Genetic Developmental Timing Revealed by Inter-Species Transplantations in Fish

Jana Franziska Fuhrmann, Lorena Buono, Juan Ramón Martinez Morales, Lázaro Centanin

Preprint posted on 2 April 2020 https://www.biorxiv.org/content/10.1101/2020.04.02.019471v1

Article now published in Development at http://dx.doi.org/10.1242/dev.192500

Take your time, it’s in your genes. Inter-species blastomere transplantations in fish reveal intrinsic and extrinsic developmental timing.

Selected by Martin Estermann

Categories: cell biology, developmental biology, evolutionary biology, genetics

Background:

Every developmental process consists of a series of synchronized events involving temporal and spatial regulation. Retinas are formed in an autonomous temporal order, showing remarkable self-organizing properties. During embryonic development, retinal progenitor cells differentiate into the neural retina and the retinal pigmented epithelium. In contrast, the lens of the eye is derived from another structure, the surface ectoderm, which is induced by retinal progenitors during retinogenesis.

Different organisms take different times to develop the same organ, directly related to their distinctive developmental timeframes. Among the teleosts, zebrafish (Danio rerio) and medaka (Oryzias latipes) belong to the most evolutionary distant subgroups. This is reflected in the time that they take to complete embryonic development, 3 and 9 days respectively. Consistent with the developmental time difference, retinal cells differentiate earlier in zebrafish (26 hours post fertilisation) than medaka (50 hours post fertilisation).

Although trans-species transplantations have been widely used in the developmental biology field, host and donor organisms tend to share not only morphological and developmental processes but also a similar timing. Through zebrafish-medaka inter-species blastoderm transplantations, this preprint provides evidence for ectopic retina formation following the intrinsic donor cells’ developmental timing, resulting in a temporal decoupling in embryonic development.

Key findings

1) Inter-species blastomere transplantation induces the formation of a retina-like organ.

Same species blastomere transplantation at blastula stage resulted in a mix of host and donor cells along the developing embryo. In the case of inter-species transplantations, the authors found that the donor cells stayed clustered together and did not mix during gastrulation. Two types of inter-species transplantations were made: transplanting blastomeres from a zebrafish to a medaka (zebraka, Fig. 1B) or the transplantation of medaka blastomeres in a zebrafish (medrafish, Fig. 1D). In both cases, the donor cells formed an ectopic organ that resembles a pigmented retina.

Fig. 1. Inter-species blastomere transplantation generated a pigmented retinal like organ. (B) Transplantation of zebrafish blastomeres in a medaka (zebraka). (D) Transplantation of medaka blastomeres in zebrafish (medrafish). (Preprint Fig. 2B and D).

2) RNA-sequencing confirmes the identity of the ectopic retina.

Taking into account the low sequence identity of homologous genes between zebrafish and medaka, the authors performed RNA-sequencing in medaka, zebrafish and zebraka 48hpf embryos. At this time, zebrafish embryos have already started neurogenesis, whereas it’s only about to start in medaka.

As expected, the zebrafish transcriptome aligned to the zebrafish genome, but not the medaka transcriptome. In contrast, only a few medaka transcripts mapped to the zebrafish genome. The zebraka transcriptome consisted of a full medaka transcriptome and a partial zebrafish transcriptome, exemplified by zebrafish reads (peaks) mapping to retinal genes, such as vsx2 and rx1 (Fig. 2 left). This indicates that the zebrafish transplanted cells in medaka embryos continue with their developmental zebrafish timing and are not slowed down by the medaka inner developmental rhythm. Cardiac genes were shown as a negative zebrafish control (Fig. 2 Right).

Fig. 2. Inter-specie blastomeres developed into an ectopic retina. Transcriptomes of medaka (upper), zebrafish (middle) and zebraka (bottom) were plotted along the zebrafish genome. The zebrafish cells in medrafish displayed retinal identity (left). Cardiac genes were shown as a negative zebrafish control (right). Lens zebrafish genes (middle) were expressed in zebrafish but not in zebrakas. (Preprint Fig. 2E and 4A).

3) Different sources for lens recruitment in zebraka and medrafish.

The vertebrate eye is composed of the neural retina and retinal pigmented epithelium, which are neuroepithelial derivatives, and the lens that derives from the surface ectoderm. Surprisingly, although retinal genes were identified, zebrafish lens transcripts were absent in the zebraka chimeras, suggesting other origins (Fig. 2, middle). If the donor is not providing the lens progenitors, is the host providing them?

To test this, zebrafish blastomeres were transplanted in a transgenic medaka expressing a reporter (CFP) under the control of the promoter of the lens specific gene cryA. The ectopic organ formed contained zebrafish (donor) retina, but a medaka (host) lens, confirming that the host is providing the lens precursors (Fig. 3B). In this case, the surface ectoderm had the potential to become a lens before the stage that is normal in medaka and demonstrated that the “zebrafish” retina induces early ectopic lens formation in the host, following zebrafish internal timing.

Surprisingly, in the medrafish (medaka donor, zebrafish host), the ectopic organ formed consisted of a medaka retina and lens (Fig. 3C). This suggests that by the time medaka retina started the lens induction program, the surface ectoderm in zebrafish was no longer competent to acquire lens identity.

Fig. 3. Different sources for lens recruitment in zebraka and medrafish. (B) Fluorescent image of a zebraka showing the recruitment of an endogenous lens (host is medaka crya:ECFP) by an ectopic zebrafish retina. (C) Fluorescent image of a medrafish that showing the ectopic medaka retina (EGFP+) contains also a medaka lens (donor is medaka crya:ECFP). (Preprint Fig. 4B-C).

Why I choose this paper:

Working in chicken as an animal model I am well aware about the transplantation experiments involving quail and chicken, especially when used to study neural crest cells migration or the gonadal development. Chicken and quail were typically used because of their developmental and timing similarity. This preprint takes a step back and uses two iconic teleost models, zebrafish and medaka, with remarkable different developmental timepoints: 3dfp and 9dpf until hatching, respectively. This research showed that the newly transplanted cells keep their developmental timing and are not susceptible to the host developmental rhythm. Interestingly, the research also showed that donor cells can influence and accelerate the development and differentiation of host cells. This preprint is the start to novel heterochronic inter-species experiments to understand how cell intrinsic timing is maintained and how changes in the internal developmental timing could affect the surrounding tissues. These results will reinforce the use of medaka and zebrafish as models, and at the same time will encourage the use of non-model organisms to uncover the timing of diverse developmental processes.

Future directions / questions for the authors:

When blastoderms were transplanted from one fish species to the other, a retinal like organ is generated. RNA-seq confirmed that those organs expressed retinal genes, but were the different retinal layers/cells present in those newly generated retinas? Was the retinal structure similar to the host, donor or both retinas?
The newly generated retinas in zebrakas were innervated, but were they functional? Can the zebraka respond to stimulus with the ectopic generated retina earlier than their medaka counterparts?
If they are functional, can they restore the vision in eyeless fish like in the case of pax6b mutants?
In medrafish, the surface ectoderm in zebrafish was no longer competent to acquire lens identity and the lens is formed from the donor cells. Where did those cells come from? Did the blastoderms first differentiate into ectoderm? Or retinal cells de-differentiated and re-differentiated into lens cells?

Tags: blastomeres, chimeras, development, differentiation, evolution, eye, medaka, retina, retinogenesis, time, transplant, zebrafish

Posted on: 6 May 2020 , updated on: 15 May 2020

doi: https://doi.org/10.1242/prelights.20114

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Author's response

Lazaro Centanin and Jana Franziska Fuhrmann shared

Q: When blastoderms were transplanted from one fish species to the other, a retinal like organ is generated. RNA-seq confirmed that those organs expressed retinal genes, but were the different retinal layers/cells present in those newly generated retinas? Was the retinal structure similar to the host, donor or both retinas?

A: This an important question as not only the onset of retinal differentiation but also the generation of each retinal cell type in the neural retina is tightly spatial-temporally coordinated. We focussed on the expression of neurogenic/differentiation markers that indicated the onset of retinal differentiation, as this is the earliest read out. As for the differentiation of retinal cell types, the presence of RGCs (the first neural cell type generated in the vertebrate retina) was confirmed by imaging and sequence analysis. We have observed transcripts corresponding to other neural cell types, but we did not confirm this by image analysis on zebrakas nor medrafish. It remains open, however, whether subsequent retinal differentiation follows the stereotypic sequence that is observed in an endogenous retina and if the structural layer organization resembles that of a wild type. We observe that the development of ectopic retinae is highly reproducible; the exact shape, form and even size can be variable, though, suggesting that there might also be variability in the spatial organization.

Q: The newly generated retinas in zebrakas were innervated, but were they functional? Can the zebraka respond to stimulus with the ectopic generated retina earlier than their medaka counterparts? If they are functional, can they restore the vision in eyeless fish like in the case of pax6b mutants?

A: The presence of RGC’s axons (presumably an optic nerve) that travel towards the host optic tecti suggests that the ectopic retina could potentially signal to the host visual circuit. The acquisition of visual information, however, requires the presence of additional cell types whose existence in the ectopic retinae we have not investigated. The experiment suggested here is certainly very interesting, but it assumes that the ectopic retina can be formed in chimeras that lack the endogenous eye. We have no indications that this is indeed the case. The suggested experiment would also provide information on permissive or even instructive cues from the surrounding host cells, which is a topic we are very interested in.

Q: In medrafish, the surface ectoderm in zebrafish was no longer competent to acquire lens identity and the lens is formed from the donor cells. Where did those cells come from? Did the blastoderms first differentiate into ectoderm? Or retinal cells de-differentiated and re-differentiated into lens cells?

A: This is a key question as well, since it deals with the possibility of alternative developmental trajectories to generate a lens. There is ample literature revealing that a lens can indeed be induced form the neural retina in newts, although only under regeneration paradigms. A scenario that appears more likely than a trans-differentiation behavior into another developmental lineage is that a subset of the donor blastomeres either acquired surface ectodermal fate or remained largely undifferentiated. We have indeed observed that donor blastomeres can differentiate into non-retinal cell types like vasculature and pigment cells, so the latter hypothesis seems more plausible in our view. But we are looking forward learning which one is the case!

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Genetic Developmental Timing Revealed by Inter-Species Transplantations in Fish

Fig. 1. Inter-species blastomere transplantation generated a pigmented retinal like organ. (B) Transplantation of zebrafish blastomeres in a medaka (zebraka). (D) Transplantation of medaka blastomeres in zebrafish (medrafish). (Preprint Fig. 2B and D).

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