Reverse transcription quantitative real-time fluorescence polymerase chain reaction, or qRT-PCR, is a widely used molecular technique to measure RNA levels.

RNA is first reverse transcribed into complimentary DNA. This complimentary DNA is then used as a template for a fluorescence-tagged amplification reaction to calculate the relative amount of a specific starting transcript.

The transcript levels are compared between genes of interest, which are all compared to a "housekeeping gene" or a transcript that has the same levels of expression across conditions. This comparison standardizes the levels of overall transcription across samples.

Thermo Fisher provides an extensive introduction to gene expression measuring technologies here: https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/gene-expression-analysis-real-time-pcr-information/introduction-gene-expression.html

The authors tested the abundance of KDM6B RNA (denoted as Kdm6b) in turtle embryos at the earliest stages of sex development (stages 12-14) to see if there was a difference between males and females.

The gonad-mesonephros complexes are the earliest sites of gonad development.

The authors compared Kdm6b gene expression between gonads at male and female temperatures throughout the temperature-sensitive stages of development (stages 15-20).

AP Biology Learning Objective LO 3.19,21:

The student is able to describe the connection between the regulation of gene expression and observed differences between individuals in a population.

The student can use representations to describe how gene regulation influences cell products and function.

AP Biology Learning Objective LO 3.5-6:

The student can explain how heritable information can be manipulated using common technologies.

The student can predict how a change in a specific DNA or RNA sequence can result in changes in gene expression.

Next Generation Science Standards HS-LS4.1:

Ask questions to clarify relationships about the role of DNA and chromosomes in coding the instructions for characteristic traits passed from parents to offspring.

AP Biology Learning Objective LO 2.42:

The student is able to pose a scientific question concerning the behavioral or physiological response of an organism to a change in its environment.

AP Biology Learning Objective LO 1.25:

The student is able to describe a model that represents evolution within a population.

AP Biology Learning Objectives LO 1.16:

The student is able to justify the scientific claim that organisms share many conserved core processes and features that evolved and are widely distributed among organisms today.

Next Generation Science Standards HS-LS4.1:

Communicate scientific information that common ancestry and biological evolution are supported by multiple lines of empirical evidence.

There are increasing concerns about reptile egg nest incubation temperatures as climate temperatures rise. Several news outlets and scientific reports detail the implications of climate change for reptile sex bias and survival.

You can read more about the concerns for reptiles that undergo temperature-dependent sex detemination, like sea turtles, alligators, and lizards, here:

1. https://phys.org/news/2021-10-animals-affected-climate.html
2. https://gizmodo.com/climate-change-could-make-american-alligators-sex-rati-1846918445
3. https://www.nature.com/articles/s41598-021-99702-1
4. https://www.nature.com/articles/s41598-019-40597-4

The genome encodes sex in many vertebrates, including humans (e.g. XX chromosomes lead to female development and XY leads male development).

But for some organisms, the ambient environment determines sex. In most reptiles, as well as some amphibians and fish, the incubation temperature of eggs directly determines whether males or females will hatch.

Here is a diagram describing the developmental trajectory of temperature-dependent sex determination in T. scripta: https://ars.els-cdn.com/content/image/1-s2.0-S0303720711006083-gr1_lrg.jpg

RNA transcripts have two major components - exons and introns. Exons stay in the transcript that gets made into a protein, whereas introns are cut out, and this is known as splicing.

Intron retention refers to a transcription event where an intron is kept in the RNA instead of being removed. This process can allow for more diversity of transcripts from the same gene.

You can visualize the splicing process here: https://www.biointeractive.org/classroom-resources/rna-splicing

Lentivirus is a type of virus that contains reverse transcriptase - a molecule that transcribes RNA into DNA to integrate into the genome and infect host cells.

Lentivirus can be used to deliver desired DNA into cells. This lentiviral transduction allows for gene expression of desired sequences in organisms of interest.

You can watch more about cloning to make the DNA for transduction here: https://www.biointeractive.org/classroom-resources/dna-cloning-plasmids, and you visualize the workflow of lentiviral transduction here: https://www.mirusbio.com/applications/high-titer-virus-production/lentivirus-production#figure1303

The authors validated the shRNAs using multiple methods.

The shRNA was infected with a fluorescence tag, called green fluorescent protein or GFP. The infected embryos were then visualized at multiple developmental stages. The treated embryos fully fluoresced green, including the gonads, which indicated that the virus successfully introduced the shRNA to the embryos.

The authors also checked the levels of transcript between the control shRNA- (a non-specific sequence) and Kdm6b shRNA-treated embryo gonads. In the green, GFP+ gonads, the shRNAs specifically reduced Kdm6b by up to 80% when compared to the controls.

RNAi describes the process in which small RNA molecules target and degrade RNA molecules to block protein expression.

shRNAs are one type of RNA that is used for RNAi.

This tutorial provides further exploration of RNAi: https://www.biointeractive.org/classroom-resources/rna-interference

The creation of an RNA molecule from DNA.

The RNA that is transcribed from DNA is commonly referred to as a "transcript".

Canonically, this RNA is later translated to make protein, as described by gene "expression".

You can visualize and learn more about transcription here: https://www.biointeractive.org/classroom-resources/dna-transcription-advanced-detail

Histones are a family of proteins that help organize and compact DNA in the eukaryotic genome. DNA wraps around histones to fit inside the nucleus, like yarn around a spool.

Here is an illustration of this organization: https://www.genome.gov/sites/default/files/tg/en/illustration/histones.jpg and a video that walks through DNA compaction: https://www.biointeractive.org/classroom-resources/how-dna-packaged

The histone proteins have exposed "tails" of peptides that can be modified with additional chemical groups, such as methyl or acetyl molecules. These molecules can alter how the histone interacts with its associated DNA.

These chemical modifications can be added or removed by specific proteins, such as KDM6B, which removes histone subunit 3 lysine residue 27 methylations.

You can learn more about histone modifications here: https://www.youtube.com/watch?v=OqRt723t33o

Meiotic is the adjective for meiosis, which is the cell division that gives rise to sex cells.

You can learn more about meiosis here: https://www.biointeractive.org/classroom-resources/meiosis

Lan et al. show that JMJD3/KDM6B demethylates H3K27. They find that this demethylation is critical to the expression of body patterning genes during animal development.

A formative study from 1979 that showed temperature directly influences sex differentiation of turtle hatchlings, rather than turtle hatchling mortality.

Pieau, Dorizzi and Richard-Mercier present what was known about temperature-dependent sex determination across species of reptiles in 1999. This review focuses on gene expression that marks temperature-dependent gonad differentiation and the regulation of hormone-producing proteins.

Navarro-Martín et al. study the mechanism underlying temperature-dependent sex determination in the European sea bass fish. They show that DNA methylation at the promoter of a gonadal aromatase (estrogen-making protein) is increased at high temperatures, causing female fish to masculinize.

This supports the idea that there are many epigenetic mechanisms that can govern temperature-dependent sex determination across species and environments.

Piferrer reviews what is known about the epigenetic mechanisms underlying sex determination in plants, invertebrates, and vertebrates. This includes a discussion of how environment can affect gene expression through changes in histone modifications, DNA methylation, and non-coding RNAs (RNA that does not make protein).

Wiles and Selker discuss what is known about the function and how H3K27 methylation is specifically deposited and defined across the genome.

This review provides good overview of histone modification interactions with gene expression.

Yatsu et al. profile the total gene expression of the American alligator with RNA-seq analysis of embryonic gonads at MPT and FPT during multiple timepoints of sex development.

One of the genes that was predicted as a regulator of sex development at MPT was KDM6B.

RNA sequencing (RNA-seq) is a high performance technique that measures which and how many transcript sequences are present in a given biological sample.

You can learn more about the technique and its analysis here: https://www.youtube.com/watch?v=tlf6wYJrwKY

A previous study identified temperature-specific Kdm6b expression changes in American alligator during gonad development.

This data supports the role of KDM6B in temperature-dependent sex determination across reptiles.

Deveson et al. reveals that a single intron change for two epigenetic-related protein transcripts (JARID2 and JMJD3/KDM6B) is specifically induced in high temperature sex reversal in female bearded dragons as opposed to normal bearded dragons. Authors propose that the intron retention changes JARID2 and JMJD3/KDM6B function and overcomes genetic sex-determining with different gene expression at extreme temperatures.

Czerwinski et al. completed sequencing for the total RNA in T.scripta embryos at MPT and FPT before gonad development and during gonad development. This allowed authors to classify male- and female-specific gene expression during sex development.

The authors previously found six genes that were enriched in T.scripta embryos at MPT compared to FPT.

Here, authors tested if KDM6B might affect the enrichment of these genes at MPT.

Ge et al., shows that Dmrt1 expression is necessary for and a driver of T.scripta temperature-dependent male sex determination.

In 2020, this laboratory published a follow-up study showing that the signal transducer and activator of transcription 3, better known as STAT3, responds to temperature and represses Kdm6b at warmer FPT.

You can read more about this exciting new study here: https://www.science.org/doi/epdf/10.1126/science.aaz4165

Chromatin immunoprecipitation, or ChIP, identifies the DNA regions were a protein binds across the genome.

First, an antibody recognizes and binds a protein of interest in the nucleus. Then the antibody holding onto the protein is isolated and any DNA bound the protein is captured for analyses.

This article describes ChIP and it's application, with some example data analysis, here: https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/gene-expression-and-silencing/chromatin-immunoprecipitation-chip

A common technique to visualize nucleotide (DNA or RNA) in cells.

A chemical or radioactive label is added to a nucleotide sequence that is complimentary to the sequence of interest. When added to the cells, this complimentary nucleotide sequence will bind to and tag the sequence of interest, allowing scientists to visualize the DNA or RNA of interest within the cell.

You can read more about in situ hybridization methods here: https://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-327/

A widely-used technique used to image proteins on or within cells. It uses antibodies to bind and tag a protein of interest.

The first antibody specifically binds the protein of interest, for example KDM6B. Then, a second antibody carrying a fluorescent dye attaches to the first antibody.

These proteins are visualized using a microscope with lasers. The lasers excite the fluorophore, which in turn emits specific light waves. Here, the KDM6B is marked by the emission of light waves in the green spectrum and beta catenin produces waves in the red spectrum.

Here is a brief introduction to immunofluorescence techniques: https://oni.bio/nanoimager/super-resolution-microscopy/immunofluorescence/

KDM6B's role in turtle temperature-dependent sex development and implications for hatchling sex bias and mortality were discussed in this BBC News article: https://www.bbc.com/news/science-environment-44071220

When Kdm6b was lost, histone methylation was higher at the Dmrt1 locus than the control at MPT. This indicates that H3K27me3 may mediate Dmrt1 expression at MPT.

H3K27me3, which is associated with silenced transcription, was more abundant at the Dmrt1 promoter at FPT compared to MPT gonadal cells.

Authors saw that KDM6B was no longer binding to the activating sites for Dmrt1 in developing Kdm6b-knockdown gonadal cells at MPT. The reduction almost brought KDM6B association with the Dmrt1 promoter to that observed in gonads at FPT.

KDM6B protein occupies the key transcriptional activation site at DMRT1 in gonads at MPT compared to FPT.

Quantitative polymerase chain reaction (qPCR) measures RNA or DNA levels for comparison among samples.

This technique is very similar to qRT-PCR (described in above annotations), but does not always require reverse transcription.

KDM6B has been shown to initiate demethylation at H3K27 in various models and systems. This demethylation is closely associated with activation of genes located near the sites of demethylation.

Depletion of Kdm6b, which encodes a histone lysine demethylation enzyme, causes overall H3K27 trimethylation levels to increase at MPT.

Cell imaging shows that the histone tail modification, H3K27 trimethylation, is specifically enriched in gonads at FPT over that of MPT.

The authors demonstrate that Dmrt1 expression relies on Kdm6b function in male gonad development, and that Kdm6b targets Dmrt1 to activate male gonad development at MPT.

The Kdm6b-depleted + Dmrt1 overexpression gonads demonstrated primarily male germ cell composition and structure at MPT.

Dmrt1 overexpression was able to restore presence of male sex development marker SOX9 in Kdm6b-depleted gonad germ cells.

Conversely, Dmrt1 overexpression also reduced the presence of female sex fate genes relative to controls.

When authors increased Dmrt1 in the Kdm6b-depleted embryos at MPT, embryos showed male gonad development.

In molecular biology, this is known as a "rescue" when addition of a molecule can correct a disrupted system.

Kdm6b is required for the expression of Dmrt1 and is active just before Dmrt1 in MPT development. Thus, authors reason that KDM6B could be required for the activation and function of DMRT1.

Authors observed that during the peak stages of gonad development, Kdm6b-deficient embryos at MPT also had lowered Dmrt1 RNA and protein levels.

In a previous paper, these authors knocked down Dmrt1 like they do for Kdm6b in this study.

They observed several female gonad features for embryos in MPT that lacked Dmrt1. When they enriched Dmrt1 in embryos developing at FPT, they saw masculinization.

This indicates that Dmrt1 regulates male gonad development, similarly to how the authors characterize Kdm6b in this paper.

This lead authors to test if Kdm6b regulates Dmrt1.

Dmrt1 is another gene enriched in embryos at MPT. The authors show here that Dmrt1 expression is activated right after Kdm6b at MPT.

The authors are hoping to reveal the molecular biology underlying Kdm6b-depletion sex reversal. They will test for the genes that KDM6B can directly bind and activate expression for in developing T.scripta gonads.

The knock-down of Kdm6b caused female gonad development at MPT, which shows that Kdm6b is required for proper temperature-dependent male gonad development.

The gonads lacking Kdm6b at MPT resembled the structure and organization of gonads at FPT.

Ectopic describes events occurring in locations that do not naturally have such events.

For example, expression of a brain-specific gene in a toenail would be considered ectopic gene expression.

Here ectopic describes the presence of aromatase in Kdm6b-depleted gonads at MPT, which produces female sex hormones.

The male sex fate gene SOX9 was depleted in gonads lacking Kdm6b at MPT.

The authors checked gene expression of male and female sex fate markers in gonads after sex determination across treatment conditions.

Next, authors wanted to see if the male-to-female sex determination shift could be detected by gene expression of additional important sex-related genes.

Embryos incubated at MPT treated with the Kdm6b shRNA developed gonads resembling females, with long and thin structure as opposed to the compact and round male gonads of the control.

It is important to have normal or "wild-type" development processes for comparison to the experimental treatment conditions.

The authors confirmed here that the control MPT and FPT embryos treated with the scrambled virus develop as expected.

A mutation is a change to the structure or sequence of a gene compared to a reference.

Loss of function mutations cause a gene not to make functional amounts or forms of its protein.

Here, the shRNA is blocking Kdm6b expression, meaning that there is not enough KDM6B to function normally in the Kdm6b-RNAi embryos.

In 2017, authors in this laboratory optimized a technique to inhibit gene expression in T. scripta (turtle) embryos.

Short hairpin RNAs (shRNAs) target complementary messenger RNA molecules for degradation, which blocks the target RNA from being able to make protein. The authors use a virus, called lentivirus, to carry and integrate RNA into the cells. This introduces the shRNA into the embryo while it is still in the egg (in ovo).

A master regulator often refers to a protein that initiates the cascade of expression for all genes involved in a specific pathway, such as cell fate and development pathways.

The specific abundance of Kdm6b expression in developing gonads at MPT, FPT-to-MPT temperatures, and hormone treatment show that KDM6B likely functions in temperature-dependent sex determination.

Kdm6b gene expression responded to temperature change and was also sensitive to sex hormone treatment.

The FPT gonads that were treated with a female sex hormone inhibitor showed similar Kdm6b expression to the male temperature gonads.

Authors checked the response of Kdm6b gene expression to changes in temperature in the gonads during sex development.

In the MPT and FPT-to-MPT gonads, authors observed high levels of Kdm6b expression, whereas the FPT and MPT-to-FPT incubated gonads maintained low Kdm6b expression.

Now that they have seen that KDM6B is specifically abundant in gonads at MPT, the authors will test if changing temperature or sex hormones will change Kdm6b expression.

The authors only detected KDM6B in the developing cells in the gonads at MPT, so they conclude that KDM6B may function in sex development.

Germ cells are an organism's reproductive cells, or the cells that go on to make gametes, like sperm and eggs.

Every organism is comprised of somatic cells and germ cells.

Seminiferous cords are one of the earliest male-specific tissues formed as the gonad develops. These cords ultimately develop into tubules which hold sperm.

The authors originally sequenced the total RNA in turtle embryos at male- and female-producing temperatures (MPT and FPT) to identify differences in transcripts that may contribute to sex development.

Kdm6b was one of six transcripts that was consistently higher at MPT compared to FPT. Therefore, the authors decided to disrupt KDM6B in turtle embryos to test if it plays a role in sex development.

KDM6B (the protein) activates genes critical for early organism development, such as gonad specification and body patterning.

Expression refers to the active process of making a protein from a gene.

H3K27me3 is a histone mark that recruits repressive factors to that region of DNA, which leads to lower expression of nearby genes.

Methylation, or the addition of methyl groups to cytosines on DNA often leads to silencing of transcription of nearby genes.

Female-producing temperatures (FPT) specifically lead to removal of methylation and the addition of transcription-associated histone tail marks at the promoter for a gene that is critical to estrogen production during female sex determination. These marks were shown across reptiles, including turtles, alligators, and sea bass.

Epigenetic describes heritable changes in gene expression, or transcription, that do not alter an organism's DNA sequence.

The region of DNA that is required for transcription initiation.

Gonad refers the organ that produces an organism's reproductive cells.

The gonad is the testis in males and is the ovary in females.

A phenotype describes the physical properties of an organism that can be observed.

A genotype defines the genetic composition of an organism, including chromosomes and DNA sequences.

Together, phenotypic plasticity is the ability of one genotype to produce multiple different phenotypes in an organism.

The authors want to know how Kdm6b expression changes based on temperature specifically in the gonads instead of all developing tissues.

It is important to note that the Kdm6b pathway is likely used by many reptiles to regulate temperature-dependent sex determination, but that the way each species has evolved to use it might be different.

Similar intron retention was found in turtles and alligators, which again points to a role for KDM6B transcription in temperature-dependent sex determination across reptiles.

Another study on the Australian central bearded dragon showed that different introns in Kdm6b transcripts at high temperatures was sufficient to reverse the genetic sex determination.

KDM6B is required for Dmrt1 activation and specifically associates with the promoter of Dmrt1.

This shows that KDM6B orchestrates male sex development early in gonadal cells by directly removing H3K27me3 at the Dmrt1 promoter to activate Dmrt1 expression.

To see if this H3K27me signature was specific to Dmrt1, the authors also checked other sex-related genes that are expressed around the same time.

Now that authors have seen that Kdm6b regulates Dmrt1 activation, they are interested in how the molecules interact in the male sex development pathway.

The opposite of knock-down or RNAi, overexpression describes a technique were an RNA transcript for a particular gene is increased above it's normal biological level.

Analysis of the sex fate gene expression showed that male markers were significantly decreased, whereas female markers were significantly increased in Kdm6b-RNAi treated gonads compared to controls.

Kdm6b-depleted gonads showed marker expression similar to the female control.

A robust ratio of the Kdm6b-depleted embryos demonstrated a shift from male to female development, despite incubation temperature. This structural data suggests that KDM6B plays an important role in sex determination.

Aromatase is an enzyme that produces estrogen, the main female sex hormone.

Differences in characteristics between males and females of the same species other than the sex cells.

Medullary describes the inside, and cords refer to the early structures that will become an organism's gonads.

A critical step in sex development of males is when medullary cord cells differentiate into Sertoli cells. If the cords degenerate, the sex cords will instead develop into an ovary - the female gonad.

Morphology is the study of the structure of an organism. This often includes observations about the size or shape of an organism.

Here the authors describe the Kdm6b-deficient embryo gonads as having a structure that looks like a developing female.

Catalysis is the initiation and acceleration of a chemical reaction.

In this case, the authors point to KDM6B as the catalyst for Dmrt1 expression and thus male sex development.

Male sex cells that are required to form testes and sperm.

Progenitor sex cells that go on to make all the reproductive cells in an organism.

All cells in an organism other than the reproductive cells.