The Mystery of Zebrafish Gonad Development
Kristopher Chu and John H. Postlethwait
Download a PDF of this comic on Zenodo.
Transcript
Overview
A colorful digitally drawn comic. On most pages, the comic background is yellow. Frequent tiny blue fish appear throughout the comic to highlight key points or add visual interest. The primary speaker is professor John Postlethwait, a middle aged man with light skin and brown hair with a few streaks of gray. He wears a blue quarter-zip sweater with a black shirt underneath. The transcript uses quotes to indicate when text is written in the comic.
Cover
The Mystery of Zebrafish Gonad Development
By Kristopher Chu, artist, and John Postlethwait, researcher. Edited by Audra McNamee.
This comic was created through the University of Oregon Science and Comics Initiative, completed July 31st 2025.
Cover description: A zebrafish, a long, thin fish with three horizontal stripes across its body, looks back curiously at a circle drawn at the lower center of its body, labeled “gonads.”
Page 1
Panel 1: “As you probably know, animals reproduce when a sperm fertilizes an egg from the same species. That process starts with gonads.” Several small sperm approach the edge of a large egg.
Panel 2: John Postlethwait: “Gonads are organs that usually develop into ovaries that make eggs, or testes that make sperm. Eggs and sperm are germ cells, cells that can become new individual animals. In many species, a major sex gene pushes gonads to become ovaries or testes. Genes are located on chromosomes, long threads of the genetic molecule DNA.”
Panel 3: “In humans, each sperm and each egg has 23 chromosomes,” Diagram: 23 red chromosomes in an egg and 23 blue chromosomes in a sperm fuse, “so a fusion of sperm and egg makes 23 pairs of chromosomes.” The fertilized embryo, a body cell, has 23 pairs of chromosomes that are alternately red and blue. “Embryo matures and begins meiosis, the cell division in germ cells producing one copy of each chromosome per each chromosome pair. The embryo’s gonads can develop into testes that produce sperm, or ovaries that produce eggs.”
Page 2
Panel 1: “22 of the 23 pairs of human chromosomes have roughly the same size, shape, and gene content. But one pair, the sex chromosomes X and Y, have different size, shape, and gene content.” A cartoon “human” says, “Whoa! My chromosomes! The Y chromosome is so small compared to X?” The 23 pairs of human chromosomes are drawn next to the human. Only the 23rd pair looks different; the X chromosome is about three times as tall as the short, round Y chromosome.
Panel 2: “human sex chromosome pairs.” Two tall XX chromosomes usually develop into ovaries. A tall X chromosome and a short Y chromosome usually develop into testes. “Most female humans have two X chromosomes, resulting in an XX chromosome pair. Most male humans have one X chromosome and one Y chromosome, resulting in an XY pair. Like many things in this comic, there are complexities beyond this simple explanation, but this is what we have room for!” The “human” character looks up with raised eyebrows.
Panel 3: “The human X chromosome has 864 protein-coding genes while the Y has only 63 protein-coding genes. Among those few 63 genes is the major sex gene that causes human gonads to become testes instead of ovaries. This major sex gene encodes the sex-determining region Y protein (a.k.a. SRY). The SRY gene determines gonads in nearly all mammals like dogs! Without SRY, their gonads become ovaries.” A flow chart, where “the embryonic gonad” wonders, “is the SRY gene present?” If yes, the embryonic gonad becomes testes. If no, the embryonic gonad becomes ovary.
Panel 4: A leaping wiener dog and strutting chicken flank the text, “birds fly in a different direction: hens have a ZW chromosome pair and roosters are ZZ. The Z chromosome has a weak testis-specifying gene (DMRTI) not on the W. Because it takes two copies of DMRTI to make a testis, ZW birds have ovaries.
Page 3
Panel 1: “Species and their gonad determining factors!” “While genes are gonad determinants in some species, environment determines gonads in other species.” A spotted brown sea turtle’s gonads are determined by temperature. A striped tiger has gonads determined by the SRY gene. The clownfish’s gonads are determined by social environment. The platyfish is polygenic: multiple genes determine their gonads.
Panel 2: “Whether an animal develops ovaries or testes is important because it affects what diseases it might get and how many babies it can make.” A female lioness sits next to a male lion.
Panel 3: John Postlethwait and collaborators Catherine Wilson, Angel Amores, John Dowd, and Yilin Yan, ponder “This brought us to our current question: What determines whether zebrafish gonads become testes or ovaries?”
Panel 4: “You might ask:” Human asks, “Why zebrafish?” while he looks down at “the simple zebrafish.”
Page 4
Page title: “Here’s a quick intro to zebrafish, a major model for gene action, development, and physiology worldwide.” In the center of the page is a zebrafish labeled, “The actually super cool zebrafish.” The zebrafish is surrounded by additional labels:
“Has transparent embryos! This makes it easy to observe internal development.” A small image of a curled fish embryo with an embryonic sac in a transparent egg is labeled with a nametag reading, “Hi, I am a: Zebrafish Embryo.”
“Has most but not all of the same organs as humans (e.g. it has a brain, liver, and intestines).”
“Develops rapidly! It only takes a couple days for embryos to develop into free living youngsters.”
“They have high fecundity (they can make a lot of children very fast). Female zebrafish can make up to ~200 babies per week!”
“Originates in India!”
“Has been domesticated in labs for ~40 years”
“Over 60,000 research papers in medical literature mention zebrafish! Basically, used for lots of research, kind of a big deal.”
An arrow pointing to the next page, with text reading, “now, let’s get into the research!”
Page 5
Panel 1: John Postlethwait: “Here’s what we know about how the gonad develops in domesticated (i.e. lab-bred) zebrafish.
Panel 2: “At 19 days post-fertilization (dpf), a juvenile zebrafish has bipotential gonads that can develop into either ovaries or testes.” A juvenile zebrafish has a circle highlighting its bipotential gonads.
Panel 3: “At 22 dpf, gonads in each lab zebrafish start to form egg cells—creating a juvenile ovary.” A simple circular bipotential gonad containing three small blue egg cells.
Panel 4: An inset panel, the closeup view of an egg cell inside the juvenile ovary. The egg has dark purple exterior support cells, a thin light purple egg cell membrane. Inside is a blue egg cell containing a dark blue egg cell nucleus.
At 25 dpf, the zebrafish can potentially develop in two different ways.
Option one, panel 5: “In some individual zebrafish, egg cells continue to increase in number and size. The support cells become estrogen-producing ‘granulosa cells.’” The gonad has developed into an ovary, with larger maturing egg cells. “The juvenile ovary continues to develop, resulting in a female zebrafish.” A female zebrafish, larger and slightly rounder than the juvenile with a gray and blue striped body.
Option two, panel 6: “In other zebrafish, young egg cells die.” A gonad with crossed out egg cells turns into testes with three large pink Sertoli cells containing many small developing sperm cells, interspersed with three small yellow Leydig cells. “In this case, support cells become Sertoli cells that nourish growing sperm. Other cells become testosterone-producing Leydig cells. The juvenile ovary has changed into a testis, resulting in a male zebrafish.” A male zebrafish, longer and thinner than the female, gray and blue striped all over its body with the exception of a yellow anal fin.
Page 6
Panel 1: “To study transformation of the juvenile ovary, we created zebrafish with a mutation in the brca2 gene. This mutation makes cells unable to repair DNA breaks, and in humans, it leads to breast cancer.” The human asks, “what happened to the fish?” “This is what happened with the mutated lab zebrafish: in the brca2 mutant zebrafish, first, the juvenile ovary developed as usual. Then, egg cells died in every mutant fish due to unrepaired DNA breaks. So, all gonads became testes. And all mutant fish became males.”
Panel 2: John Postlethwait considers a diagram. The diagram is a close-up of an egg. “Based on this result, we hypothesize that in normal lab zebrafish: 1. The egg makes a signal” Triangular ‘signals’ appear inside the egg, next to the nucleus. “2. The signal goes to the support cells.” The signal touches the rim of light purple support at the edge of the egg’s interior, and the signal is received. “3. Support cells make estrogen.” Pink estrogen rhombi are created outside the egg. “4. Estrogen maintains the egg cells and thus the ovary.” The estrogen enters the cell, supporting it, and the process is repeated. “In contrast, in the brca2 mutant, all egg cells die so there’s no estrogen to maintain the ovary and its egg cells. Without estrogen, the ovary becomes a testis, resulting in only male zebrafish. This is ‘sex reversal’: juvenile females change into males.” Human raises his hand and asks, “In normal lab fish, why aren’t they all female? There’s no DNA breaks, so…” Caption: “Well, just like some people are taller than others, some normal lab zebrafish don’t make as many eggs as others. Thus, these fish don’t make enough estrogen to maintain the ovary, so their gonads become testes and those fish become males.
Page 7
Panel 1: “We wondered if the sex reversal found in lab zebrafish happens in zebrafish in the wild or if a major sex gene causes testes to develop directly without first making a juvenile ovary. To find the answer, we got wild zebrafish from India.” Two people stand near a river, catching zebra fish.
Panel 2: “We separated the males and females.” A group of male zebrafish and a group of female zebrafish. “Then, we extracted their DNA to identify DNA sequences that were present in one sex but not in the other.” A test tube with a light substance. Inside is a multicolored DNA double-helix.
Panel 3: “Finally, we mapped the likelihood that a DNA sequence is associated more often with one sex and not the other vs. the location of that DNA sequence on the fish chromosomes.” A bar graph of the likelihood a genetic variant is sex-linked. There are 25 zebrafish chromosomes. There is a peak in likelihood at chromosome 4 that is three times as high as most of the other bars. “Results showed that a single place along all 25 chromosomes of wild zebrafish was statistically linked to sex: at the tip of chromosome-4. We called it ‘sex-associated region on chromosome-4,’ or sar4. When doing the same analysis in lab zebrafish, we found no site in the genome linked to sex. We concluded that wild zebrafish have a major sex gene, but lab zebrafish lost it in domestication.” Zebrafish in the wild amidst greenery at the bottom of a stream have sar4, but a zebrafish in a bowl doesn’t have sar4. John Postlethwait and two other researchers contemplate the likelihood graph.
Page 8
Panel 1: “The data showed that: wild zebrafish males have two Z chromosomes, and wild females have one Z and one W chromosome.”
Panel 2: “So, wild zebrafish sex chromosomes are similar to those in chickens.”
Panel 3: “ZW wild fish, like all lab zebrafish, have bipotential gonads that become juvenile ovaries. Gonads in ZZ wild fish always develop directly into testes and ZZ fish always become males. Most ZW fish become females but some experience sex reversal and their juvenile ovaries become testes.”
Panel 4: Header with block letters: “We concluded that either:” Caption: “1. The W has a gene that is necessary to make a female but that gene is not sufficient to make a female (since some ZW fish undergo sex reversal), or 2. Two Zs are necessary to make a male, like in birds. Regardless of which hypothesis is correct, we figured out lab zebrafish are different from wild zebrafish because lab zebrafish lost the Z chromosome during domestication and are now chromosomally WW.”
Page 9
Panel 1: Header with block letters: “Why do some ZW juvenile ovaries change to testes?” A small sea turtle, with the caption: “We thought it might be environmental, like how turtle gonad development depends on temperature. So we raised some wild zebrafish at a high temperature, (98F, 36C) during the period of sex determination and others at the normal temperature (83F, 28C) as a control.” Graph of temperature against time. The high temp group is kept at 83 degrees until 20 days post-fertilization, when they are brought up to 98F for 10 days. They return to 83F at 30 days post-fertilization. The control group’s temperature is steady at 83 days over the whole time.
Panel 2: “The results!” A diagram showing ZW and ZZ zebrafish at the control temp and the high temp. At the control temp, two out of three ZW zebrafish developed into female while one is male, with a label “mostly female”. The control temp ZZ zebrafish developed into “all male.” At the high temp, ZW zebrafish developed into “mostly male.” There are two male fish and one female fish. The ZZ zebrafish developed into “all male.”
Panel 3: “We observed that most ZW zebrafish developed ovaries at the control temperature: meanwhile, ZW zebrafish raised at the higher temperature underwent sex reversal and developed testes. In contrast, ZZ zebrafish developed testes under both circumstances.”
Panel 4: A sticky note on the page, labeled “stressors.” An image of many fish, and a crossed out burger. A ZW zebrafish looks at these images, sweating with a tight face. “Other environmental stressors (like population density or lack of food) also caused sex reversal in developing ZW zebrafish.”
Page 10
Header: “So here’s how zebrafish sex genes likely control gonad development.” The next three panels contain diagrams of the edge of a developing germ cell.
Panel 1: In ZW zebrafish, “1. A W gene in support cells causes them to send a feminizing signal to germ cells.” Yellow signals from the W gene enter the egg. “2. Germ cells send a second signal to support cells.” Blue signals move inside the egg to the support cell at its perimeter. “3. Support cells make estrogen.” Pink estrogen are created outside the egg, and circle around to enter it. “4. Estrogen maintains the egg cells and thus, the ovary.”
Panel 2: In ZZ zebrafish, “1. Without the W gene, there’s no feminizing signal.” No signal enters the egg. “2. There’s no second signal.” The support cells don’t receive a signal. “3. Support cells make testosterone.” tTstosterone circle around to enter the germ cell. “4. Testosterone allows germ cell to begin sperm development.”
Panel 3: In ZW zebrafish at a high temp, “1. Heat blocks either: A. The feminizing signal to the germ cell or B. The second signal to the support cells.” Either the circular signal from the W gene or the triangular signal from the egg are blocked by a red X. “2. Without the signal, there is no estrogen. 3. The egg cells die. 4. Support cells make testosterone. 5. Germ cells begin sperm development.” An illustration of three round germ cell surrounded by a larger round sertoli cell, supported by spirals of testosterone.
4. A whiteboard. Two zebrafish, the Human character, and John Postlethwait contemplate four questions written on it. “Questions for the future: If climates warm, will all wild zebrafish become male? How does high temperature block signals? What is the feminizing gene on the W or the masculinizing gene on the Z? How does the major sex gene act?”
Page 11
Panel 1: A portrait of John Postlethwait. “John Postlethwait is a professor of biology and a zebrafish researcher. He’s been cited over 50,000 times with 103 publications cited more than 103 times (Google Scholar)! You can find his research on zfin.org/ZDB-LAB-970408-67.”
Panel 2: A portrait of the artist, Kristopher Chu. He has dark short hair, rectangular glasses, and a short beard. He wears headphones. “Kristopher Chu is a recent UO Art and Tech graduate. He drew 36 zebrafish (and five zebrafish researchers) in this comic! You can find more of his work at kristopherchu.carrd.co.”
Panel 3: A portrait of the editor, Audra McNamee. Audra has long curly brown hair, light skin, and big glasses. “Audra McNamee is a cartoonist, editor at Crucial Comix, and program coordinator for the University of Oregon Science and Comics Initiative.”
Panel 4: “This material is based upon work supported by the National Science Foundation under Grant No. 2232891 and the National Institutes of Health Grants No. R35GM139635, R01GM085318, and R01OD011116. Key primary lab work performed by Angel Amores, John Dowd, Catherine Wilson, and Yilin Yan.”
Literature: Kanamorei A, Sugita Y, Yuasa Y, Suzuki T, Kawamura K, Uno Y, Kamimura K, Matsuda Y, Wilson CA, Amores A, Postlethwait JH, Suga K, Sakakura Y. (2016) A Genetic Map for the Only Self-Fertilizing Vertebrate. (Bethesda). 6:1095-106. doi: 10.1534/g3.115.022699.
Pan Q, Anderson J, Bertha S, Herpin A, Wilson C, Postlethwait JH, Schartl M, Guiguen Y. (2016) Vertebrate sex-determining genes play musical chairs. C R Biol. 339(7-8):258-62. doi: 10. 1016/j.crvi.2016.05.010.
Valdivieso A, Wilson CA, Amores A, da Silva Rodrigues M, NoXXbrega RH, Ribas L, Postlethwait JH, Piferrer F. (2022) Environmentally-induced sex reversal in fish with chromosomal vs. polygenic sex determination. Environ Res. 213:113549. doi: 10.1016/j.envres.2022.113549. Epub 2022
Wilson CA, High SK, McCluskey BM, Amores A, Yan YL, Titus TA, Anderson JL, Batzel P, Carvan MJ 3rd, Schartl M, Postlethwait JH. (2014) Wild sex in zebrafish: loss of the natural sex determinant in domesticated strains. Genetics. 198:1291-308. doi: 101.1534/genetics.114.169284.
Wilson CA, Batzel P, Postlethwait JH. (2024) Direct male development in chromosomally ZZ zebrafish. Front Cell Dev Biol. 12:1362228. doi: 10.3389/fcell.204.1362228.
Yan YL, Titus T, Desvignes T, BreMiller R, Batzel P, Sydes J, Farnsworth D, Dillon D, Wegner J, Phillips JB, Peirce J, Dowd J; Undiagnosed Diseases Network; Buck CL, Miller A, Westerfield M, Postlethwait JH. (2021) A fish with no sex: gonadal and adrenal functions partition zebrafish NR5A1 co-orthologs. Genetics. 217(2):iyaa030. doi: 10.1093/genetics/iyaa030.
About the authors
name: Kristopher Chu
Kristopher Chu is a recent UO Art and Tech graduate.
name: John Postlethwait, Ph.D.
institution: University of Oregon
John Postlethwait is a professor of biology and a zebrafish researcher. He’s been cited over 50,000 times with 103 publications cited more than 103 times (Google Scholar)!