May 13, 2003


Several people, well... actually only J-4, has/have been asking about what I am doing in school these days. I am in the middle of my final rotation at the moment and am in the process of trying to decide on a lab to settle in for the next few years.

I am currently working on trying to establish zebrafish as a model organism for studying the roles of intestinal microflora in gut development. In humans, the number of bacteria living in the digestive tract is at least an order of magnitude greater than the number of cells in the human body, so it isn't surprising that this "normal flora" plays many important roles, including aiding in digestion, protecting us from all sorts of nasty pathogens, and is necessary for normal development. Historically, "germ-free" mice, rats, hamsters, and even kooky animals like cows and goats have been used to study the interactions between hosts and their microflora. Zebrafish have several advantages over the traditional model organisms used for studying these interactions. Namely, fish embryos and larvae are clear, lots is known about their development, and they reproduce frequently and mature quickly making them useful for all kinds of fun genetic techniques.

So, I have been looking at conventional and germ-free fish, attempting to see differences in cell proliferation (using BrdU labeling and/or antibodies to phosphorylated histone H3) and glycoconjugate expression on the surfaces of epithelial cells that line the lumen of the gut (using fluorescently conjugated lectins). All of my stains/antibodies are working, but raising germ-free fish has been a colossal pain in the ass. I either don't kill all the bacteria, or I kill both the bacteria and the embryos. I need to find a happy medium in my killing.

Here is a picture I took last week. Red is fluorescently conjugated soybean agglutinin (SBA), green is anti-BrdU, and blue is autofluorescence.

5-5 Brdu SBA 4 day CV 40X merge 03.jpg

P.S. Anyone who can figure out why "Gordon" is the title of this entry gets a gold star.

Posted by doug at May 13, 2003 07:00 PM

Heh. As I was reading through this post I was thinking, "Wow, that sounds really cool, but how the hell do you raise germ-free fish?" And that's when I got to the part about it being a pain in the ass.
How do you even start to get an embryo germ-free? Do you have to autoclave the tank?

And why does is the BrdU staining so weird? (or is that normal BrdU for a zebrafish?)

Posted by: Jacob at May 13, 2003 08:11 PM

Most importantly, though, which lab are you leaning towards at the moment?

Posted by: Jacob at May 13, 2003 08:11 PM

What is weird about the BrdU stain? The way I am interpreting it is that the green dots are cells that were synthesizing new DNA during the 15 minutes that the larvae were soaked in the BrdU solution. Then, I fixed the poor bastards, sectioned them, and labeled the cells that were proliferating during the 15 min with anti-BrdU-fluorescein antibody.

Currently, I am leaning towards the Johnson Lab. All three PI's that I rotated with have given me their recruitment sales pitches, and I am still seriously considering working in the lab I am in now. It is a tough call. Eric has lots of cool proteomic stuff lined up that I could work on, and that is sort of what I want to get in to. However, Karen works with bacteria, and I have liked bacteria for a super long time. Both labs have fun atmospheres and plenty of funding. Karen and Eric also happen to be married, so which ever lab I choose, I will still get to go to the social functions for both labs. It is a hard decision to make.

Posted by: Doug at May 13, 2003 08:59 PM

Oh yeah, the germ-free fish... I have been putting embryos (still in their chorions) through a series of dilute bleach solutions. Supposedly the protocol has worked in the past, but I don't know if I believe it.

Posted by: Doug at May 13, 2003 09:04 PM

i remember the sad part of your thesis presentation when someone asked about the zebrafish and you explained that you killed them.

but the part your advisor did with the pictures sort of made up for that. ha ha! tom cruises' body!

flash/flesh gordon?

Posted by: michele at May 13, 2003 09:54 PM

*looks back at my original comment*
Hmmm. Maybe if I had proofread that...what I mean to say was, "Why does the BrdU stain look so weird?" I was wondering what's up with that pattern of the cells actively replicating (or lack of pattern, as the case may be)? Maybe I'm just not orienting myself this part of a grown fish, or a developing embryo on top of the yolk sac?

Dilute bleach? No wonder you're skeptical. Has anyone tried just hitting them with all the old favorite antibiotics (kan, amp, chlor, and rif)? You could homogenize the little buggers and plate out the mix to see if you killed everything...

Posted by: Jacob at May 13, 2003 11:35 PM

The picture sort of resembles the Bat-symbol, used by Gotham City Police Commisioner Gordon?

Posted by: sean at May 14, 2003 01:04 AM

Gordon :
I assumed it was a reference to Gorton's, the huge Gloucester based fish company.


Posted by: gene at May 14, 2003 08:23 AM

to the bat cave!

Posted by: michele at May 14, 2003 08:35 AM

I don't really know what is up with the lack of a pattern of cell division. Perhaps I could explain it if I had more of a background in zebrafish development. The picture is a cross section of a 4 days post-fertilization larvae. The yolk sack is on the bottom. You can sort of see a pectoral fin on the left side of the picture. If it were an adult, you might expect to see a pattern of cell division similar to what is seen in mammals, where stem cells in the intestinal crypts continually divide, however, we don't know for sure if it works that way in zebrafish, and we are nowhere near being able to raise germ-free adults.

No gold stars have been awarded is a hint: PNAS

Posted by: Doug at May 15, 2003 05:22 PM

Hmmmm....I'm going to need a bigger hint than the name of a journal...

Posted by: Jacob at May 15, 2003 08:03 PM

sounds like 'penis'?

Posted by: michele at May 15, 2003 08:36 PM

I have decided to award gold stars to all who participated in the Gordon Guessing Game. It seems that Jeff Gordon is only well known among people who are into gnotobiology. He is pretty much THE big name among scientists that study germ-free animals. His lab published a cool paper in PNAS last month that showed that germ-free mice have less developed intestinal blood vessels than conventional mice. Exciting stuff in the lab I am working in, but probably not for the rest of the world.

Posted by: Doug at May 16, 2003 03:04 PM