DEVO-7: Gradients & Morphogens –1–
Review and move on… DEVO-7
• Complete Left-right asymmetry paper • On to morphogensis (gradients, responses, and patterns)
Q: A question (from you) about the myoD1 paper? Types of myosins –2–
Q: what types of myosin are you familiar with?
Q: How might these different myosins have arisen (evolutionarily)?
DEVO-7 Question from the past –3–
Q: why does muscle contract Q: why is it that MTs can support rather than expand (or both)? movement in both directions?
DEVO-7 Cellular handedness –4–
Q: how do microfilament and microtubule systems interact?
DEVO-7 Cellular handedness –5–
Q: Does the microtubule system have a handedness?
DEVO-7 Thinking about myosin 1D / 1C paper –6–
we will be reasonably superficial
• What did they know before they started? • What was the author’s assay system - what were they looking at? • What did they do? • What did they find?
DEVO-7 Types of myosins –7–
myo1D is (one of a number of) situs inversus genes: its absence leads to the full reversal of organ positioning along the LR axis, with organs adopting a mirror-image orientation.
DEVO-7 (A) Control larvae showing bilateral symmetry. The trachea are indicated, with anterior tracheal spiracles indicated by a red (left) or blue (right) (Bottom) Front view of the larvae (ventral down, dorsal up).
MH, mouth hooks; ALS, anterior left spiracle; ARS, anterior right spiracle; PLS, posterior left spiracle; PRS, posterior right spiracle.
A – anterior; P – posterior; L – left; R – right; D – dorsal; V– ventral. “we ectopically expressed the protein in different naïve tissues (i.e., tissues devoid of LR asymmetry). We found that Myo1D expression in the larval epidermis induces dextral twisting of the whole larval body
Q: what is ectopic expression and how did they do it. Q: what does it mean that “phenotype is 100% penetrant and is specific to Myo1D”?
DEVO-7 Linear morphology of a control (btl>srcGFP) third myo1D expression in the instar larvae trachea. tracheae (btl>myo1D) increases their dextral twisting with Q: What does GFP multiple loops. expression control for? Co-expression of myo1D and an RNAi against myo1D completely suppresses the twisting phenotype
Q: what does this experiment “control” for? is a positive or negative control?
DEVO-7 myo1D mRNA levels (by qPCR) in the epidermis of control (tsh>), myo1D-GOF (gain of function) (tsh>Myo1D) or myo1C-GOF larvae (tsh>Myo1C). Error bars indicate standard deviation, RNA levels in arbitrary units (AU).
Q: how did they get these data and why did they include it? Myo1C is a sinistral myosin, an antagonist to Myo1D.
Control larvae (A) and larval-twisting phenotype (B) induced by over- expression of myo1D or by the over-expression of (C) myo1C or the co- expression of (D) myo1D & myo1C.
DEVO-7 Q: What is a chimera? Myo1D:Myo1C chimeras indicate that head/neck domain provides directionality to the proteins. Domains designated D & C derived from Myo1D and Myo1C, respectively.
chimeras
DEVO-7 myosin handedness –17–
Q: How would the presence or absence of ATP influence this experiment?
DEVO-7 • At the adherens junction (↑) MyoID–β- catenin interaction is stabilized cadherin. MyoID triggers dextral development. • B: MyoIC over-expression (→) disrupts MyoID-β-catenin interaction and MyoID’s function leading to sinistral development. • D: In the absence of DE-cadherin (→), there is no MyoID over-expression rotation phenotype. • What is missing from the “model”? Q: What does a morpholino do?
Organ situs of wild-type (A) & myo1d morphant tadpoles displaying situs inversus (B) and heterotaxia (C) at stage 45. D: Quantification of organ situs analysis. Numbers of analyzed specimens, derived from 3 (D) independent experiments. Your question –20–
Q: Major questions (from you) about the morphogen paper?
DEVO-7 FIRST
Q: what is going on here? what do the arrows and bars mean?
Q: what would you need to know to predict the behavior of such a DEVO-7 system? Morphogens –24–
https://www.ibiology.org/development-and-stem-cells/enhancers/#part-1
background through 7:29 Next : Complete morphogens and: Genikhovish & Technau. 2017 On the evolution of bilaterality