Continuing the discussion from Why do we have two of some things but only one of others?:
So I have like a high school education in biology (but not chemistry), and for the heck of it, I’m trying to read and understand this paper about kidney development.
Fetting, J. L., Guay, J. A., Karolak, M. J., Iozzo, R. V., Adams, D. C., Maridas, D. E., Brown, A. C., & Oxburgh, L. (2014). FOXD1 promotes nephron progenitor differentiation by repressing decorin in the embryonic kidney. Development (Cambridge, England), 141 (1), 17–27. https://doi.org/10.1242/dev.089078
The part I understand so far is that they bred mutant mice to lack the FOXD1 gene. They took tissue samples of the developing kidneys from mutant and normal mouse embryos. Next they dipped the tissue slices in a solution of fluorescent molecules that only stick to decorin proteins. Then they stuck the slices under a microscope to observe the concentration of decorin, and found that there’s a lot more decorin in mice that lack the FOXD1 gene.
The rest… I’m having some difficulty with. I’ve tried to bold the actual questions.
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How did they come to suspect decorin? As I understand it medullary interstitial kidney cells are daughters or descendants of cortical interstitial kidney cells, and whatever Foxd1 proteins do happens in the cortical interstitial cells because it hasn’t been seen floating around in medullary cells. So they took the genetic sequence (?) of both cells and made a list of the differences. (Does this mean a cell’s DNA is different depending on what role it takes on? I always thought every cell contained a complete and identical copy of your whole genome.) Then they did the same for cells from the mutant embryo, which lacked FOXD1. Then they compared the lists of differences, to see what changed in the normal medullary DNA that didn’t change in the mutant medullary DNA - this is the list of 47 genes they suspect Foxd1 regulates. Then it says they screened all the target gene candidates for FOXD1-binding sites within 5kb upstream of the promoter. I have no idea what that means or why they did it.
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A couple times the paper mentions transfecting cells with Foxd1 expression constructs, or luciferase constructs. I think I understand the concept of transfection - they take a mouse papillomavirus or cytomegalovirus and stuff it with some DNA or RNA or protein, then inject the virus into the tissue. But what exactly did they put into the vector, what is the ‘expression construct’? What is the luciferase construct, exactly? How did they know what payload to use, and how did they acquire it?
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The entire section where they show that FOXD1 directly represses Dcn is over my head. I remember from biology that a DNA sequence is composed of a list of ACGT pairs, and so the sequences of A, C, G, and T represent genetic code (with the paired nucleobase being implied). I remember the concept of protein binding sites, but I don’t know if that is what is meant by a binding site in this section. I don’t understand what a residue or consensus site is. I don’t understand what chromatin immunoprecipitation is, and trying to learn more about it, I don’t understand the concept of crosslinking… If anyone knows the relevant chapter to an online textbook or something for these, or if you want to explain it that’s cool too.
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Some notations I am a little unclear about. Could someone clarify the meaning of E14.5, E15.5, etc? I believe the E in E15.5 refers to “embryo” (contrast P for postnatal), and 15.5 is some measure of time after fertilization (days? hours?). I am also a little unclear as to the meaning of “Foxd1-/-” v “Foxd1+/-” v “Foxd1” v “Foxd1 null” v “FOXD1”. I believe the all-caps refers to a gene and the italics refers to a protein, but I don’t know what the superscript +/- means, possibly dominant vs recessive alleles?
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What does CD mean? With that abbreviation Google isn’t very helpful. Example usage: “cross-section through a CD tip with a visible lumen” “cells per CD tip” “50 CD tips from three non-adjacent sections” “staining surrounds CD trunks in all three genotypes”
~Max