A Genome Wide Search for Proteins that Control the Balance Between Proliferation and Differentiation in the Drosophila Ovary, an in vivo model System

By Avanti Jakatdar
2008, Vol. 5 No. 1 | pg. 2/2 |


Unfixed, dissected ovaries were screened directly for naturally fluorescent GFP expression. The ovaries were from females containing the P{GFP} insertion. Possible expression patterns that could have been detected are shown below (Fig. 3, provided by Johnie Chau).

Figure 3. Possible GFP expression patterns. A) snf148 ovaries were screened for GFP expression throughout the germline. B-D) Expression patterns that could have been detected in wild type ovaries are B) expression in undifferentiated, early germ cells, C) expression in differentiated cells and D) no expression in the germline.

Figure 3

To generate lines containing a single GFP insertion, different genotypes of flies were selected and mated based on eye color and shape until females with the insertion were obtained. First, males containing the P{GFP} transposon and transposase were mated to females homozygous for snf148. Progeny in which the transposon had jumped to an autosome were then selected and mated to homozygous snf148 females. Finally, females with the insertion were selected and screened for GFP expression. The screen is shown to the right in a figure created by the author. Once a line expressing GFP was identified, the line was expanded to obtain a sufficient number of flies for confocal microscopy and inverse PCR.

Confocal Microscopy

10 wild type flies and 10 mutants were selected from each GFP positive line in order to compare expression in wild type and tumorous ovaries. A three day antibody staining procedure with rabbit anti-GFP was applied to increase GFP expression for easier identification of expression patterns in the ovary. Slides were prepared by teasing apart the ovarioles and fixing them in Vectashield Mounting Medium. High resolution microscopy was then used to capture images of GFP expression in the ovarioles.

Inverse PCR

To locate the protein-trap insertion, the DNA flanking the insertion was recovered and sequenced using inverse PCR. The process of inverse PCR is shown in figure 5 (next page), provided by Johnie Chau. DNA was extracted from 30 mutants per line by treating them with buffer, a LiCl/KAc solution and TE. Then the DNA was digested with restriction enzymes HinP1 and Msp1. The transposon was ligated to the endogenous DNA using ligation buffer and T4 Ligase. Finally, inverse PCR was performed with Pwht1 and Plac1 as 5' primers and Pry4 and Pry1 as 3' primers.

Figure 4. The GFP Screen

Figure 4

Gel Electrophoresis

This procedure was used to determine if the PCR products would be sufficient for obtaining good sequencing results. A 1.2% agarose gel was used to run the samples and the 100 bp marker.


By analyzing the sequencing results, the endogenous gene was identified by its presence between the P{GFP} transposon and the restriction site. BLAST alignments were performed on the endogenous sequence and the Drosophila genome to identify the location of the insertion within the genome.

Figure 5. Inverse PCR. 1. Genomic DNA from a P{GFP} line is digested with restriction enzyme MspI or HinPI. 2. Fragments are made to self-ligate using T4 ligase. 3. 1-4 listed by the small arrows are the primers; red and purple bars represent ends of the transposon; black line represents endogenous DNA sequence.

Figure 5


Confocal Microscopy

Shown to the right are high resolution pictures of lines that were used (Figure 6).

Figure 6. Confocal Imaging a) Wild type ovary from line AJ87. GFP expression is seen in the stem cells in the germarium, and in the follicle cells, b) Wt ovary from line AJ39. GFP expression is seen in ring canals, the region where interconnected germ cells connect, c) Wt ovary from LSK991. GFP expression is seen in the stem cells and follicle cells, d) Wt ovary from LSK1093. GFP expression is seen in the ring canals, e) Tumorous ovary from AJ87. Strong GFP expression is seen throughout the ovary. In (a), (b) and (e), DNA is colored red and GFP is green. In (c) and (d), DNA is colored blue and GFP is green, and 1B1 antibody causes the red color.

Figure 6

Inverse PCR and Gel electrophoresis

Shown to the right are results from the inverse PCR of lines LSK 1261 and 1335 (Figure 7). The products are different in size because they were digested with different restriction enzymes, either Msp1 or HinP1.

Figure 7. Inverse PCR. On the left are results obtained from 5' PCR primers Wht1 and Plac1. On the right are results from 3' PCR primers Pry4 and Pry1. The bright bands at the bottom of the gel are most likely primer dimers.

Figure 7


Sequencing results from PCR products showed the location of the transposon and the restriction site, whose sequences are known. In between the two was the sequence of the endogenous gene. Aligning the endogenous gene with the Drosophila genome showed us the approximate location of the gene within the genome. The 5' PCR product of LSK1093 digested with Msp1 aligned with a region outside the 5' region of the ̳Cheerio' gene. The 5' product of LSK991 digested with HinP1 aligned with part of the sequence of the ̳Apontic' gene. The approximate locations of the P element insertions are shown above (Figure 8).


Figure 8. Location of the endogenous gene. A) from line LSK1093, digested with Msp1 B) from line LSK991, digested with HinP1.

Figure 8

In the mutant AJ87, GFP expression was seen throughout the ovary (Figure 6), in the stem cell-like cells arrested in an intermediate stage of development. This may show that the ̳trapped' protein is involved in arresting differentiation and restarting proliferation. GFP expression was seen in proliferating germ cells in the AJ87 and LSK991 wild types, which may show that the trapped protein has a role in early germ cell proliferation. Although the intention of the screening was to identify expression in proliferating cells, expression was unexpectedly found in ring canals in AJ39 and LSK 1093. Ring canals are cytoplasmic bridges that allow the flow of nutrients from nurse cells to oocytes. They are necessary for proper oocyte and egg chamber formation (Robinson D.N, Cant, K and Cooley, L., 1994). Therefore, expression in the ring canals may show that the trapped protein is involved in differentiation and proper cell development.

In LSK991, the GFP insertion occurred in Apontic, which is thought to be involved in transcriptional activity, mRNA binding and DNA binding. This gene may not be directly involved in stem cell proliferation. Cheerio is known to be involved in ring canal formation, but ring canals may only be required for proper differentiation and not for making the transition between proliferation and differentiation. Therefore, Cheerio may not have a direct role in controlling the balance between proliferation and differentiation.

Sequencing of lines AJ87 and AJ 39 is currently in progress, and it will be interesting to see if the insertions occurred in Cheerio and Apontic, as the expression patterns in AJ87 and AJ39 are consistent with LSK991 and LSK1093 respectively. The goal was to find genes that are highly expressed in tumor cells and may play a role in the stem cell's decision to proliferate or differentiate. In all four lines, GFP was highly expressed in tumor cells, and looking at expression patterns in the wild type offered a clue as to what type of role the trapped protein may have. Further analysis to determine the identity of the trapped proteins will lay the groundwork for understanding their role in the pathway that controls the choice between proliferation and differentiation.


I would like to thank L.S. Kulnane for her help in conducting the screen and collecting data, J. Chau and M.L. Johnson for figures and helpful discussions, and H.K Salz for introducing me to this project and mentoring.


Buszcak M, Paterno S, Lighthouse D, Bachman J, Planck J, et al. (2007). The Carnegie protein trap library; a versatile tool for Drosophila developmental studies. Genetics 175: 1505-1531.

Morin, X., R. Daneman, M. Zavortink, and W. Chia. 2001. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. PNAS 98, 15050-15055.

Robinson, D.N., Cant, K. and Cooley, L. (1994). Morphogenesis of Drosophila ovarian ring canals. Development 120, 2015-2025.

Suggested Reading from Inquiries Journal

Parkinson’s disease (PD), a progressive neurodegerative disorder most prevalent in the elderly and for which there is currently no cure, selectively targets nigrostriatal Dopaminergic (DAergic) projection neurons in the substantia nigra pars compacta (SNpc), which generates a consequential loss of Dopamine (DA) in the striatum (ST) (1). Because of the involvement of DA in voluntary movement, the resulting alteration in basal ganglia circuitry... MORE»
The discovery of adult neurogenesis (the endogenous production of new neurons) in the mammalian brain more than 40 years ago (Malcolm R. Alison, 2002) has resulted in a wealth of knowledge of this branch of neuroscience. Today we know that the continuous production of new neurons is facilitated by adult neural stem or progenitor cells (NSC/NPCs) (Cattaneo & McKay, 1990; Gage, 2000; Temple... MORE»
The Ras/Raf pathway is a crucial cell signaling pathway utilized by eukaryotic cells for growth and proliferation, and it is highly conserved amongst all eukaryotic organisms. Mutations in this pathway lead to uncontrolled growth and proliferation of cancerous cells, effectively giving them a major advantage over normally functioning... MORE»
Gene therapy has ceased to be a futuristic dream and instead become a treatment for the present. Somatic cells can be treated with gene replacements or gene fragments to express required proteins and whilst gene therapy was originally aimed at inherited disease, it is now capable of treating a number of acquired problems such as... MORE»
Submit to Inquiries Journal, Get a Decision in 10-Days

Inquiries Journal provides undergraduate and graduate students around the world a platform for the wide dissemination of academic work over a range of core disciplines.

Representing the work of students from hundreds of institutions around the globe, Inquiries Journal's large database of academic articles is completely free. Learn more | Blog | Submit

Follow IJ

Latest in Biology

2021, Vol. 13 No. 10
Sociobiology is a sub-discipline of biology that aims to examine and explain social behavior in terms of evolution. It is interdisciplinary in nature, drawing from disciplines including psychology, ethology, anthropology, evolution, zoology, archaeology... Read Article »
2020, Vol. 12 No. 12
Transposable elements (TEs), also referred to as ‘jumping genes’, are sequences of DNA located in the eukaryotic genome that have the ability to mobilize. This functional mobilization allows TEs to insert at random positions throughout... Read Article »
2016, Vol. 8 No. 08
The Ras/Raf pathway is a crucial cell signaling pathway utilized by eukaryotic cells for growth and proliferation, and it is highly conserved amongst all eukaryotic organisms. Mutations in this pathway lead to uncontrolled growth and proliferation... Read Article »
2015, Vol. 12 No. 1
Published by Discussions
Electronic cigarettes (E-cigarettes) are devices that effectively deliver vaporized liquid nicotine to the lungs and are commercially available as a nicotine replacement therapy that is safer than conventional tobacco smoking. However, there is... Read Article »
2015, Vol. 11 No. 2
Published by Discussions
To maintain sodium (Na+) homeostasis in a hypotonic environment, freshwater teleosts must constantly absorb Na+ through their gills. Teleosts in temperate climates have the extra challenge of living in an environment in which ambient temperatures... Read Article »
2015, Vol. 11 No. 2
Published by Discussions
The increasing level of obesity in the general population of industrialized nations is a major public health concern. While obesity increases morbidity and mortality, increasing body habitus also impacts the utilization and analysis of medical imaging... Read Article »
2015, Vol. 11 No. 2
Published by Discussions
With the advent of antiretroviral therapy, suppressing the HIV-1 virus and stopping the progression of the disease are now possible. Even with long-term antiretroviral therapy, HIV reservoirs remain in individuals. These individuals experience an... Read Article »

What are you looking for?


"Should I Go to Graduate School?"
5 Tips for Publishing Your First Academic Article
The Career Value of the Humanities & Liberal Arts