C elegans development Flashcards
(12 cards)
Development overview
Development Recap: cytoplasmic molecules can bring about differentiation+ re-programming. MyoD TF= master regulatory gene for muscle differentiation. +ve feedback loops ensure stable maintenance/ gene exp-> differentiation. Cell fate determinants in egg cytoplasm- maternal factors allow rapid early cell/nuclear divisions, can be instructive; found essential for fly+ frog dev. Screens for zygotic mutations (flies)-> genes req to segment embryo. Vegetal cells in frog can induce mesoderm. ‘Zones’ in limb drive pattern formation.
Maternal determinants switch on zygotic genes (subdivide body). Homeotic genes assign distinct IDs along body axis. Hox gene clusters highly conserved. Animal homeotic genes encode TFs w/ v conserved homeo-domain, arr in clusters along chr, linear arr on chr matches exp domains along body (collinearity). Plant homeotic genes encode TFs w/ conserved MADS box DNA-binding seq w/ no clustering/collinearity.
Mouse embryogen: zygotic genes active early, little maternal contribution; IPS cells-> therapy opportunities?
Specified vs determined, cell fate factors and modes of action
Specified: @ blastula/ early gastrula stage, animal cap cells and specified to become epidermis- meaning if pieces of animal cap removed, put in neutral env-> grow into epidermis.
Determined- by late gastrula stage, determined to become either epidermis (ventral cells) or neutral tissue (dorsal cells), meaning putting them in ANY env-> grow epidermis/neutral cells.
Cell fate depends on intrinsic (segregation of cell fate determinants-> diff fates after cytokinesis) and extrinsic (induction- interactions between non-equiv cells- cells-> diff fates through exposure to local signal. Gradient- diff conc thresholds of signal-> distinct cell fates. Combinatorial- distinct cell fates via exposure to multiple signals-> unique cell type from action of both. Lateral inhibition- interactions between equiv cells, small differences in levels of signals (stochastic) amplified by feedback mech-> distinct fates) factors.
C elegans as a model: reproduction, early embryogenesis
C elegans as a model: ’63 first proposed as model for neural dev. 2002 Nobel genetics of organ dev in animals. 2006 Nobel- RNAi. 2024 Nobel miRNA role in post-transcriptional gene reg. Lineage tracing enables cell fate maps/ all cells followed over time. Adult has 959 cells, adult male 1031. Embryo transparent+ easy to image.
Reproduction: 2 oviducts, mirror image in adult- internal self-fertilisation (make sperm too). Embryos dev internally, then deposited through vulva. Sperm crawl along walls like amoebas- not swimmers like in mammals. Botha anterior+ posterior involved in reproduction.
Early embryogenesis/divisions:
1) fertilisation-> pronuclear migration-> pronuclear breakdown->1st mitosis
2) 2 cell stage (AB+P1)-> AB cell division (ABa+ABp)
3) P1 div (P2+EMS)- 4 cell stage
4) EMS div into MS and E cells. P2 into C and P3, P3 further into C and P4
dev regular, can name+ follow individual cells. Lineage mapping shows that only a subset of cells dev from AB (hypodermis, neurons, pharynx, some other) but all cell types dev from P1. Asymmetric divs+ localisation/ cytoplasmic+ mem factors-> asymmetry. Early processes est 3 principal axes of body, set fates of 6 founder cells: AB, E, MS, C, D, P4- each produce specific subset/ cell types. E (intestine), D (muscle) and P4(germ line) only produce 1 tissue type. Ablation of founder cells-> loss of those tissues- suggest cell fate determined by cell intrinsic mechs. Moving cells around to create new contacts-> diff outcomes, shows that dev also depends on cell interactions.
Posterior pharynx development: Skn-1 mutants
Part of digestive+ respiratory sys. Muscular walls-> swallowing. Pharynx from ABa+ MS lineages. But only P1, EMS, MS cells can autonomously dev pharyngeal tissue if isolated.
Skn-1 mutant- EMS produces only hypodermis+ muscle (found via genetic screen), posterior pharynx lost+ has epidermis xs. Skn-1 encodes TF 61kD DNA-binding protein w/ basic Leu zipper-like motif. Antibody stain of SKN-1 shows it exp specifically in EMS+ P2 (after 1st embryonic division, low levels in both AB+ P1- strengthens in P1 and lost in AB).
Posterior pharynx and Mex, par and Pie mutants
Mex-1+ par-1 mutants: SKN-1 protein accum in ABa/p (genetic screen). Mex-1 encodes cytoplasmic protein of germ line req to restrict segregation+ activity of SKN-1 to P1. Par-1 req for coordination of spindle orientation+ asym dist/determinants such as segregation/SKN-1 to P1.
Pie-1 mutants- P2 converts to EMS (P1 produces 2EMS), lineage duplicated- suggesting that in pie-1 mutants, SKN-1 inappropriately active in P2, converting fate into EMS. Antibody stain shows PIE-1 (transcriptional repressor) segregates to germline-> P1-> P2. Normally represses SKN-1 activity in P2 (not directly acting on SKN-1 itself, present in P2, as it’s supplied maternally, but rather repressing exp of its targets). Overall, pie-1 represses somatic cell fate in P lineage (germline determinant)
MS and E cell lineages
Isolation+ cell contact tests: When isolated, EMS or E-> intestinal and pharyngeal cells). @ 4-cell stage, each cell can be disaggregated and recombined in pairs-> test which cell influences fate of EMS progeny. An inductive, time-sensitive signal from P2 to EMS establishes E/intestine fate- if cells separated completely >10 min before EMS cleavage-> 2MS, no intestine. If contact with an AB cell (but not P2) is kept, still no intestine. If P2 contact kept, normal dev (also if separation <10 min before EMS cleavage).
Screen for MS/E lineage mutants-> phenotypes affect pharynx+ intestine. (probe mech of P2 action on EMS)
Pop-1 and Mom-2 mutants
Pop-1 mutants overproduce E-derived intestine, lack pharynx (from MS)- MS fate switched to E. antibody stain shows POP-1 more in nucleus of MS that E. Ablation of 1 cell in 8-cell embryo- single presumptive MS cell that’s a pop-1 mutant becomes E, showing pop-1 inhibition-> E fate. Pop-1 encodes TF that represses E fate. A signal from P2 must repress POP-1 activity, allowing E fate.
Mom-2 req only in P2 for intestine/E dev. Recombination/cells from mutant+ wt embryos-> only when P2 mutant for mom-2-> intestine loss. In double mutants for mom-2 and pop-1, MOM-2 signal from P2 not required to repress POP-1 activity, both cells-> E, confirming interaction. In mom-2 -/- cells, POP-1 immunostain signal equally strong in both MS+E nuclei.
Wnt signalling
Wnt signalling: Wnt-> frizzled/ LRP5/6-> disheveled-> GSK3->beta-catenin-> transcription. MOM-2= wnt ligand. MOM-5 is a wnt receptor (frizzled homologue). POP-1= TCL/LEF (wnt-reg transcriptional repressor). P2 MOM-2 signalling to E cell MOM-5-> low nuclear POP-1, so it can’t repress E genes. No signalling to MS-> high nuclear POP-1-> E genes blocked.
MS+ E fates depend on both intrinsic+ extrinsic factors. Skn-1+ receive no signal, -> MS. Skn-1- cells receive MOM-2 signal, -> E.
Anterior pharynx development: EMS/MS contacts and Apx-1/ glp-1
AB is unable to produce pharyngeal tissue autonomously, req additional input.
EMS/MS contacts: Laser/needle ablation of EMS-> both anterior+ posterior pharynx lost, req for AB cell fate. In early embryo, EMS contacts ABa+ p, MS cells contact their descendants- but only ABa progeny-> anterior pharynx. MS shown to signal to bot ABa+ p, but only a responds. Reversing ABa and ABp positions doesn’t perturb dev, indicating they’re initially equiv.
Apx-1/glp-1: apx-1 ID’d as P2 signal to ABa/p in screen for xs pharynx. Gpl-1 mutants fail to dev anterior pharynx+ show germline expansion in screen for pharynx loss. Both integral mem proteins in Delta-Notch signalling pathway. APX-1= Delta-ligand. GLP-1= Notch-receptor. Signal breaks ABa/p symmetry. GLP-1 actiated by MS signalling only in ABa progeny. If no contact w/ P2, MS signal-> ant pharynx. If contact w/ P2, MS signal->no pharynx. Combinatorial inputs est cell fate- allocation/ cells to construct pharynx depends on network/ interactions involving determinant segregation+ inductive signals.
Vulva development and the anchor cell
Mutant phenotypes easy to ID (multivulva/ ‘bag of worms’ (no vulva)
Anchor cell- req for vulva dev. Ablation-> no vulva. Established from 1 of 2 initially equiv cells that -> anchor cell+ ventral uterine precursor- ablation of either cell-> other becomes anchor, suggesting 2 equiv cells interact to inhibit AC fate, small differences in signalling break equivalence (lateral inhibition). Small diff in initial AC/VUP signalling amp by feedback loops-> fate.
Lag-2 and lin-12, vulval construction
Lag-2+ lin-12- ID’d in screens for mutants w/ 2 ACs. Exp of these initially identical, gradually LAG-2 (ligand) increases in AC+ LIN-12 (receptor) increases in VUP. Lin-12 mosaic show cell receiving signal always-> VUP.
Vulva construction: from 3 cells under APC: P3.p to P8.p vulval precursor cells lie in row under AC, w/ P6.p directly under it, usually takes on primary cell fate; P5.p+P7.p cells either side of it-> secondary cell fate. Others tertiary cell fate. Ablation shows all 6 cells can take on 1o/2o/3o cell fate- equiv in dev potential.
2 models for assigning fates to cells underlying the anchor cell
2 models for assigning fates:
1) Graded morphogen from AC. Test: ablate all but one outlying VPC- fate of remaining cell depends on distance from AC, suggesting AC secretes morphogen. Screen for vulvaless mutant- LIN-3= EGF ligand. LET-23= EGF receptor. Anchor cell signals through conserved receptor Tyr kinase/MAP kinase (ras/raf) cascade. Fate of remaining cell depends on distance from AC. LIN levels determine cell fate.
2) Sequential cell interactions AC-> 1o->2o->3o. Test: mosaic analysis/let-23 mutants- when let-23 only present in P6.p, wt vulva. Ablation in VPC lin-15 mutants: lin-15 mutants have multivulval phenotype due to constitutive let-23 signalling, suggesting VPC equivalence group deciding by lateral inhibition. Screen for mutant in lateral signalling-> lateral signalling medicate by receptor lin-12- both necessary and sufficient for 2o cell fate, member of Notch fam/ receptors+ ligands. Primary specification- receive high LIN-3 signal, LET-23 receptor high, more DSL ligand, less LIN-12 receptor. Secondary specification: receive less LIN-3, LET-23 receptor low, LIN-12 receptor high, low DSL ligand.
Combined action for multiple signalling pathways: inductive signal (LIN-3) from AC-> primary fate. Lateral signal LIN-12 between induced VPCs-> secondary fate. Negative signal proposed based on other data.
