitment to different types of skeletal muscle. Pax3 is not muscle- specific, but critical for induction of MyoD. Nature couples transcription factors promoting migration and differentiation. Myogenin and MRF4- transcription factors in positive feedback loop with prior transcription factor. They also induce transcription of proteins to make muscle cells. They can also induce cell cycle arrest. Knockout MyoD- normal skeletal muscle. Some functional redundancy or compensation. More to story was other transcription factors. Double knockout- no muscle embryonic lethal. Myogenin knockout- no differentiation of myocytes to myotubes. Pax3 is secreted from notochord due to Wnt signaling from ectoderm in chick. Myf5/Pax3 double knockout is lethal.
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See papers. 5-azacytidine blocks DNA methylation. Could get some fibroblasts to become muscle cells. Later found myoD expression, and used it to turn cells into muscle cells.
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Skeletal muscle differentiation is a one-way street. They do not de-differentiate.
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What controls migration?
Met receptor is an RTK expressed on myoblasts when migrating. Activated by hepatocyte growth factor. Signalling critical for migration. Rhabdomyosarcomas express a lot of muscle proteins.
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What is basis of differentiation?
Cell has to undergo biochemical and morphological change. What starts process?
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whether is autonomous or not is not clear. Certain cues have to happen in vivo.
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bHLH has domains you should know. Does not interact with RNA pol 2 directly, but withHATs, etc. No TAD, unable to make muscle cell. Transcription factors must bind DNA at E boxes. CANNTG genome- E box every 250 nt. MyoD does not bind every one. Basic region essential for specificity. 4 are myogenic, many other bHLH transcription factors are not myogenic. 3AAs in basic region are essential. What they do exactly is unknown.
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E proteins interact with MyoD thrugh HLH domain. MyoD cannot bind DNA by itself- needs E box.
16 Carboxyterminus is large. Can be lopped off and still cells form muscle cells in vitro. May control stabilization, etc.
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Myoblasts- MyoD is inactive. One critical way to control MyoD is heterodimer formation. MyoD can form homodimers.
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Dominant negative inhibitors of differentiation- dominates another protein to turn it off. Id has no basic domain for DNA binding. Shuts off MyoD. Id means inhibitors of differentiation. There are 4 Id proteins.
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MEF2 is involved in cardiac muscle and skeletal muscle. MyoD and Mef 2 cooperate functionally.E boxes have Mef2 binding sites adjacent to them.
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Mef2 and MyoD attract CBP/p300 with HAT activity.
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cultured cells- if you grow to confluence or add mitogens, they start to differentiate. Arrest causes differentiation.
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E2F induces genes involved in DNA synthesis. Rb is complex. Guides lineage specification. Rb prevents G1/S transition. Phophorylated by cyclin/cdk. Proliferation signals induce cyclins. Cdk2 inhibitors also work.
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Rb is essential for muscle cell development. Cells can become commited, but not become normal muscle cells without it. Without it, cells do not arrest and become post-mitotic.2 defects: lack of muscle specific genes and lack of cell cycle arrest.
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3 D type cyclins expressed early in proliferating cells. Kinase is constant .
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In the presence of cyclin D 1- MyoD phosphorylated.
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In vivo differentiation signals unknown.
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Transcriptional machinery regulated at multiple levels.
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Twist encodes a bHLH like protein with properties like Id proteins. Blocks MyoD-E heterodimer formation and functional activity Mef 2.
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Mapk involved. Positive feedback activates p38 MAPK.
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How do we study this?
Take fibroblasts- cells undergo changes in medium with MyoD. Stain for muscle proteins and see them.
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Reporter gene on plasmid driven by muscle-specific promoter can be manipulated quantitatively.
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Review

Similar bHLH are essential for other developmental processes.
Email him questions- test from chapter and these slides. Make sure you understand molecular biology of these transcription factors and how they work . Challenging question about how to find this and what to do with results.