Bio Seminar 2 29.06

Presented by:Jaganmoy ChoudhuryJRF, Biological SciencesIISER-Kolkata

Collective cell migration

Cells can move either individually or in a group or sheet where they have to maintain some sort of cell adhesions. This kind of cell migration is called Collective Cell Migration.

Collective cell migration is critical in various physiological aspects, like

Embryo development Wound healingAngiogenesis

Defective migration may causes fatality too. Like,

--- Cancer metastasis

It has been less extensively studied in comparison to individually moving cells.

But…

…Lateral line primodia in Danio rerio.

…Border Cell Migration in Drosophila melanogaster.

stage 10

ovariole

ovaries

uterusspermetheca

germarium

1 3 5 7stage 8

stage 9

125 µm

Nurse cells

oocyteBorder cells

ovaries

Border cells are easily genetically tractable.

Maintaining of Drosophila is cheap.

This provide us ex-vivo study system, where cells lies almost in their native environment.

Generation time is less.

This is invasive migration, so can be a nice model of cancer metastasis.

Lots of established tools, various reagents and genetically modified flies are available in Drosophila research community.

Can be studied in fixed tissue or in live condition.

Forward genetics approach: To identify a gene or a set of gene that are responsible to produce a particular phenotype in an organism.

Reverse genetics approach: To look at the phenotypes generated by alteration of a particular gene.

In forward genetics random mutagenesis and subsequent mapping is very significant step to locate and identify the gene.

Using Biological means- Transposons

Using Physical means- UV ray, Gamma ray X ray radiation.

Using Chemical means- Ethyl methanesulfonate (EMS).

EMS induced mutagenesis on X chromosome to identify the gene(s)

that may play some role in Border Cell Migration

IUPAC Name: 1-Methylsulfonyloxyethane.

Other Names: Ethyl mesylate.

Chemical StructureThe point mutation is created via nucleotide substitution by guanine alkylation.

The ethyl group changes the guanine into unusual base O-6-ethylguanine.

During DNA replication instead of cytosine, thymine is frequently placed in opposite of O-6-ethylguanine. By subsequent rounds of replication the G:C pair becomes A:T pair.

Mechanism of Action:

THE BENEFITS AND LIMITATIONS

Benefits: - EMS mutagenesis gives a point mutation. - There is no intrinsic bias for mutation sites. - On principle it may occur throughout the genome.

Limitations: - The mapping is difficult and tedious. - EMS-induced mutation may recover by DNA repairing.

But EMS-induced mutagenesis is better option than others, as it gives more specific result.

Inducing mutation in males

Cross with balancer fly to generate stable mutant lines

Immuno-staining of egg chambers and screening for border cell migration defect

In homozygous viable cases

In homozygous non-viable cases

MARCM

Identification of prospective mutant line showing migration defect

Deficiency and recombination mapping

Identification of the gene

Confirmation of the gene

Subsequently experiments will be done to identify the role of the gene in Border Cell Migration.

10 Males of genotype FRT19A (Bl 1709)

Kept them fasting, without dehydration

EMS is mixed with 1% sucrose solution (final EMS conc. will be 25mM)

Some tissue paper is soaked with this mixture and kept in a vial (treatment).

The starved flies are transferred into the treatment vial. Kept overnight.

Next morning the treated flies are transferred into a new empty vial for 1 hour.

Mutagenized males are crossed ndividuallywith virgin balancer fly to get progeny (generating stable line).

EMS

FRT19A Y

FRT19A* Y

FRT19A*FRT19A*

FM7a FM3

FM7a Y

FRT19A* FM7a

FRT19A* FM3

XX

X

FM7a Y

FM7a Y

FRT19A* Y

FRT19A* FM7aX FM7a

FM3FM7aFM7a

X

Crossed to get stable mutant lines

Go for screening. If not available (hemizygous lethal) go for MARCM.

Progeny

FRT19A* FM7a

hsflp FRT19A Tub GAL80 Y

; Slbo GAL4 UAS mCD8GFP CyO

X

hsflp FRT19A Tub GAL80 FRT19A *

; Slbo GAL4 UAS mCD8GFP +

Heat shock for 3 day at 370C. 3 heat shock per day. Each heat shock for 1.5 hours with minimum 2.5 hours gap between two heat shocks.

Dissection and Immunohistochemistry for further screening

Mosaic Analysis with a Repressible Cell Marker or MARCM is done when homozygous flies are lethal.

Mosaics are generated to evaluate the role of a particular gene in homozygous condition.

Only homozygous mutant cells will be marked against a background of wild type and/or heterozygous cells and easier to identify the phenotype showing by the homozygous mutant cells.

Schematic diagram of MARCM technique

Phenotype No of Fly LineMale lethal 49

Male Sterile 2

Heldout Wing (Male) 1

We have generated 312 prospective mutant fly lines and currentlyscreening is going on. Interestingly we got some lines where males are sterile and some (males only) with “Held out wing” phenotype. In several lines the males (carrying prospective mutant X chromosome) are not viable.

10-12 mutant females (6 to 8 days old ) were taken.

Fattened with yeast paste at 250C for O/N

Ovaries are dissected out in FBS added Schneider’s media

Ovaries are fixed in PBS buffered 4% paraformaldehyde (PFA) solution

PFA is removed by washing with PT (PBS+ 0.3% TritonX-100)

Ovaries are resuspended into individual egg chambers in PT

Egg chambers are blocked with PBT (PBS+BSA+TritonX-100).

Primary antibody is added, shake for sometime and kept in cold for O/N.

Washing with PT 3 times, with shaking. (20 minutes each)

Secondary antibody added, shake for 1 hour at room temperature

Washing with PT 3 times, with shaking. (20 minutes each)

In last wash samples are treated with DAPI

Mounting media is added to the samples.

Samples mounted and checked under fluorescence microscope

0% 50% 100%

<50% >50%100%

0%

Complete Migration Defective Migration

Border cells Border cells

Line No Total Complete DefectiveB1-1 64 63 1B1-2 98 98 0B1-3 83 81 2B1-4 119 117 2B1-5 82 80 0B1-6 87 87 0B1-7 97 97 0B1-8 78 78 0B1-9 91 89 2

B1-10 84 82 2B1-12 93 93 0B1-13 114 114 0B2-4 124 123 1B2-5 101 98 3B3-2 121 121 0B3-5 113 112 1B7-1 24 24 0B7-3 59 57 2B7-9 67 64 3

B7-10 38 37 1B7-13 63 63 0C5-3 113 113 0C5-1 128 127 1

Line No Total Complete DefectiveB8-1 29 29 0B8-2 58 57 1B8-3 29 28 1B8-4 17 17 0B8-6 41 39 2B8-7 15 15 0B8-9 19 19 0

B8-11 29 29 0B6-1 37 31 6B6-2 98 97 1B6-9 104 102 2

B6-10 45 43 2B2-3 44 42 2B4-2 29 29 0B9-5 39 39 0B9-9 42 41 1C6-2 91 89 2C9-3 83 83 0C6-3 27 27 0C6-8 99 95 4C6-1 21 21 0C7-5 78 78 0C7-2  80  80  0 

Continued…

Line No Total Complete DefectiveC6-12 36 35 1C7-7 31 27 4C7-4 101 97 4

C10-1 35 35 0C7-10 73 73 0C8-10 102 102 0

Continued…

To identify the gene present on 984 allele (3R Chromosome) playing role in collective cell

migration

I would like to express my sincere gratitude to Dr. Mohit Prasad for his supervision and guidance throughout this project.

I would like to give special thanks to Mrinal, Amit, Abhinoy and Arunabha for their immense help and support.

I would like to thank other members of Biological Sciences Department.

THANK YOU