UNDERSTANDING COLLECTIVE CELL MIGRATION USING BORDER CELLS AS MODEL SYSTEM

Presented by: Jaganmoy Choudhury JRF, Biological Sciences IISER-Kolkata

WHAT IS 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

IMPORTANCE OF COLLECTIVE CELL MIGRATION

Collective cell migration is critical in various physiological aspects, like

Embryo development

Angiogenesis

Wound healing

Defective migration may causes fatality too. Like,

--- Cancer metastasis

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

DIFFERENT MODELS FOR STUDY

Border

Cell Migration in Drosophila melanogaster.

Lateral line primodia in Danio rerio.

WHAT ARE BORDER CELLS?

ovaries

ovaries

ovariole

spermetheca

uterus

125 m

germarium

Nurse cells 5 7
Border cells

1 3

stage 8 oocyte stage 9 stage 10

WHY BORDER CELLS?

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.

HOW TO STUDY:FORWARD AND REVERSE GENETICS

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.

DIFFERENT METHOD FOR MUTAGENESIS

Usi g Bi l gic l Usi g P ysic l UV r y, r y X r y r i ti Usi g ic l

s- Tr s-

. s- Ethyl thanes lf nate (EMS).

EMS induced mutagenesis on X chromosome to identify the gene(s) that may play some role in Border Cell Migration

ETHYL METHANESULFONATE (EMS)

IUPAC Name: 1-Methylsulfonyloxyethane. Other Names: Ethyl mesylate.
The point mutation is created via nucleotide substitution by guanine alkylation.
Chemical Structure

Mechanis

f Acti n:

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.

WHY EMS? 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.

BASI W

AN
Inducing mutation in males

ross with balancer fly to generate stable mutant lines

In homozygous viable cases Immuno-staining of egg chambers and screening for border cell migration defect

In homozygous non-viable cases MARCM

Identification of prospective mutant line showing migration defect

BASIC WORK PLAN continued

eficienc and reco ination a in

Identification of t e ene

Confir ation of t e ene

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

METHOD FOR INDUCING MUTATION

10 Male of genotype FRT19A (Bl 1709) Kept them fa ting, without dehydration EMS i mixed with 1% ucro e olution (final EMS conc. will be 25mM) Some ti ue paper i oaked with thi mixture and kept in a ial (treatment).

The tar ed flie are tran ferred into the treatment ial. Kept o ernight. Next morning the treated flie are tran ferred into a new empty ial for 1 hour.

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

GENERATING STABLE MUTANT LINES

EMS FRT19A Y
FM7a X Y

FRT19A* X Y
FRT19A* FM7a

FM7a FM3 FRT19A* X FM3 FM7a Y

FRT19A* X Y

FRT19A* FM7a

FM7a Y Progeny

FM7a FM3

FM7a FM7a

r ssed t

et sta le FRT19A* FRT19A*

tant lines

G f r screening. If not availa le (hemizygous lethal) go for MAR M.

MARCM CROSS AND SCREEN

FRT19A* FM7a

h flp FRT19A Tub GAL80 ; Slbo GAL4 UAS mCD8GFP CyO Y

h flp FRT19A Tub GAL80 ; Slbo GAL4 UAS mCD8GFP + FRT19A *

Heat hoc for 3 day at 370C. 3 heat hoc per day. Each heat hoc for 1.5 hour with minimum 2.5 hour gap between two heat hoc .

Di

ection and Immunohi tochemi try for further creening

MARCM
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.

GAL4/UAS SYSTEM

FOR TARGETED GENE EXPRESSION

FLP/FRT SYSTEM

Schematic diagram of MARCM technique

GENERATED PROSPECTIVE MUTANT LINES

We have generated 312 prospective mutant fly lines and currently screening 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.

Phenotype
Male lethal Male Sterile Heldout Wing (Male)

No of Fly Line
49 2 1

IMMUNOSTAINING & SCREENING FOR MIGRATION DEFECT

10-12 mutant female (6 to 8 day old ) were ta en.

Fattened with yea t pa te at 250C for O/N

Ovarie are di

ected out in FBS added Schneider media

Ovarie are fixed in PBS buffered 4% paraformaldehyde (PFA) olution PFA i removed by wa hing with PT (PBS+ 0.3% TritonX-100) Ovarie are re u pended into individual egg chamber in PT

continued
Egg chamber are bloc ed with PBT (PBS+BSA+TritonX-100). Primary antibody i added, ha e for ometime and ept in cold for O/N. Wa hing with PT 3 time , with ha ing. (20 minute each) Secondary antibody added, ha e for 1 hour at room temperature Wa hing with PT 3 time , with ha ing. (20 minute each) In la t wa h ample are treated with DAPI Mounting media i added to the ample .

Samples mounted and checked under fluorescence microscope

MEASURING MIGRATI N EFFICIENCY

0% 50% 100%

0%
<50% >50%

100%

Complete Migration

Defective Migration

Border cells

Border cells

RESULT OF SCREENING
Line No B1-1 B1-2 B1-3 B1-4 B1-5 B1-6 B1-7 B1-8 B1-9 B1-10 B1-12 B1-13 B2-4 B2-5 B3-2 B3-5 B7-1 B7-3 B7-9 B7-10 B7-13 C5-3 C5-1 Total 64 98 83 119 82 87 97 78 91 84 93 114 124 101 121 113 24 59 67 38 63 113 128 Complete 63 98 81 117 80 87 97 78 89 82 93 114 123 98 121 112 24 57 64 37 63 113 127 Defective 1 0 2 2 0 0 0 0 2 2 0 0 1 3 0 1 0 2 3 1 0 0 1

Continued
Line No B8-1 B8-2 B8B8-4 B8-6 B8-7 B8-9 B8-11 B6-1 B6-2 B6-9 B6-10 B2B4-2 B9-5 B9-9 C6-2 C9C6C6-8 C6-1 C7-5 C7-2 Total 29 58 29 17 41 15 19 29 37 98 104 45 44 29 39 42 91 83 27 99 21 78 80 Complete 29 57 28 17 39 15 19 29 31 97 102 43 42 29 39 41 89 83 27 95 21 78 80 Defective 0 1 1 0 2 0 0 0 6 1 2 2 2 0 0 1 2 0 0 4 0 0 0

Continued
Line No
C6-12 C7-7 C7-4 C10-1 C7-10 C8-10

Total
36 31 101 35 73 102

Complete
35 27 97 35 73 102

Defective
1 4 4 0 0 0

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

ACKNOWLEDGEMENT
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