Developmental Biology

Question 1

Techniques for analysing development

Answer 1

1. Description
   - Cellular description = direct observation of living embryos, needs transparency
2. Physical manipulation
   - Removal of cells/tissues like cell isolation
3. Genetics/molecular experiment
   - Forward genetics
   - Reverse genetics
   - Temp sensitive/epistasis

Question 2

Developmental principles

Answer 2

1. Development occurs by epigenesis
2. Development starts with a single cell
3. Progressive restriction in developmental potential
4. Proliferation vs cell death
5. Oriented + asymmetric cell divisions
6. Epithelial sheets importance
7. Developmental induction
8. Developmental fields
9. Boundaries (fields result in boundaries)
10. Signalling between cells
11. Choice of fate
12. Morphogenesis and cell affinities

Question 3

Benefits of using C elegans

Answer 3

• Simple but has all major cell types
• Easy to grow
• Transparent
• Different anatomy for XX vs XO
• Genetic analysis can be used to study development
• Vulva formation is a good example of tissue remodelling

Question 4

Steps in vulval development

Answer 4

1. Generating VPCs (3 fates 1o,2o,3o
2. Vulval precuror patterning (in L3, signal from the gonad specifies 3 VPCs to generate vulval cells, typically 3-3-2-1-2-3)
3. Generation of adult cells (can be VulA-F)
4. Anchor cell invasion (forms hole in epidermis)
5. Morphogenesis (forms 7 distinct toroids)

Question 5

Generating VPCs

Answer 5

• 11pnp cells are formed during L1 Laval stage

- How gene lin-39 = major determinant of VPC group + is expressed in p3p-p8p

Question 6

VPC 1o, 2o and 3o pattern formation

Answer 6

• 2 systems cooperate :
  1. graded signal of Lin-3 acting via Let-23
  2. sequential signal of DSL ligand acting via LIN-12

Question 7

Anchor cell

Answer 7

• Ablation prior to L3 stage completely blocks vulval development
• AC produce Lin-3, whose action is graded
• Vulvaless/multivulvaless mutations
• Lin3-let23-sem5-let60-lin45-lin-1
• Signalling terminates with modulation of TF like lin-1 that controls morphogenesis
• Lin-31/lin-1 ETS complex are phosph. by MAPK → no inhibition of vulva
• P6p receives most Lin-3, , p3p + p8p have negligible

Question 8

Lateral inhibition

Answer 8

• Stops adjacent cells also adopting primary fate
• ## Genes involved in lin-12 activation are unregulated by Ras in p6p, lead to activation of lin-12/notch in p5p/p7p, helping p5p/p7p adopt 2o fate

Question 9

Negative regulation of induction

Answer 9

• 3 classes of SynMuv gene
• Need mutation in 1 class A + 1 class B
• Prevents wrong LIN-3 expression

Question 10

Formation of axes

Answer 10

• asymmetric divisions establish 3 major axes

- 5 asymmetric divisions produce 6 founder cells: AB, MS, E, C, D, P4

Question 11

Establishing A/P axis

Answer 11

1. Breaking symmetry (sperm assembles PCM, CYK-4, 1st cleavage → Ab1 + P1)
2. PAR proteins (PAR 3+6 form complex w/ PKC-3, needs cdc-42 to maintain polarity, PAR1+2 localise in posterior cortex)
3. P granules (form germline, start in cytoplasm, move to posterior of cell)

Question 12

Spindle positioning

Answer 12

• Heterotrimeric G protein acts ds of PAR to transduce polarity
• GOA-1 + GPA-16
• GRP1/2
  Dynein

Question 13

Formation of D/V axis

Answer 13

• 1st division → big AB, smaller P
• AB = symmetrical division, controlled by PAR
• P1 = asymmetric division, spindle oriented on AP axis
• 2nd division → ABa, Abp, P2 + EMS
• D + V axis different (only AB daughter cell has GLP-1 receptor, P2 has APX-1)

Question 14

Formation of L/R axis

Answer 14

• Determined by division of ABa/ABp
• LH daughter are ↑ anterior to RH, causes cell to contract
• EMS divides 1st to make E + MS
• Then P2 divides to make C + P3 which divide again to make D + P4

Question 15

Breaking symmetry

Answer 15

• All cells have same developmental potential

- However end point = differentiation

Question 16

1st step of symmetry breaking

Answer 16

1. Maternal specification

2. De novo program

Question 17

Drosphilia Origins of polarity

Answer 17

• A-P polarity of embryo
• Maternal effect genes
• Cooperation btw nurse and follicle cells

Question 18

Movement of oocyte nucleus

Answer 18

• Nucleus moves from central posterior → asymmetrical anterior
• Involves GRK, EGFR
• Gurken mRNA

Question 19

Mutagenesis study

Answer 19

• Heidelberg screen
• 1. Maternal
• 2. Zygotic

Question 20

Following fertilisation

Answer 20

• Bicoid + hunchback gradients along AP axis
• 3’UTR of nos can be replaced w/ 3’UTR of bcd mRNA
• Nos inhibits translation of hb + bcd mRNA
• BCD gradient

Question 21

Embryo segmentation

Answer 21

• Mitosis
• Syncytium
• 13th division, 600 nuclei

Question 22

Gap genes

Answer 22

• Mutant screens (3 points)
• Regulated by maternal TF
• Hunchback, Kruppel (7 total)
• Gap proteins = TF, diffuse into syncytial cytoplasm
• Regulate expression of pair-rule genes

Question 23

Pair rule genes

Answer 23

• Transcribed in 7 broad strips
• Some genes (even-skipped) are controlled directly by GAP proteins
• Each stripe = gene activation
• Regulatory elements = fused to LacZ reporter
• Each stripe of pair rule protein defines a stripe of segment polarity expression
• Each segment polarity gene is induced by 2 pair rule proteins → 14 segment polarity stripes
• Nucleus → Cytoplasm
• Makes a gradient

Question 24

Segment polarity genes

Answer 24

• Overlaps of pair rule gene pattern causes activation of segment polarity
• TF like engrailed
• Mediate cell interactions

Question 25

Body patterning

Answer 25

• Segments need to take on individual identities
• ANT-C (head + anterior thorax)
• BX-C (posterior thorax)
• Homeodomain protein, homeobox
• Hox genes

Question 26

Let-23 signalling

Answer 26

• Originally ‘graded’ signal model (P6p most, decreases for others)
• But, Let23 only needed in p6p
• Sequential signal model