Invertebrate Development

Question 1

How is germline specified in C elegans

Answer 1

pie-1 in P-granules segregates with P-lineage.

Question 2

Isolated P1, EMS, MS cells autonomously develop pharynx, but not AB cells

Answer 2

Mex1 Par1 mediates asym seg of skn1 into P1 cell

Skn1 mutants –> loss of pharynx

mex1 par1 mutants –> AB cell accumulates Skn1, forms excess pharynx

Question 3

P-lineage does not form pharynx, even though Skn1 is present.

Answer 3

Pie1 suppresses Skn1.

pie1 mutants –> P2 cells become EMS cells, forming excess pharynx

Question 4

EMS cell divides into MS and E cell

Answer 4

pop-1 represses E-cell fate in MS-cells, and is localised in the nucleus only MS-cells but not MS-cells

(Transcription level shown to be the same using a reporter)

pop1 mutants result in formation of 2 E-cells
MS-cell takes on E-cell fate even when all other cells are ablated, shows that it is cell-autonomous

Question 5

How is EMS division polarised

Answer 5

mom2 on P2 cell binds mom5 on EMS cell, which represses pop1 in destined E-cell

Dissociation of 4-cell embryo >10min before division = both MS cells.
<10min before division = E+MS cell

MUST reassociate EMS with P2 >10min before division for normal development of E+MS.

Mosaic - WT P2 cell replaces P2 cell of mom2- 4-cell embryo can result in normal development

mom2 represses pop1: mom2 mutant has POP1 nuclear localisation in both cells

Double mutants take on E-cell fate

Question 6

How AB cell can develop pharynx?

Answer 6

Isolated AB cell does not form pharynx autonomously - requires signal from EMS cell

Ablation of EMS cell results in no pharynx formation at all.

Reversing position of ABa and ABp - no effect; suggests developmentally equivalent

Question 7

How ABa and ABp are made different

Answer 7

P2 cell contacts ABp - supplies it with APX1 ligand, which binds and inactivates GLP-1 (Delta-Notch)

ABp progeny is made insensitive to the MS-derived signal that induces anterior pharynx formation - so only ABa progeny makes anterior pharynx

APX-1- have excess anterior pharynx
GLP1- do not form anterior pharynx

Question 8

How anchor cell is chosen?

Answer 8

Anchor cell - VUP lateral inhibition
lag2 is the Delta-ligand, lin12 is the Notch receptor

Cell lineage analysis –> 50% of either possibility

Mosaic organisms with one lin12- mutant and one WT cell –> lin12 mutant always becomes anchor cell

lag2 lin12 double mutants –> Two anchor cells

Question 9

How is vulva formed using morphogens?

Answer 9

AC secretes LIN3, binds to Let23 on VPCs - morphogen gradient

Moving 3o VPC to a central position makes it take on 1o cell fate

All VPCs are ablated except 1 - still takes on correct fate

Lin3 under a heat shock promoter - all cells ablated except P7.p, fate corresponds to level of heat shock

Question 10

Lateral inhibition / induction of VPCs

Answer 10

P6.p VPC sends a signal to adjacent VPCs to take on 2o fate via lin-12 receptor

In mosaics with all VPCs being Let23- mutants except the central VPC, vulva develops normally.

lin15 mutants (constitutive let23 signalling) - alternating 1o and 2o fates

lin12 GOF mutation - 2o cells

lin12 LOF mutation - no 2o cell

Question 11

Formation of neuroblast

Answer 11

Proneural cluster - a single cell delaminates and becomes neuroblast, which contributes to the ventral nerve cord while others become epidermal cells

AS-C induces Delta expression –> Notch receptor –> Represses AS-C activity

Question 12

Division of neuroblast

Answer 12

Asymmetric division - Prospero, NUMB, BRAT are segregated into GMC by Par complex

Limits GMC division to just one more cell division

Question 13

Neuroblast clock

Answer 13

Fate of every GMC is different despite coming from the same parent neuroblast

Neuroblast clock - temporal regulation of transcription factors expressed in the NB, changing every cell cycle - hb then Kruppel

Hb mutants affect the first GMC, Kr mutants affect the second.

Hb or Kr overexpression mutants affect all the following GMCs from the neuroblasts

Question 14

A-P axis specification in the oocyte

Answer 14

gurken mRNA transported from nurse cells to oocyte, localised near the nucleus, translated

Signals to posterior follicle cells through torpedo, which signals to the oocyte to form MTs with ‘+’ ends in the posterior pole

Question 15

Localisation of mRNA and proteins in the oocyte

Answer 15

Bicoid mRNA and protein in the anterior

Oskar mRNA and protein the anterior

Nanos mRNA (bound by oskar protein, promotes translation) and protein in the anterior

Caudal protein in the posterior (translation repressed in anterior)

hb protein in the anterior (translation repressed in the posterior)

Torso activated in the termini

Question 16

Gap genes

Answer 16

Giant / hb / Kruppel / Knirps / Giant / hb

Tailless in the termini

Gap genes undergo mutual inhibition to sharpen expression domains

Giant induced by high bicoid or by high caudal - so expressed in two broad domains

Question 17

Pair-rule genes

Answer 17

In syncytial blastoderm

Eve stripe 2 - repressed by Giant and Kruppel, induced by bicoid and hb

Ftz and eve - out-of-phase expression

Primary pair-rule genes mediate expression of secondary pair-rule genes via zebra enhancers (not stripe-specific)

Question 18

Segment polarity genes

Answer 18

Even-number engrailed aligh with front of ftz

Odd-numbered engrailed with front of eve

Maintenance of expression uses feedback loops

Engrailed induces expresion and secretion of Hh on neighbouring cells, activates Wingless expression and secretion, which activates Engrailed expression

Question 19

Hox gene expression

Answer 19

2 regions of chromosome 3 - co-linear with A-P axis

Posterior ‘dominates’ over the anterior Hox genes - LOF of Hox genes can lead to expansion of segment identity of the more anterior segment

Polycomb –> Maintains H3K27me3

Trithorax -> maintains H3K4me3

Question 20

D-V patterning

Answer 20

Loss of deticles –> dorsalisation

Gurken polarises dorsal follicle cells - which inhibits Pipe synthesis of dorsal follicle cells

Pipe signal from follicle cells induces expression of Dorsal which activates twist and snail expression while repression Dpp and Tld

Dpp and Tld localised to the dorsal side. Antagonism by Sog ensures Dpp remains in the Dorsal pole