Growth factors in Drosophila:

Characterization of two novel families of growth factors,

IDGFs and ADGFs

IDGFs = Imaginal Disc Growth FactorsIDGFs = Imaginal Disc Growth Factors

• secreted glycoproteins

• isolated from conditioned medium, where they are abundantly secreted by various Drosophila cell lines exhibiting macrophage properties (Cl8+, S2, Kc) (Kirkpatrick et al. 1995, Kawamura et al. 1999)

Detection of DS47 secreted by Dm cell lines1 - M3 media + serum2 - supernatant from S2 culture, serum-free3 - supernatant from S2 culture, serum+4 - supernatant from KcO culture, serum-free5 - supernatant from 1006-2 culture, serum+6 - supernatant from 1006-2 M culture, serum+7 - supernatant from Ox736-3 M culture, serum+8 - supernatant from 5697-5 culture, serum+9 - supernatant from 5697-7 culture, serum+

IdgfIdgf genes in genes in Drosophila melanogasterDrosophila melanogaster

~ 55 % seq. similarity at cDNA level~ 50-60 % seq. similarity at aa level

IDGF = Imaginal Disc Growth FactorsIDGF = Imaginal Disc Growth Factors

• IDGF proteins in cooperation with insulin are able to stimulate proliferation, polarization and motility of Cl8+ cells in culture (Kawamura et al.1999 – recombinant IDGF1 and 2)

Cl8+ in CM

Human homolog HC-gp39Human homolog HC-gp39

• stimulate proliferation of fibroblasts and synovial cells in culture (Recklies et al.2002)

• in vivo is connected with differentiation of peripheral macrophages (Kirkpatick et al. 2007)

• is highly expressed in human body during inflammation (autoimmunity deseases like arthritis, final stage of cancer, viral infections)

IDGFs probably act as lectinsIDGFs probably act as lectins

• binding domain is slighly modified

• Do they bind chitin or different oligosaccharide motive?

A – IDGF2 binding domainB – Chitinase A binding domainC – Ym1 binding domain (murine homolog of insect IDGFs)Varela et al. 2002

IDGFs can be specialized to different functionIDGFs can be specialized to different function

Western blot (Bryant and Zurovec)

2 main groups: IDGFs with constitutive and induced expression

IDGFs 1-3 were identified IDGFs 1-3 were identified as a part of as a part of DrosophilaDrosophila immune response immune response

(DeGregorio et al. 2001, 2002, Irving et al. 2001, Levy et al. 2004,…)

• various MA and proteomic analysis identified IDGF 1-3 as a part of Drosophila immune response in both larvae and adults

• IDGF 1 and 3 are induced with septic injury

• IDGF3 expression is affected with Toll and Imd pathway, however IDGF1 expression is independent on both of them.

• IDGF2 expression is upregulated early after Gram-positive bacterial infection as well as after infection with yeasts.

• All IDGFs were found as a part of haemolymphal clots

17650 17660

ADG F-D ADG F-C CG 9599

9460 9465 9470

ADG F-E

ADA CG 18005

P-EP

P-EP

3L;74E2

3R;87F12

2R;51C1

3R;85C7

ADG F-A ADG F-B

96409635

4870 4872

M SI

ADA-RELATED GENES IN DROSOPHILA

The effect of recombinant ADGF-A on the growth and morphology of the Cl.8+ imaginal disc cells

Dying cells Living cells

It is possible to mimic the effect of ADGFAon cell growth by removal of adenosine

The mutant ADGF has no effect

Expression of different ADGFs ADGF-A is expressed in embryo, larva and adult (arrows)

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Gene targeting by homologous recombination

Mutagenesis of the gene ADGF-A17650 17660

3L;74E2

ADGF-A ADGF-B

MSI

17650 17660

ADGF-A ADGF-B

MSI ADGF-B

MSI ADGF-B

MSI

ADGF-A

3L;74E2

2L;

ADGF-A White

WT

The ADGFA mutant

A rescue construct

Stop kódón

Tests for somatic mutagenesis inDrosophila based on a recessive mutation in a marker gene (left), or a mutant tumor suppressor gene (right)

y = yellow, yellow bristles instead of black normal ones;wts = warts, warts-like patches of overgrown tissue

Heterozygous wild type cells at four-chromatide stage

Mutagen Mutagen

Tumor cell Normal cell

Clone with yellow bristles

Tumorous clone

Wild type homozygousclone

Surrounding heterozygous tissue, phenotypically normal