The Imaginal disc growth factor 3 interacts with a Planar Cell Polarity component during dorsal-appendage tube formation in Drosophila
posterposted on 20.04.2020 by Claudia Espinoza, Celeste Berg
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I am interested in studying the genetic basis of biological tube formation. For this, I am using the Drosophila egg chamber and laid egg as a model system. Specifically, I am using two appendages located on the dorsal side of the egg called dorsal appendages. The dorsal appendages are not tubes themselves, but tubes mold their shape. Therefore, looking at their shape, I can assess whether tube formation happened successfully or if there were problems during the process.
Using this model system, in my lab, we have been able to identify different genes involved in tube formation. We recently identified a gene family, called the Imaginal disc growth factors to be involved in dorsal appendage formation. Specifically we found that one member of this family, the Idgf3, plays a role in dorsal appendage formation. The overexpression of the Idgf3 causes dorsal appendage defects (Zimmerman, et al. 2017). We do not know much about this gene family, especially, it is unknown in what pathway this gene plays a role in.
Wanting to identified the pathway that this gene play a role in, we run a modifier screen, taking advantage of the fact that overexpressing Idgf3 causes dorsal appendage defects 50% of the time (Zimmerman, et al. 2017). We used this Idgf3 overexpression phenotype to look for regions of the genome that, when removed by half, enhance or suppress the phenotype.
We used the deficiency kit available at the Bloomington Stock Center that cover most of chromosome 3L and cross the deficiency lines with an Idgf3 overexpression stock. We identified one region that enhanced the Idgf3 overexpression phenotype. Using smaller overlapping deficiencies, we narrow down that region into a few candidate genes. By using RNAi alleles and mutant alleles of the candidate genes and by crossing them with the Idgf3 overexpression stock, we identified the combover (cmb) gene to be responsible for the genetic interaction.
Since cmb interacts with the Planar Cell Polarity (PCP) pathway, the PCP pathway is involved in cell intercaltion, and cell intercalation takes place in dorsal appendage formation, we hypothesized that the Idgf3 - cmb interaction had an effect in cell intercalation in our system. Wanting to test this hypothesis, we stained the cells that made the dorsal appendages, also known as dorsal appendage patches, by using an antibody against a nuclear dorsal-appendage-patch-marker called Broad. We measured cell intercalation in our system by measuring the aspect ratio of the dorsal appendage patch before cell intercalation and after cell intercalation in four different genotypes: A control group, a +/cmbKO group, an Idgf3 overexpression group, and a +/cmbKO with Idgf3 overexpression in the background group. We found that cell intercalation is not affected in our system.
How is the Idgf3 and cmb interaction affecting dorsal appendage formation? Looking at E-cadherin staining of the egg chamber used to delineate the cells of the egg, we found differences of the apical area of the tubes among the genotypes tested. We quantified that area and normalized it for the migration of the tubes. We found that overexpressing Idgf3 results in a larger area compared to the control and +/cmbKO. This effect was enhanced when one copy of cmb was removed in addition to overexpressing Idgf3.