Scholar’s Week Spring 2016

Last Spring the labrats put together a couple of posters and presented them at our local Scholar’s Week showcase. Both posters related to the DMAP1 project and give me a chance to see where we are at. I’m going to put up the poster slides here, mishing them together as they overlapped on some background stuff. True to form, I let the students play with powerpoint, illustrator and photoshop, to try and assemble a poster, but told them in advance I would be control freaky about requiring a very specific style, which I prefer (since the posters go up outside my lab and they MUST be clear).  I am always amazed by how my social media and computer savvy students don’t actually know how to put a poster together in powerpoint. It is always a brand new experience (and given the amount of time I spend using powerpoint, photoshop and the like, a useful one.)

When students come up with good design ideas (and some of them are really amazing) I do my best to make it so, together with them. So while the appearance of the poster and all the illustrations are mine (the fly pics typically have 50-100 component parts!) many of the layout and design ideas come from the students.

So, five students, two posters. Group 1: Genetic analysis of recovered lesions. Group 2: single embryo PCR analysis. What they had in common of course was the gene (DMAP1) and the model (Drosophila) and the male recombination scheme performed by students in Dr. Kathleen Fitzpatrick’s class at Simon Fraser University.  But I asked one group of students to focus on the genetic complementation tests that were used to define the limits of the deficiencies we obtained, and the other group to concentrate on the single embryo PCR (AKA getting DNA out of pieces of lint). Here are the abstracts for each poster. Poster 1: the genetics poster, has a blue dominant theme, while poster 2, the single embryo PCR poster, is green dominant (because, well, GFP, as you will see).

abstractsThe links to stem cell identity  and the immune system  are discussed here. Common to both posters was a description of the genomic landscape in which DMAP1 resides, and an outline of the male recombination scheme used to generate the deletions removing DMAP1 (and downstream flanking genes), more information about which can be found here.

So, a summary of the male recombination scheme carried out at SFU under Dr. Fitzpatrick’s supervision: this was included as part of the Single Embryo PCR poster (hence the green dominant theme).

male-rc-composite-slide

And a description of the genetic landscape where DMAP1 resides on the right arm of chromosome 2 (this slide appeared in both posters so there the blue version):

slide-4-poster-1-spring-2016

For the genetics poster, poster 1, we began by presenting evidence that DMAP1 might be essential, using transgenic RNAi.  A more detailed discussion about whether being essential matters or not can be found here.

transgenic-rnai-slidesHaving established that DMAP1 is likely essential using RNAi, we wanted to figure out how many genes were deleted by our male recombination-induced lesions. So we ordered a few overlapping deficiencies from the Drosophila stock center in Bloomington Indiana and performed complementation tests. Very cool, since some of the kids were in my genetics class at the time and so this was a practical application (“see? It does happen in the real world!”) The red lines in slide 5 below indicate two of those overlapping deficiencies (note that the red line indicates the DNA MISSING in the deficiency ) – one (BSC 402) takes out DMAP1 and MANY additional upstream and downstream genes, while the other (BSC 404) appears to break just upstream of a gene called “exu” (exuperantia). Slide 6 shows the results of the complementation test: all pairwise tests between K8-15 and 10-14 (the two male recombination deletions we made) and BSC 402 and 404 (the deficiencies of known limits). The bottom line is that deficiencies that remove DMAP1 and flanking genes fail to complement our male recombination mutants (10-14 and K8-15) whereas a deficiency that begins downstream of DMAP1 somewhere in the vicinity of exu complement our male recombination mutants. These data tell us that both lesions we made using male recombination are pretty large, and theoretically should end in the vicinity of exuperantia.

complementation-slidesBest laid plans, and all that. Sometimes, the molecular breakpoints of these deficiency fly stocks that one orders from a center like Bloomington have been mapped, sometimes not. Theoretically, Df(2R)404 has been molecularly mapped, and does in fact delete exuperantia. So based on the complementation data, our male recombination mutants should include exuperantia, right? So we’ve mapped the breakpoint, right?

Wrong. Lethality is one phenotype that results from a failure to complement. Lethality means there is an overlap between the known deficiency and our male recombination mutants. By sheer chance (and curiosity) one of Kathleen’s students tested the progeny from a cross between Df(2R)BSC404 and 10-14 or K8-15 for fertility. SURPRISE: progeny are both male AND female STERILE. So in fact, 10-14 and K8-15 do NOT complement Df(2R)BSC404. Take a look at the region deleted by this deficiency (the red bar highlighted in yellow on the map below):

df2rbsc404-map

Exuperantia is the tiny little blue triangle near the top of this figure on the left hand side. Our male recombination mutants in fact fail to complement Df(2R)BSC404, because progeny heterozygous for this deficiency and either K8-15 or 10-14 are sterile. Sterility, from an evolutionary point of view, is just as bad as lethality, and therefore is just as much a failure to complement. So what is exu? What does it do? Is sterility a predictable phenotype?

Exuperantia has an illustrious pedigree. It was isolated as a maternal effect gene necessary for the normal anterior-posterior patterning in the Drosophila embryo. Exu was one of an elite suite of genes isolated and studied in the mid 1980’s by Trudi Schüpbach and Eric Wieschaus in a screen to study pattern formation in development. This work revealed a highly conserved genetic determination of body patterning and opened up the fascinating field of developmental genetics. Oh yes, and resulted in a Nobel Prize in 1995.  Bottom line: if the mothers are homozygous for loss of function mutations in exu, the embryos fail to develop, and they show anterior patterning defects. How does the exu gene product do this? Presumably by controlling some of the other mRNA’s that mum packs into the cytoplasm of her unfertilized egg, providing the egg with patterning coordinates (head end, tail end, dorsal side ventral side etc.)

exu-fcn-flybase

A number of mutations in exu have been isolated since then. Most are female sterile, one is male sterile. A couple are lethal (but we have determined that at least one of those lethals is due to a second side hit on the same chromosome). So, yes, theoretically, male and female sterility is a reasonable phenotype for our male recombination lesions, and tells us that they both extend at least out to exuperantia.

Buggah.

Can we confirm these data using PCR? Certainly we know our lesions take out DMAP1 and do not disrupt the region upstream (see here), but how far downstream do these lesions extend? More primer design for the students,  targeting genes in the vicinity of exu.

slide-7-poster-1-spring-2016And our results? Using DNA from adults means using balanced flies, with the wild type genetic region on the balancer. (Remember what a balancer is? No? Explanation to follow). Thus, the data below show the results using a primer to the P element which we know is still present upstream of DMAP1 (at least half of it is) and gene-specific primers. P-out is the P element primer, in combination with the upstream gene CG33785 we have a product, but nadda in combination with anything down stream, including exuperantia.slide-8-poster-1-spring-2016Now, these data make a bold assumption, that so much DNA has been deleted in  10-14 or K8-15, that the region between the P element and the exu gene-specific primer is now small enough to be spanned by garden-variety PCR (usually a limit of about 2 to 3 kilobases of DNA). What if that’s wrong? Also, what if the priming site in the P element isn’t there anymore, for down stream amplification? Really the only way to find that out is to try PCR on single K8-15 or 10-14 homozygous individuals. And as those are dead, we may be stuck.

Or are we? We put both K8-15 and 10-14 over a balancer chromosome that also carried a transgene expressing the Green Fluorescent Protein (GFP). What’s a balancer? Recall that if you want to maintain a recessive lethal mutation in a population of individuals, meiosis will eventually purge the population of the mutation – lethality rather interferes with fitness, after all. Drosophila geneticists have developed a useful tool called a balancer chromosome, which has essentially all the right DNA in it, but not necessarily in the right order, having been scrambled somewhat by radiation (which reminds me of the famous Morecambe and Wise sketch with Andre Previn: “I am playing all the right notes, just not necessarily in the right order” – see around 11:00 in the sketch). The balancer is usually marked by a mutation that has a dominant phenotype (curly wings, stubbly bristles, messed up eyes etc) that is recessive lethal. That way you can instantly see if your stock is balanced. What this means is that during meiosis, when a chromosome with a lethal mutation on it attempts to pair with a balancer homolog, well, it can’t, and all recombinant products are lost. Thus, the population consists entirely of individuals that are heterozygous for the balancer and the chromosome with the desired mutation. A marvellous tool, so far unique to Drosophila.

So we balanced our male recombination mutations over a balancer with GFP on it, which allowed us to do two things. First, we could identify the lethal phase, or when homozygotes for K8-15 and 10-14 died. Second, we could isolate individuals, dying, to be sure, that were homozygous for the lesions, which would be GFP minus, and extract their DNA for PCR analysis.

slide-4-poster-2-spring-2016In fact the graphic above is not entirely accurate: the GFP -/- individuals never made it to adult hood, as implied. They died as first instar larvae – fresh out of the egg.

single-emb-pcr

Panel C in slide 5 above shows a first instar larva that is homozygous for 10-14 or K8-15 – both lesions behaved the same way. Slide 6 shows how we used GFP fluorescence to select against all the genotypes we did not want, and select only those that did not fluoresce. Those larvae were placed individually into eppendorf tubes for DNA extraction and subsequent single embryo PCR. We tested a number of genes in the vicinity of exu – here are some data for CG13437: clearly it is absent from the male recombination lesion 101-14.

slide-7-poster-2-spring-2016

I can tell you we also tested using primers to exu, and to galla-1, which is even further away.  The Galla-1 results are puzzling because Galla-1, which may play a role in chromosome segregation through establishment of sister chromatid cohesion (PMID 25065591), is supposed to be lethal (same paper). But our “complementing” progeny from a cross between Df(2R)BSC404 (removes galla-1 completely) and our male recombination lesions (also remove Galla-1 – enough of it to be considered gone based on where the primers target) are alive. Sterile, but alive. What could this mean? Who knows.

Buggah, buggah, these are large deficiencies we made, when what we really wanted were small lesions confined to DMAP1.

Do we have ANY idea where the break point is? Both K8-15 and 10-14 are viable but sterile over Df(2R)BSC404.  Are there any essential genes, in terms of viability, included within the genomic territory defined by this deficiency? Aside from Galla-1? Yes, but perhaps we have to stick to the brut force single embryo PCR approach at this point. The next victims in my lab will design more primers getting further and further away from exu, Galla-1…until we get a PCR result that tells us a gene is actually present in our male recombination induced lesions.

We will have to map these breakpoints. We want to know how big these lesions are. The next step is to use them as known deficiencies over which to carry out a “local hop” – try to get the P element just upstream of DMAP1 to move out of its current location and into DMAP1, hopefully knocking the gene out. K8-15 and 10-14 may well be huge, but they are much smaller than the stock deficiencies available, like Df(2R)402 or 404. They may yet be useful. Fingers crossed.

Here are the concluding slides for the two posters my students presented last spring, and a couple of shots of students by their posters.

conclusion-slides

 

 

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