BIL455 extra bullshit (signaling pathways + Macho1 + experiments and methods)

just like. everything else thats not specified in the other flashcard sets who knows. im so fucking tired

What is the universal signaling framework?

Extracellular ligand → receptor → intracellular signal transduction → transcriptional regulator(s) → altered target-gene expression → context-dependent developmental response.

What is a ligand?

A signaling molecule that binds a receptor and initiates a cellular response.

What does extracellular mean?

Outside the cell.

What does intracellular mean?

Inside the cell.

What is the RTK/Ras/MAPK sequence?

Ligand → Receptor Tyrosine Kinase (RTK) → Guanine Nucleotide Exchange Factor (GEF) → Ras-GTP → Raf → Mitogen-Activated Protein Kinase Kinase (MEK) → Extracellular Signal-Regulated Kinase (ERK) → transcription-factor regulation.

What are the major RTK receptor regions?

Extracellular ligand-binding domain → transmembrane region → intracellular/cytoplasmic tyrosine-kinase domain.

What is Ras?

A small GTPase molecular switch: Ras-GDP is inactive; Ras-GTP is active.

What does GEF do?

Activates Ras by promoting GDP-to-GTP exchange.

What does GAP do?

Promotes GTP hydrolysis by Ras, returning Ras to the inactive GDP-bound state.

What does Raf do?

A protein kinase activated downstream of Ras-GTP that initiates the Raf → MEK → ERK kinase cascade.

What does MEK do?

A kinase downstream of Raf that activates ERK.

What does ERK do?

A kinase downstream of MEK that can regulate transcription factors and thereby alter target-gene expression.

RTK cytoplasmic domain deleted: prediction?

Ligand may bind, but intracellular signaling is lost/strongly reduced.

Ras cannot interact with GAP: prediction?

Ras stays active longer → high/prolonged downstream signaling.

GEF cannot activate Ras: prediction?

Ras remains mostly GDP-bound → low/absent downstream signaling.

Constitutively active Raf: prediction?

MEK/ERK output remains high even without normal upstream RTK/Ras activation.

What is the Wnt/beta-catenin sequence?

Wnt → inhibition of beta-catenin destruction → beta-catenin stabilization/accumulation → nuclear entry → cooperation with DNA-binding regulators → altered target-gene transcription.

What is the key beta-catenin exam trap?

Beta-catenin is a transcriptional co-regulator; it does NOT directly bind specific cis-regulatory DNA sequences as the sequence-specific DNA-binding factor.

What is the TGF-beta/SMAD sequence?

TGF-beta-family ligand → heteromeric serine/threonine kinase receptor → SMAD proteins → nuclear transcriptional regulation → developmental response.

Which ligands are in the TGF-beta superfamily?

BMP, Activin, DPP, Vg1, and Nodal.

What does heteromeric/heterodimeric mean?

The receptor complex contains different kinds of subunits/components.

What is one developmental TGF-beta-family example?

BMP4 signaling from the optic vesicle contributes to final lens differentiation.

What is the vertebrate Hedgehog sequence?

Hedgehog ligand → Patched/Smoothened regulation → Gli transcription factors → altered target-gene transcription.

What happens without Hedgehog?

Patched inhibits Smoothened, preventing the normal Hedgehog-dependent Gli response.

What happens when Hedgehog binds Patched?

Patched's inhibition of Smoothened is relieved, allowing downstream regulation of Gli and transcription.

What is the key nuclear regulator in Hedgehog signaling?

Gli transcription factors.

What is the Notch/Delta sequence?

Delta on signaling cell → Notch on adjacent responding cell → Notch cleavage → Notch Intracellular Domain (NICD) → nucleus → transcriptional regulation.

Why is Notch/Delta juxtacrine?

Both ligand and receptor are membrane-associated, so signaling requires direct contact between neighboring cells.

What is lateral inhibition?

A Notch/Delta patterning mechanism in which small differences between neighboring cells are amplified so they adopt different developmental states/fates.

How can you distinguish the pathways instantly?

RTK = Ras/Raf/MEK/ERK kinase cascade; Wnt = beta-catenin stabilization; TGF-beta = SMADs; Hedgehog = Patched/Smoothened/Gli; Notch = Delta contact + receptor cleavage/NICD.

Do these pathways have one universal developmental outcome?

No. Their molecular machinery is characteristic, but the target genes and resulting fate/behavior depend on developmental context and regulatory state.

What organism is studied in the Macho1 work?

An ascidian, a marine invertebrate commonly called a sea squirt.

Why are ascidians useful for studying autonomous specification?

Localized maternal cytoplasmic determinants are partitioned into particular blastomeres, producing strongly lineage-associated developmental fates.

What is ooplasmic segregation?

Post-fertilization rearrangement of egg cytoplasm that relocates pre-existing developmental materials before cleavage partitions them into particular blastomeres.

What is Macho1?

A localized maternal zinc-finger transcriptional regulator/determinant that promotes muscle fate in the ascidian embryo.

Why is macho1 maternal?

Its mRNA is supplied in the egg by the mother and is present before the relevant embryonic transcriptional program.

Why is macho1 localized?

Its mRNA is not uniform; ooplasmic segregation positions it so future muscle-lineage blastomeres preferentially inherit it.

What developmental mechanism does Macho1 exemplify?

Autonomous specification through inheritance of a localized intracellular maternal determinant.

What protein domains does Macho1 contain?

Five C2H2-type zinc-finger repeats.

What do the zinc fingers suggest?

DNA-binding capability and a role as a transcriptional regulator.

What is the mechanistic logic from macho1 mRNA to muscle?

Localized macho1 mRNA → Macho1 protein → transcriptional regulation → downstream muscle gene-expression program → muscle differentiation.

How was macho1 mRNA localization tested?

RNA in situ hybridization.

What does macho1 localization provide?

Correlation evidence: the mRNA is positioned where the muscle determinant is expected, but localization alone does not prove causation.

How was Macho1 necessity tested?

Loss of function using an antisense oligonucleotide against macho1, followed by assessment of muscle differentiation.

What was the Macho1 loss-of-function result?

Muscle differentiation/markers were strongly reduced, supporting that Macho1 is necessary for normal muscle development.

Which markers indicate muscle differentiation in this context?

Myosin and acetylcholinesterase.

What was HrWnt5 used for after macho1 depletion?

A specificity/control-type test: preserved HrWnt5 localization showed the treatment did not simply destroy general ooplasmic segregation or localized maternal-RNA organization.

How was Macho1 sufficiency tested?

Gain of function by adding/ectopically expressing macho1 mRNA and asking whether muscle differentiation appears or increases where it normally would not.

Why express Macho1 in a non-muscle/endodermal context?

A true sufficiency test asks whether Macho1 can promote muscle fate in cells that would normally adopt another fate; adding it only to normal muscle progenitors would be much less informative.

What was the Macho1 gain-of-function result?

Extra/ectopic muscle differentiation occurred, supporting that Macho1 is sufficient to promote muscle fate in the tested context.

Why can extra muscle be accompanied by reduced endoderm?

Some cells normally contributing to endoderm can be redirected toward muscle when ectopic Macho1 alters their developmental program.

What does alkaline phosphatase mark here?

Endoderm.

Macho1 versus myosin: what is the difference?

Macho1 is an upstream regulatory determinant; myosin is a downstream muscle differentiation product/marker.

What is the three-experiment Macho1 logic?

Correlation: locate macho1 mRNA. Necessity: inhibit macho1 and see whether muscle decreases. Sufficiency: add/ectopically express macho1 and see whether muscle appears/increases.

Does macho1 antisense produce extra muscle?

No. Antisense is loss of function and reduces muscle differentiation.

Does macho1 overexpression produce extra/ectopic muscle?

Yes. This is the gain-of-function result supporting sufficiency.

Why does the Macho1 study connect Chapters 2 and 3?

It links autonomous specification/localized determinants to differential gene expression through a DNA-binding transcriptional regulator.

What are the three core experiment types?

Correlation → necessity/loss of function → sufficiency/gain of function.

What does correlation ask?

Is candidate X present/active in the correct place and time to be associated with outcome Y? Association alone does not prove causation.

What does necessity ask?

Is X required for Y? Remove/block/reduce X; if Y decreases or disappears, X is necessary for normal Y.

What does sufficiency ask?

Is X enough to promote Y in the tested context? Add/activate/ectopically express X where Y is normally absent; if Y appears, sufficiency is supported.

What is loss of function (LOF)?

Reduction or elimination of a gene product/pathway function; commonly tests necessity.

What is gain of function (GOF)?

Increased, ectopic, or constitutive activity/expression; commonly tests sufficiency.

What is ectopic expression?

Expression in a place/cell/time where the gene is not normally expressed.

What is the reusable Exam 1 design structure?

Testable hypothesis → correlation experiment → necessity/LOF experiment → sufficiency/GOF experiment.

How should the hypothesis be phrased?

State a plausible causal relationship suggested by the observation, e.g. 'Molecule X produced by Tissue A promotes/induces fate Y.' Do not automatically claim both necessity and sufficiency.

mRNA location: method?

RNA in situ hybridization.

Protein location: method?

Immunostaining/immunofluorescence with an antibody against the protein.

Genomic binding site of a transcription factor: method?

Chromatin Immunoprecipitation Sequencing (ChIP-seq) or targeted ChIP.

Whole-sample transcript abundance: method?

RNA sequencing (RNA-seq).

Individual-cell transcript profiles: method?

Single-cell RNA sequencing (scRNA-seq).

RNA-seq versus scRNA-seq?

RNA-seq averages expression across a sample; scRNA-seq preserves individual-cell heterogeneity, cell states/types, and inferred trajectories.

What is HCR-FISH?

Hybridization Chain Reaction Fluorescence In Situ Hybridization: amplified, potentially multiplexed spatial RNA detection.

What is direct immunofluorescence?

Fluorescent primary antibody binds target directly.

What is indirect immunofluorescence?

Unlabeled primary binds target; fluorescent secondary binds primary, providing flexibility and signal amplification.

What is a reporter construct?

Regulatory DNA linked to a basal promoter and detectable reporter gene; reporter expression tests regulatory activity.

Enhancer + basal promoter + reporter tests what?

Whether the enhancer is sufficient to drive the observed expression pattern in the tested context.

Enhancer deletion followed by loss of expression supports what?

That the enhancer is necessary for the tested normal expression pattern.

Basal promoter deletion: prediction?

Transcription is abolished/strongly reduced even with an enhancer because transcription initiation requires a functional promoter.

Does the basal promoter normally provide tissue specificity?

Not like an enhancer. Enhancers/cis-regulatory modules provide much of the spatial/temporal specificity; the basal promoter supports initiation.

What does spatial expression mean?

WHERE expression occurs.

What does temporal expression mean?

WHEN during development expression occurs.

Can one endpoint establish temporal expression?

Usually no. One endpoint shows expression at that time; onset/change requires multiple stages or time-resolved data.

What does 'based on available data' mean?

Conclude only what was actually measured; do not overclaim causation, timing, location, or mechanism.

What cue means necessity?

Words such as required/necessary → perform LOF/blocking

What cue means sufficiency?

Words such as sufficient/enough/can induce → perform GOF/ectopic addition.

Neutral environment tests what?

Specification: isolated tissue follows its prospective fate without new instructive signals.

Transplant to a different embryonic environment tests what?

Determination: determined tissue retains its original fate despite a new environment.

What do antisense Morpholinos usually do?

Block translation or alter splicing; they do not usually act by degrading the target mRNA.

What does constitutively active mean?

A component remains active without its normal upstream activating signal.

RTK cytoplasmic-domain deletion: logic?

Mechanism/LOF: ligand binding may remain, but downstream signaling is lost.

Ras cannot bind GAP: prediction?

Ras remains GTP-bound longer → high/prolonged output.

GEF cannot activate Ras: prediction?

Ras stays mostly GDP-bound → low/absent output.

Constitutively active Raf: prediction?

High downstream MEK/ERK output independent of normal upstream RTK/Ras activation.

IP3 injection into an unfertilized egg tests what?

Sufficiency-type logic: if IP3 triggers the calcium wave/activation component, IP3 is sufficient for that response.

EGTA injection tests what?

Necessity of calcium: EGTA sequesters Ca2+; loss of the response supports that Ca2+ is required.

Voltage clamp in sea-urchin eggs tests what?

A causal role for membrane potential in the fast block to polyspermy.

What does +20 mV versus -70 mV show?

+20 mV prevents fertilization; a strongly negative potential permits polyspermy, supporting membrane potential as the fast-block mechanism.

Macho1 experimental logic?

Correlation: RNA in situ. Necessity: antisense LOF → muscle decreases. Sufficiency: ectopic macho1 mRNA → extra/ectopic muscle.

Why use developmental markers?

They provide measurable evidence of a cell fate/differentiation program rather than relying only on gross morphology.