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Deterministic and probabilistic fate decisions co-exist in a single retinal lineage

Elisa Nerli, Jenny Kretzschmar, Tommaso Bianucci, Mauricio Rocha-Martins, Christoph Zechner, Caren Norden

Preprint posted on 11 August 2022 https://www.biorxiv.org/content/10.1101/2022.08.11.503564v1

To be or not to be a photoreceptor: cell fate choices in the developing zebrafish retina are deterministic and probabilistic.

Selected by Laura Celotto

WHY I CHOSE THIS PREPRINT

Many studies in developmental biology have investigated the development of the vertebrate retina in zebrafish, thanks to the transparency of the larvae, which are suitable to live imaging microscopy. However, this preprint by Nerli and colleagues represents the first study that combines in vivo imaging of the developing retina with in silico simulations to explain cell fate choices of retinal progenitor cells (RPCs) that express the transcription factor Atoh7. I selected this preprint because I enjoyed the accurate characterization of fate commitment at a single cell level, a novelty in the study of retinogenesis, since previous works have dissected fate choices only at RPC population or clonal level. Moreover, there is a plot-twist in the story: Atoh7-RPCs have the potential to generate more neuronal types than previously thought… Do you want to know more? Keep reading then…

KEY FINDINGS

Atoh7-RPCs generate a photoreceptor and a second cell with variable identity across time

The authors used a transgenic fish line expressing a fluorescent reporter under the control of atoh7, one of the earliest neurogenic markers during retinogenesis. In this way, they could track cell divisions of single Atoh7-positive RPCs (Atoh7-RPCs) using live imaging of zebrafish embryos between 28 hours post-fertilization (hpf, the beginning of neurogenesis), and 60 hpf. They found that Atoh7-RPCs divided and generated two daughter cells with different fates. One daughter cell was always a precursor of photoreceptors, the light-sensing cells of the retina. Photoreceptor precursors further divided symmetrically to produce two photoreceptors. The second daughter of the Atoh7-RPCs, instead, could be a retinal ganglion cell, which is the output neuron of the retina, or a horizontal or an amacrine cell, the main inhibitory neurons in the retina. The probability that the Atoh7-RPCs would produce a retinal ganglion cell rather than an inhibitory neuron changed across time in neurogenesis. Indeed, RPCs during early (28-42 hpf) neurogenesis generated a photoreceptor precursor and most likely a retinal ganglion cell. Conversely, RPCs during late (36-60 hpf) neurogenesis generated a photoreceptor precursor and most likely an inhibitory neuron. Hence, Atoh7 RPCs differentiate along two branches: a deterministic branch that always produces photoreceptors, and a probabilistic branch that generates different cell types (a retinal ganglion cell or an inhibitory neuron) with different probabilities across time.

Faithful photoreceptor genesis is necessary for correct lamination of the retina

Next, the authors perturbed the outcome of Atoh7-RPC divisions by using morpholinos (short, antisense oligonucleotides) to knock down the expression of distinct fate determinants of the deterministic and probabilistic branches. Knocking down of Prdm1a, which is a transcription factor necessary for photoreceptor determination, impaired the deterministic branch of the Atoh7 lineage, so that photoreceptors were never produced at any time in retinogenesis. Indeed, interfering with the deterministic branch of the Atoh7-RPC division resulted in the production of a cell that initially looked like a photoreceptor precursor, but never divided further, nor differentiated to photoreceptors. Moreover, severe impairment of retina lamination occurred, with reduction of the photoreceptor layer and of the retinal ganglion cell layer. Conversely, perturbation of the probabilistic branch resulted in changed probabilities of the retinal neurons produced by Atoh7-RPC divisions, without affecting retinal lamination. Indeed, knock down of Atoh7 depleted retinal ganglion cells, but increased genesis of inhibitory neurons as well as of photoreceptors, with no significant effect on the overall thickness of the retina. Moreover, knock down of Ptf1a, a transcription factor necessary for the development of inhibitory neurons, resulted in decreased genesis of horizontal and amacrine cells, but increased production of retinal ganglion cells as well as of photoreceptors (Figure 1).

So, what do you think would happen if both Atoh7 and Ptf1a were knocked down?

Plot twist: Atoh7 RPCs have the potential to generate all retinal neurons, even bipolar cells!

Previous work1 and the current preprint show that bipolar cells, the last-born neurons in the retina, usually come from RPCs that never express Atoh7. Indeed, right before neurogenesis, a RPC divides to generate an Atoh7-positive lineage, including the probabilistic and deterministic branches, and an Atoh7-negative lineage that generates precursors of bipolar cells expressing the transcription factor Vsx1. However, the present study shows that knock down of Atoh7 and Ptf1a proteins in Atoh7-RPCs produced photoreceptor precursors as well as bipolar cells throughout the neurogenic window (Figure 1). If Atoh7-RPCs were not multipotent, they would never produce any bipolar cells upon Atoh7 and Ptf1a depletion. Hence, the authors concluded that Atoh7-RPCs potentially can generate all retinal neurons, including bipolar cells, but are eventually competent to produce only retinal ganglion, horizontal, amacrine and photoreceptor cells.


Figure 1. Interference with the probabilistic branch of Atoh7-RPCs. In control retinae, Atoh7-RPCs always generated a photoreceptor precursor (PRpr, deterministic branch) and another cell that could be either a retinal ganglion cell or an inhibitory neuron (IN) (probabilistic branch). In the morphant retina, where Atoh7 had been knocked down (Atoh7 MO), PRpr and IN production increased as compared to control during early and late neurogenesis. In Ptf1a MO retinae, RGC and PRpr genesis increased as compared to control during early and late neurogenesis. In the Atoh7+Ptf1a double MO retinae, PRpr genesis increased and BC production appeared. Figure modified from the original preprint.

A simple network of factors governs RPC fate choices

Finally, the authors developed a computational model to investigate what type of gene regulatory network involving Atoh7, Ptf1a, Prdm1a and Vsx1 would be sufficient to predict cell fate choices of Atoh7-RPCs (Figure 2). They tested two possible in silico scenarios. In the first one, it was assumed that a single transcription factor was enough to effectively inhibit the expression of a downstream factor, and, consequently, the related fate acquisition. The fate outcomes predicted by this first model, however, did not match the observed outcomes of cell divisions: indeed, the first simulation predicted the genesis of bipolar cells even in the single knock down for Atoh7 or Ptf1a, which was never detected by live imaging experiments. In other words, if it were true that, for instance, Atoh7 alone would effectively inhibit Vsx1, then we would expect bipolar cell production upon Atoh7-single knock down (Figure 2, scenario A). This simulation did not match the live imaged fate outcomes observed in the retinae of the single knock downs, nor the neuronal proportions observed in wild type embryos. In the second scenario, it was assumed that a combination of transcription factors have to exert a summed inhibition on a downstream factor to prevent the acquisition of a certain fate (Figure 2, scenario B). For instance, Atoh7 and Ptf1a were predicted to sum their inhibitions against the downstream factor Vsx1, preventing the bipolar cell fate acquisition by the Atoh7-RPCs. Indeed, it was necessary to experimentally knock down both Atoh7 and Ptf1a to observe bipolar cell genesis, while neither the Atoh7-single knock down, nor the Ptf1a-single knock down were enough to generate bipolar cells in the Atoh7-RPC, probabilistic branch (compare Figure 1 ad Figure 2, scenario B).

Figure 2. A summed inhibition is required to prevent the acquisition of a certain cell fate. In the first simulations scenario (scenario A), each transcription factor alone is able to effectively inhibit a downstream transcription factor and the acquisition of the cell fate specified by the inhibited factor. This model predicts bipolar cell (BC) production in the Atoh7-single morphant (MO) retinae, an outcome never observed with live imaging experiments (see Figure 1). In the second simulations scenario (scenario B), the effects of inhibition from more than one transcription factor are summed. This model predicts increased probability of photoreceptor (PR) and inhibitory neurons (IN) production in the Atoh7-single MO retinae, of photoreceptor and retinal ganglion cell (RGC) production in the Ptf1a-single MO retinae and of photoreceptor and bipolar cell production in the Atoh7+Ptf1a-double MO retinae. The predictions made by the simulation scenario B match the observed outcomes of live imaging experiments. Figure modified from the original preprint.

QUESTIONS FOR THE AUTHORS

1. You use morpholinos to prevent the expression of transcription factors during retinogenesis, but how specific is the action of morpholinos? Did you consider the potential off-target effects of these morpholinos? Did you think of using a conditional knock out approach to answer your questions?

2. Do you have a hypothesis about the molecular signal that starts the Atoh7 lineage? That is, did you obtain any indication about what molecular cue triggers the “firing” of Atoh7 in RPCs?

3. Did you further characterize the “aberrant” photoreceptor precursor upon Prdm1a knock down, for instance using immunohistochemistry or other approaches?

4. How do you explain the thickness reduction of the ganglion cell layer upon Prdm1a knock down?

REFERENCES
1. Vitorino, M., Jusuf, P.R., Maurus, D., Kimura, Y., Higashijima, S., and Harris, W.A. (2009). Vsx2 in the zebrafish retina: restricted lineages through derepression. Neural Develop. 4, 14. 10.1186/1749-8104-4-14.

Tags: development, live-imaging, retina, zebrafish

Posted on: 15 November 2022

doi: https://doi.org/10.1242/prelights.33096

Read preprint (No Ratings Yet)

Author's response

Elisa Nerli shared

  1. You use morpholinos to prevent the expression of transcription factors during retinogenesis, but how specific is the action of morpholinos? Did you consider the potential off-target effects of these morpholinos? Did you think of using a conditional knock out approach to answer your questions?

We decided to use morpholinos because they are more versatile than mutants, they can be injected into different transgenic lines and their dosage can be modulated depending on the degree of knockdown needed. These morpholinos used in this study have been extensively used and been validated by the respective mutants. It should be noted that we were not interested in studying the gene function per se, which was already known in all cases, but we used the morpholinos as a tool to prevent the emergence of different cell types.

  1. Do you have a hypothesis about the molecular signal that starts the Atoh7 lineage? That is, did you obtain any indication about what molecular cue triggers the “firing” of Atoh7 in RPCs?

It is possible that this is related to Notch signalling. Our lab and many others have shown, directly or indirectly, and in different model organisms, that Notch inhibits the onset of Atoh7 expression. In previous work (Nerli et al 2022 elife) we showed that, during the asymmetric divisions that form Atoh7+ progenitors, Notch signalling is asymmetrically inherited by the Atoh7-negative sister cell through the endocytic pathway. In this way, Notch signalling is promoted in the Atoh7-negative progenitor and depleted in the cell that will become Atoh7+ progenitor. Therefore, it is possible that multiple rounds of asymmetric inheritance of Notch signalling are needed to decrease the levels of Notch and therefore activate Atoh7 in one part of the lineage.

  1. Did you further characterize the “aberrant” photoreceptor precursor upon Prdm1a knock down, for instance using immunohistochemistry or other approaches?

We are combining different transgenic lines that are specific for different cell types, but to date we had confusing results. Most likely only a transcriptomics approach would clarify what these cells are and what they could become, however it is possible that this cell type remains immature, expressing different markers but never differentiating nor integrating into the neural circuitry.

  1. How do you explain the thickness reduction of the ganglion cell layer upon Prdm1a knock down?

We think that this might be due to the overall reduction in the rate of Atoh7+ progenitor differentiation. When we inhibit the emergence of the photoreceptor precursors we indeed observed defects in lineage progression. It is possible that overall less progenitors manage to enter the Atoh7 lineage, and as RGCs are the second most abundant cell types generated by this lineage, this would lead to their reduction . While we could not directly assess the rate of neurogenesis, all the embryos analysed showed a reduction in the number of neurons with respect to control. At the same time, this perturbation does not seem to affect the Atoh7-negative lineage, giving rise to Bipolar cells, as the thickness of the layer occupied by these cells is not dramatically affected by the perturbation.

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