Modular control of time and space during vertebrate axis segmentation

Ali Seleit, Ian Brettell, Tomas Fitzgerald, Carina Vibe, Felix Loosli, Joachim Wittbrodt, Kiyoshi Naruse, Ewan Birney, Alexander Aulehla

Posted on: 24 June 2024

Preprint posted on 31 August 2023

Article now published in The EMBO Journal at http://dx.doi.org/10.1038/s44318-024-00186-2

and

Natural genetic variation quantitatively regulates heart rate and dimension

Jakob Gierten, Bettina Welz, Tomas Fitzgerald, Thomas Thumberger, Oliver Hummel, Adrien Leger, Philipp Weber, David Hassel, Norbert Hübner, Ewan Birney, Joachim Wittbrodt

Posted on:

Preprint posted on 2 November 2023

F2 segregation analysis: the ‘Formula 1 grand prix' of experiments to establish genotype-phenotype correlations

Selected by Girish Kale, Jennifer Ann Black

Categories: clinical trials, developmental biology, evolutionary biology, genomics, physiology, systems biology

Background

As humans, we know that most of our genome is conserved, and yet it is quite obvious that we are not all the same: we don’t look the same, we don’t think the same way, and this individuality is something we cherish. Since our genome is almost completely conserved, we need to focus on the subtle differences that form the basis of our individuality. Trying to understand these genomic differences, and linking that understanding to the differences in individual characteristics and susceptibility is an active area of research.

The subtle differences in our genomes are heritable. Again, this is a piece of common knowledge, as looking at our parents or grandparents we can tell where our characteristics come from. Of course, this also extends to various diseases: often doctors ask for a history of certain illnesses in our family. However, such correlations are only limited, as many subtle mutations in our genomes often produce a unique combined output, forming the basis of our individual differences. Linking any subtle mutation to a characteristic or disease susceptibility is really challenging. To tackle this, scientists can exploit high-resolution whole-genome sequencing to pick up even the most subtle mutations, and combine it with unique experimental systems where these mutations in the genome can be easily tracked over generations. The two preprints highlighted here share a unique experimental system where this approach is applied: the inbred lines of Japanese rice fish, also called medaka.

Inbred lines are an interesting resource for the scientific community and exist for other model organisms as well, such as mice and fruit flies. Essentially, an inbred line has individuals with extremely similar genomes: even the most subtle mutations are the same. As a result, they all have the same features and characteristics, including their susceptibility to various diseases, almost as if they are all clones of each other. If the individuals from such an inbred line are mated with others from a different inbred line, then in the next generation (called F1), we will see a difference in characteristics and susceptibilities due to a mixture of the parental genomes (the parents are called F0). In the F1 generation, meiotic recombination shuffles the genomes, and when these individuals mate with each other, the following generation (called F2) will include individuals with genomes that randomly segregate the subtle mutations present in the F0 inbred lines. Now the effects of various subtle mutations in the genome can be teased apart using whole genome sequencing and correlated with quantifications of various characteristics and susceptibilities.

The two preprints highlighted here use this same technique to address two completely different questions. One study tries to identify genes involved in the timing and size of segment formation during embryo development, while the other tries to identify genes leading to heart defects. Both of these studies used medaka species due to various additional experimental advantages including, the possibility to have genetic cross-species hybrids (besides in-bred lines), their small genome size and genetic tractability, and ease of in-vivo imaging during embryo development.

The preprint on Segment formation

Background

Segment formation, or segmentation, is an important process during embryo development. The process is regulated by hundreds of genes. Here, individual somites, the precursors of segments, are derived from pre-somitic mesoderm, and are added one after the other. The number of somites, the initial size of somites and the rate of somite addition is specific to a species. As we can imagine, the number, size, and rate of somite addition will depend on each other: fewer somites will require less time to form, larger somites will need more time to form, etc. Due to such interdependence, it is hard to tease apart how a certain gene influences somitogenesis. In their manuscript, Seleit and colleagues use inbred lines from 2 closely-related medaka species Oryzias sakaizumii and Oryzias latipes to understand the spatial and temporal control of somite formation in these species.

Key Findings

In their experiments, the authors decided to exploit the spatial and temporal differences in somitogenesis between the medaka species. They imaged the process of somitogenesis in live embryos in both Oryzias sakaizumii and Oryzias latipes and found that Oryzias sakaizumii has smaller somites which are added faster than Oryzias latipes. After mating the individuals from these species, in the next F1 generation, the pace of somitogenesis was intermediate to the F0 parent. Then the authors quantified PSM, somite size and somitogenesis pace using quantitative imaging of endogenous her7-venus oscillations in-vivo in about 600 F2 individuals and obtained an expanded (and continuous) phenotypic space for all three traits. They found that while PSM size and somite size are corelated in the F2s (with the same slope as was present in the F0s!), the pace of somitogenesis was not correlated with either PSM or somite size in F2s. This data argues that a developmental constraint mechanism underlies the link between PSM and somite size, while there is modular control of timing and size. The authors used the same phenotyped individuals for whole-genome sequencing, to identify regions of the genome which correlated with somite size or somitogenesis pace. This allowed them to using devQTL mapping to identify novel candidate genes, which might be regulators of the tempo of the somitogenesis clock/ the size of the tissue giving rise to somites.

After identifying interesting candidate genes, the authors introduced CRISPR-Cas9-mediated mutations to functionally test the activity of these candidate genes and to check whether/how they affect somitogenesis. The authors could show that the candidates associated with somite size don’t affect the pace of somitogenesis and vice versa, clearly demonstrating the independence of their genetic regulation. This demonstrated, for the first time in vivo, that somite size and the pace of somitogenesis can be uncoupled.

What we liked about this preprint

This preprint really demonstrates the power of the use of inbred lines. The authors could clearly demonstrate that the regulation of spatial and temporal aspects of somitogenesis are independent, and their genetic basis can be uncoupled. The study also uses strong quantitative and statistical approaches to test their hypothesis, allowing them to make clear statements about how somitogenesis is spatially and temporally regulated. With their approach, the authors can further comment on regulation of time and space in embryonic development in general, as similar mechanisms are expected to also regulate the developmental tempo of a species, which varies similarly across species.

The preprint on Heart defects

Background

In this study, the authors use inbred Medaka fish to examine early cardiac changes in the embryo and how these impact upon adult fish. Specifically, they examined changes to the heart rate of these fish by examining defined features of their hearts and their genomes. They find similar changes to those found in studies performed in humans but additionally, report novel genes that affect heartbeat and could have implications for managing and detecting heart problems in humans.

Key Findings

Changes in embryo heart rate impact the health of the adult heart

The authors predominantly used two inbred Medaka lines for their studies: HdrR and HO5 (species: Oryzias latipes). By measuring the dynamics across development from the embryonic heart to adulthood, they revealed notable differences between the two inbred fish lines. These two fish lines represent two extremes; slowest heartbeat (HrdR) and fastest heartbeat (HO5). HdrR fish with the slow heart rate performed well in swimming tests, whereas HO5 fish, which have a fast heart rate and show signs of heart alterations early in development, swam poorly in comparison. Thus, the early heart changes affected both the fish’ heart performance and fitness when they developed into adults, with fast heart beat correlating with pathology.

59 loci in Medaka embryos correlated with alterations to heart rate

Next, the authors looked for changes in the genomes of these two inbred fish lines when environmental conditions changed. In this instance, they opted for water temperature, which coincides with higher heart rate when temperatures increase. They then crossed individuals from these two lines (HdrR X HO5) to generate a representative spectrum of heart changes, and examined the genomes of over 1000s individuals from this F2 generation. Overall, they identified 59 loci associated with heart rate changes. Within these 59 loci, they identified genes previously associated with pathogenic heart changes in humans.

To confirm their genome-wide studies and to ask if some of the genes identified played a role in heart disease development in these fish, they selected a representative cohort of previously known and novel genes and generated individual CRISPR/Cas9-modified Medaka fish. They show that inactivating genes within this cohort resulted in changes to fish’ heartbeats.

What we liked about this preprint

This preprint, like the other one discussed earlier, shows the power of using inbred (fish) lines. In this specific case, to identify complex pathologies that can be translated to human health. We found it interesting that the authors identified a cohort of genes that previously were not linked to changes in human heart health. Future studies will hopefully reveal if they contribute to human pathologies.

Questions to the authors

specifically for Segment formation manuscript

1) The segmentation and axis elongation process is supposed to be highly robust, as it occurs during the phylotypic stage of embryonic development. How is it that you can see so much variability in the F2? wouldn’t you have expected the “dominant” species to drive the process?

2) You have reported differences in the size and timing of segmentation. Do you think the dataset also includes genes that have changed the onset of when the segments begin to form during the time course of embryonic development?

specifically for Heart defects manuscript

1) Do you think the higher BMI observed in Ho5 is a cause or a consequence of hypoplastic ventricles and faster heart rate?

2) Galectins are promiscuous – how easy is it to target them in terms of translating your findings into the clinic?

for both

1) How important is it to see that the phenotypic spread in the F2 goes beyond the F0 parental extremes? How would the interpretation of the data change if the F2 extremes are the same as, or within, the F0 extremes?

2) How easy is it to generate CRISPR-modified medaka?

3) Why use fish for these models? What about mice or fruit flies, which also have inbred lines?

4) We (the preLighters) assume that all of you (the authors) believe in open science. Are there plans to create a searchable database of the candidate genes, as a resource for the scientific community?

5)What are the challenges of crossing these organisms – what’s the success rate? how does it affect their fitness overall?

doi: https://doi.org/10.1242/prelights.37723

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Author's response

Ali Seleit shared about Modular control of time and space during vertebrate axis segmentation

specifically for Segment formation manuscript

1) Indeed the developmental hourglass model posits that while early development is quite variable among vertebrate species (most likely due to differential reproductive strategies/ecological niches), this diversity gets funneled into a developmental bottleneck known as the phylotypic stage of embryonic development. During this stage two processes are tightly spatially and temporal controlled. The first is the segmentation clock that builds the body axis by sequentially forming somites and the second is the Hox timer which patterns and regionalizes the body plan laid down by the process of somitogenesis. As embryos exit this bottle-neck they start showing more diversity in forms and shapes.

As conserved and tightly regulated as this developmental bottleneck is, it is interesting to note that different vertebrate species have vastly different tempos of the segmentation clock and PSM tissue/somite size. Yet within a species timing and size are tightly controlled. This suggests that the differences are likely genetic and heritable. It therefore comes as no surprise to see the expanded phenotypic space of both timing and size in our F2 cross as we introduce quite a strong shuffling of the genetic variants, and since the F0s differed in both traits we expected to see an expanded variation in the F2s as well.

2) This is a very interesting question, I think that it could in theory be possible to change to onset of somitogenesis as compared to other developmental processes, a sort of induced heterochrony if you will. I however do not think our data-set includes any of those putative regulators since we always measured the pace of the clock already at the 10-11^th somite stage. In theory what you are proposing could happen and would involve a temporal decoupling or delay at the level of the process as a whole, compared to other developmental processes. But since somitogenesis is so central to the developmental progression of an embryo I think it will rather be hard to completely delink it from other developmental processes.

for both

1) The phenomenon of the F2 phenotypic spread exceeding the F0 parental extremes is known as transgressive segregation and it is quite common for many traits (though not by any means ubiquitous) in F2 crosses, especially in plants. So I would not say it is important to see that, it is certainly trait-specific. Some traits will likely show evidence of transgressive segregation in an F2 cross and some others will not.

2) CRISPR/Cas9 knock-outs in medaka are quite easy to generate and F0 KOs have been shown to replicate phenotypes in stable mutant lines (as has been shown in zebrafish). CRISPR/Cas9 Knock-ins are also relatively straight-forward to generate in medaka, with a slew of reports continually improving efficiency and accuracy of KIs. Base-editing is also possible in medaka, what is definitely laborious and still a technical challenge is large-scale multiplexed base-editing.

3) The ability to genetically hybridize these fish and the short generation time of fish as compared to mice offers a real advantage. Additionally, more embryos can be obtained from an F2 fish cross in a shorter amount of time facilitating high-throughput screens. Being a vertebrate model is an advantage in fish as compared to fruit flies. Lastly, the genome of medaka fish is quite compact in comparison to other vertebrates (and even to other fish) with an approximate size of 800Mbs only which makes mapping easier. That said each system has its benefits and drawbacks, and both mice and fruit fly inbred lines have been invaluable in unraveling the genetic basis of complex traits.

4) We certainly share the enthusiasm and support of open science and have sent our work to Review Commons for a transparent, open and fair review process. The paper will also be open access once published. And all candidate genes will be provided as supplementary material.

5) The north/south crosses are fairly simple and do not require any additional steps. Interestingly I had the feeling that adult north/south F1 hybrids had an even higher fecundity than the F0 parents, but I did not quantify this, so do not quote me on it. In general, though there was no apparent reduction in overall fitness or fecundity in F1 hybrids. An interesting and open question is how does the fecundity and overall fitness of F2 adults look like? Unfortunately for logistical reasons we could not grow all 600 F2 embryos to adulthood but I think that would be a very interesting experiment indeed.

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Modular control of time and space during vertebrate axis segmentation

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Background

The preprint on Segment formation

The preprint on Heart defects

Questions to the authors

Share this:

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