Manuscript II - planning

I'm about to start writing my next manuscript. It's going to deal with patterns of gene expression in the placenta of F1 hybrids. I'm stalled a bit as I try to figure out exactly what questions I will be addressing and how I will go about discussing them in the manuscript. So, I'm hoping that writing this will help me flesh out my thoughts on what questions I'll be asking, why they are important, and how to address them.

First, all the questions I can think of that I can address with my data:

Is the histology of placentas abnormal in large or small hybrids? males or females?
Is gene expression disrupted in hybrid placentas?
How many genes show differential expression (DE)?
How many genes show DE between P. campbelli and P. sungorus (evolutionary changes)?
How many genes show DE between parents and hybrids (hybrid breakdown)?
How many genes show DE between cxs and SxC hybrids (could explain POE growth)?
How many genes are DE between males and females within each parent species?
How many genes are DE between males and females with the hybrids?
Are sex-specific effects prevalent in hamster hybrids?
For all these categories: what do those genes do? / Is there any functional enrichment for DE genes?
Do imprinted genes show DE?
Is imprinting disrupted (DI) in hybrids? Large, small, or both?
Does DI correlate with POE growth?
How often does disrupted imprinting correlate with differential expression?
Does the simple model of 'disrupted imprinting=2x increase in expression' hold?
Do paternally expressed genes show higher expression in large hybrids than small hybrids?
Do paternally expressed genes show higher expression in large hybrids than the parents?
Do maternally expressed genes show lower expression in large hybrids than small hybrids?
Do maternally expressed genes show lower expression in large hybrids than the parents?
What is happening on the X chromosome in hybrids?
Is X-linked expression disrupted?
Is X-linked expression correlated with abnormal growth?
Is there imprinted X inactivation (XCI) in hybrid placentas?
Is XCI disrupted?
Do any/many genes escape XCI? Which?
Does escape of XCI seem to have anything to do with POE growth?


Ok, that was more than I expected so that's good.
Now some organization.

My first thoughts are that the main question is what are patterns of expression in hybrid placentas and do they reflect a possible basis for parent-of-origin growth? This has three main sub-questions which are (1) what are the patterns of differential expression in hybrid placenta? (2) what is the pattern of imprinting in hybrid placenta? and (3) what is the overlap between DE and DI in hybrids?

So far, this sounds really descriptive and one of the strengths of my project is that I have a couple really strong a priori predictions about disrupted expression and imprinting and the size of the hybrids. Therefore, I think I should be able to motivate this in a more hypothesis-driven manner, rather than just as a simple description of expression patterns.

To do that, I need to lay out the prediction of gene expression.

To be continued...

Denver Museum of Nature and Science

K~ and I visited the Denver Museum today and were given a private tour of the collections. J. D~ was kind enough to spend his entire Friday afternoon showing us around and letting us see all the cool stuff. Fortunately, we got the grand tour despite the fact that museum is currently moving it's entire collection to a brand new housing area with fancy state-of-the-art rolling cabinets.

We spent most of the time with the bird collection and we got to see about 40 passenger pigeons (extinct), an ossified goose trachea (shout out to M. J~), and articulated skeletons of a bunch of birds including a kestrel and three different owls (which K~ identified on sight as a saw-whet, burrowing, and pygmy).

They also had two complete elephant bird eggs (these are watermelon size, the hand for scale doesn't actually do it justice). Elephant birds are from Madagascar and went extinct a couple hundred years ago. The fact that any eggs stil exist is amazing - not to mention two complete, unbroken ones.

 The Denver Museum had a collection trip to the Galapagos in 1960, one year before they stopped allowing collections, and returned with a number of Geospiza finches.

Most excitingly, they had a male and two female speciments of the infamous Ivory-Billed Woodpecker (unfortunately, the photo is a bit blurry*):

















We also got to see a bunch of the beetles, scorpions, spiders, camel scorpions (which I've never even heard of before but looked bad-ass - apparently the mouthparts for predator in the movie Predator were based on these guys) and whip scorpions (which gained fame in the movie Harry Potter and the Goblet of Fire as the spider that Moody used to demonstrate the unforgivable curses to the defense agains the dark arts class). Also we were able to see some more beetles, butterflies and moths as they were being moved to the new shelves (the staff were fantastic and took a break from moving specimens to let us look at the cool beetles).

After the birds and bugs we headed over to the fossils - there were two dire wolves (smaller than I expected), an irish elk skull (with antlers spanning further than I could reach from toe to fingertip), a bunch of mammoth and mastodon skulls, leg bones, and vertebrae, a petrified tree stump we helped move, and a Stockoceros (which is like a pronghorn with two horns).

One really cool specimen we saw was a giant ground sloth.

Then we got to the mammal cabinets and looked mainly at the chipmunks, but we got to see some lions, tigers, a gorilla, and bats too. Didn't see any Phodopus but I guess I've seen plenty of them.

All in all it was a great afternoon. Major thanks to J. D~ for showing us around and answering all our questions. Also to the staff of the Denver Museum for taking time to show us some cool biology!

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*I'm told all ivory-billed photos are blurry - it's a tradition. 

Parent-of-Origin Effects Paper is out!

http://authorservices.wiley.com/bauthor/onlineLibraryTPS.asp?DOI=10.1111/evo.12500&ArticleID=1354661

Capture design

One project that I'm working on is to map the genetic factors that cause parent-of-origin growth in dwarf hamsters. This project will end up being the final chapter in my dissertation and has a number of angles that hopefully will all come together in the end to forma  complete story. One of these angles is a mapping panel made out of ~200 backcrossed hamster individuals. I'm using a RAD-seq approach which has recently been sent for sequencing. This will use anonymous markers across the genome to find regions associated with growth. The shortcoming of this approach is that the markers are anonymous and not necessarily associated with known genes. In order to place known genes on the map I am designing a capture.

The capture will pull out ~10,000 gene regions (parts of exons) that are known to contain snps from each individual. I will be able to sequence those specific regions in a subset of individuals and then place those genes on the genetic map. In the end I will have actual genes that associate with growth.

I've never designed a custom capture before but J~ has and so with some guidance I'm figuring it out. My goal is to have a list of sequences, each around 200bp long (I should check that length - it needs to be long enough to sequence on Illumina) that have a diagnostic snp in the middle somewhere.

The first step is to find genes that have snps in them and I have done this earlier for my F1 placental transcriptome project.
Now that I have a set of genes with known snps, I need to make sure that none of these fall near exon boundaries. To do this I am using a Blat approach. Blat is nice because it will align my transcriptome to the Mesocricetus auratus genome and split up the genes into different exons. The output of a blat gives me the exact breakpoints in each gene so that I can choose snps that are far from those areas.

Then, once I have the list of genes that have snps far from breakpoints, we send that in to agilent (I think we're using agilent) who will actually design the capture for me. Then it just remains to do the labwork and sequence it.

Parent-of-Origin Effect paper was just accepted!!

My very first first-author paper has just been accepted by the journal Evolution!

It's now going through the typesetting process. They publish the pre-formated articles here and while it's not up now, it should appear soon.

They said expect ~12 weeks for the final proofs to be ready and then I expect there is a queue before it shows up in print.

Generally, it's a description of the parent-of-origin growth effects in dwarf hamsters and the first dalliances of mine to understand the genetic basis and answer the question: is imprinting involved or not?

I'm really excited. Now on to the writing of my second paper...

Parent-of-Orign Growth Effects Paper

J~ and I recently submitted my first first-author manuscript to Evolution. The reviews came back fairly positive, but there were some things that they would like changed. In total, I thought the reviews were really reasonable and I've already started collecting some extra data.

The main issue that they had was that I present evidence that some genes are properly imprinted in hamsters, some are polymorphic in their imprinting status within cross types, and some are expressed from two alleles in both hybrids. None of these patterns can explain POE growth as in all cases the two reciprocal hybrids show the same patterns. The reviewers were concerned that as I don't know the imprinting status of these alleles within each species, it is hard ("impossible" was the word they used) to interpret whether imprinting is breaking down in the hybrids. To address this apparent shortcoming, I'm planning on doing two things. First, making the writing clearer. I need to make it clear that if we see the same pattern of expression in hybrids, it cannot explain POE growth, regardless of whether the gene is imprinted in the parentals or not. While I think that should be sufficient, I am also planning on collecting more data in the form of a qPCR experiment. If I can show that the expression level is the same between parentals and hybrids, that is good evidence that the gene is not disrupted in the hybrids, and is therefore not imprinted in the parents. This should be easy(-ish) data to collect and so I'm going forth with some qPCR experiments.

There were other minor things, but all can be addressed in the text fairly easily. Hopefully I can turn this around and get it back to the reviewers soon.

DDIG

It's been a while since my last post. I've been working on a couple things recently that I should start writing about, but first, I should announce that I was awarded the NSF DDIG. The award money will go towards mapping the maternally inherited factors that result in POE overgrowth in dwarf hamsters.
The crossing scheme is this: make a small F1 (cam x sun) and then back cross those females to a campbelli male. See the figure.

I am focusing only maternally inherited factors because F1 hybrid males are sterile so I cannot do an F2 cross or a sun x F1 male backcross. As should be clear in the figure, the variation occurs due to recombination in the F1 female between P. campbelli and P. sungorus chromosomes, but that variation is only inherited through the mother, I cannot send variation through the father.

While it's kind of a bummer that I will be blind to anything that causes POE overgrowth when inherited from the paternal line, it's not the end of the world. We expect that maternally inherited things that are silenced are growth factors and their failure to be silenced is what causes overgrowth. In other words, we don't have an a priori expectation that paternally inherited factors are involved.

The grant money will support a RAD digest (Peterson et al. 2012) and Illumina sequencing in order to build the genetic map and map the causative factors. The shortcoming of using RADs is that they are anonymous markers - I don't know what genes are near them in the genome. In order to actually localize genes, I will do two things: (1) use a custom-designed array capture to sequence exons from the mapping panel and associate them with the anonymous markers  on the genetic map and (2) use RNAseq on the placentas of small and large backcross offspring to associate genes that are mis-expressed in those individuals with the genetic map.

I'm pretty excited about this whole approach as I think that it will give me the ability to get almost down to the gene level without fine-mapping. We'll see if I'm right or not, but it's pretty exciting that NSF is impressed enough to give me monies for it.

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Peterson, B. K., J. N. Weber, E. H. Kay, H. S. Fisher, and H. E. Hoekstra. 2012. Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLOS ONE 7:e37135.