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Cephalopod Retinal Development Shows Vertebrate-like Mechanisms of Neurogenesis

Francesca Napoli, Christina M. Daly, Stephanie Neal, Kyle J. McCulloch, Alexandra Zaloga, Alicia Liu, Kristen M. Koenig

Preprint posted on November 18, 2021 https://www.biorxiv.org/content/10.1101/2021.10.28.466353v2

Not so unique anymore? Similar cellular mechanisms exist between cephalopod retinal neurogenesis and vertebrate neurogenesis

Selected by Niveda Udaykumar

Background 

The nervous system is one of the most complex systems of the vertebrate body and is formed by precise and tightly regulated processes of cell proliferation and cell differentiation. However, the lack of high-resolution imaging techniques has limited our understanding of the evolution of the nervous system to date. The coleoid cephalopods, comprising species of cuttlefish, octopus, and squid have the largest nervous system in invertebrates. In addition, cephalopods also possess camera eyes that have convergently evolved [1,2], making them a good model to study the comparative evolution of the nervous system between vertebrates and invertebrates.

The adult cephalopod retina comprises two layers separated by the basal membrane (Fig 1A), the nuclei are pseudostratified and exhibit interkinetic migration, making it a classic model to study neurogenesis. The cell bodies of the photoreceptors are found in the layer further from the lens, while the support cells reside in the layer closer to the lens. The squid retinal progenitor cells, like vertebrate neurogenesis, undergo interkinetic nuclear migration along the apicobasal axis [3,4]. Observations on fixed squid retinal tissue show that in the developing retina, mitosis is initiated in the anterior side, thereby prompting the authors to investigate whether retinal progenitor cells undergo interkinetic nuclear migration and how they differentiate within the epithelium.

In this preprint, Napoli et.al., have pioneered high-resolution imaging systems, in the squid Doryteuthis pealeii retina, to compare the cellular mechanisms of retinal neurogenesis to that of vertebrates. The authors demonstrate that squid retinal neurogenesis shows comparable hallmarks to vertebrate neurogenesis, wherein progenitor nuclei show interkinetic nuclear migration regulated by Notch. This suggests a common proliferative neurogenic primordium between the invertebrate and vertebrate nervous systems.

Key findings

Developing squid retinal cells show interkinetic nuclear migration 

To understand if squid retinal progenitors show interkinetic nuclear migration, a 10-minute BrdU pulse labeling was performed on Doryteuthis pealeii retina, followed by fixing and immunostaining with phospho-histone 3 (PH3). BrdU positive nuclei were observed in the posterior retina, while PH3 positive nuclei were observed in the anterior retina, corroborating previous observations of the cell cycle in the retina. This10-minute BrdU pulse was chased for 110 minutes, sampling the embryos every ten minutes to assess for nuclear migration from the basal to the apical side (from posterior to anterior) of the retina. On fixed tissues, after 80 minutes, BrdU positive cells were seen in the anterior retina, thereby indicating that the BrdU positive nuclei migrate apically for cell division (Fig 1B). This observation was further strengthened by live-cell imaging using fluorescent-labeled dextran, to label the cell membrane, and imaging for more than 9 hours. Over this time course, individual cells were observed to migrate apically, undergo mitosis, and migrate basally again, confirming that the squid retinal cells undergo interkinetic nuclear migration (Fig 1C).

Fig 1: A and B- Cell cycle analysis of squid retinal cells using BrdU and PH3. C-Squid retinal nuclei showing interkinetic nuclear migration by live-cell imaging. (Adapted from Fig 1, Napoli et.al.,2021)

Further, the contribution of actomyosin and microtubules to the interkinetic nuclear migration of squid retinal cells was assessed, by treating stage 23 embryos with cytochalasin D and nocodazole respectively. This was followed by examining the location of PH3 positive nuclei in the retina. In both cases, the PH3 positive nuclei were displaced from the mitotic zone (MZ), the epithelial region consisting of nuclei abutting the apical membrane, indicating that both actin polymerization and microtubules are essential for proper nuclear migration.

Molecular marker analysis of the retinal cells 

Next, the molecular identity of the retinal cells was investigated by a candidate gene approach for thirteen markers of neurogenesis. Spatiotemporal expression profiling showed that at stage 23, DpSoxB1 expression is enriched on the basal side of the retina while DpEphR expression is enriched on the apical side. At stage 25, in addition to the maintenance of the segregated DpSoxB1 and DpEphR expression, the cells start expressing terminal differentiation markers DpRetinochrome and DpRhodopsin. Marker analyses suggested that the progenitor cells can follow two differentiation trajectories: photoreceptor differentiation and simple differentiation trajectories. For the photoreceptor differentiation trajectories, DpEphR positive cells undergo interkinetic nuclear migration, become post-mitotic, and start expressing terminal markers, probably through asymmetric cell division. In contrast, the simple differentiation trajectory allows DpSoxB1 positive cells to self-renew by symmetric cell division. At stage 27, the DpSoxB1 cells migrate to the anterior retina, remain in the cell cycle, and start expressing DpRetinochrome, thereby maintaining a long-term stem cell population in the squid retina.

Notch signaling regulates the retinal progenitor state

Data from this study correlates BrdU positive cells with DpSoxB1 expression and post-mitotic cells with DpEphR expression (Fig 2A, B). Also, over time, the DpSoxB1 cells become DpEphR positive, a classic case of exit from the cell cycle and cell differentiation. One of the major regulators of this process is Notch signaling. To understand if Notch signaling regulates cell differentiation with proliferation, mRNA in situ hybridization for members of Notch signaling was performed in stage 23 embryos. In situ hybridization showed that retinal progenitors expressing DpSoxB1 showed enriched expression of DpNotch and DpHes-1, suggesting the role of Notch signaling in maintaining progenitor identity. Also, DpNotch was expressed in a small number of nuclei on the apical side. Immunostaining with PH3 after in situ hybridization correlated the expression of DpNotch with nuclei undergoing nuclear migration, and that inheritance of Notch mRNA is necessary for progenitor identity (Fig 2C).

To concretely establish the role of Notch signaling in maintaining cell fate identity, embryos were treated with gamma-secretase inhibitor DAPT to inhibit Notch signaling and subjected to RNA sequencing. Analysis of RNA sequencing showed downregulation of rhodopsin, cell cycle genes, and significant changes in cell signaling genes, indicating that Notch signaling may either work alone or crosstalk with other signaling pathways like BMP and Wnt signaling in the squid retina (Fig 2D). Additionally, in situ hybridization of the genes identified from the RNA sequencing on DAPT-treated embryos showed significant downregulation of DpNotch expression, complete loss of DpSoxB1 expression, and ectopic expression of DpEphR when compared to control, suggesting that Notch signaling is required for maintaining the fate of the squid retina (Fig 2E).

Fig 2: A and B-Expression of SoxB1 in basal retinal progenitors and EphR in post-mitotic apical retinal cells at stage 23. C- Expression of Notch and Hes-1 mRNA in the squid retina progenitor population at stage 23. D-Transcriptomic profiling of signaling pathways between control and Notch signaling inhibited embryos. E- Downregulation of Notch signaling pathway players, and its interaction with other signaling pathways, in Control and DAPT-treated embryos. (Adapted from Fig 4, Napoli et.al., 2021).

Why I chose to highlight this preprint

I chose to highlight this preprint as the authors have coupled high-resolution live-cell imaging techniques, gene expression analysis, and transcriptomics, to demonstrate that squid retinal cells are capable of interkinetic nuclear migration, and that Notch signaling is critical for maintaining the progenitor identity in the retinal progenitor cells. Further, this preprint also sheds light on the mechanisms of neurogenesis between invertebrates and vertebrates, permitting us to better understand the evolution of the nervous system.

 Questions to authors

1)    Why was this squid species, Doryteuthis pealeii, particularly chosen as a model system for the study? Does this species have certain characteristics/advantages over other coleoid cephalopods?

2)    How do you think Notch signaling may be working with other signaling pathways to regulate retinal progenitor identity?

References

  1. Gagnon, Y. L., Sutton, T. T., & Johnsen, S. (2013). Visual acuity in pelagic fishes and mollusks. 463 Vision Research, 92, 1–9. https://doi.org/10.1016/j.visres.2013.08.007
  2.   Packard, A. (1972). CEPHALOPODS AND FISH: THE LIMITS OF CONVERGENCE. In Biological Reviews (Vol. 47, Issue 2, pp. 241–307). https://doi.org/10.1111/j.1469- 525 185x.1972.tb00975.x
  3. Koenig, K. M., Sun, P., Meyer, E., & Gross, J. M. (2016). Eye development and photoreceptor differentiation in the cephalopod Doryteuthis pealeii. Development, 143(17), 3168–3181.https://doi.org/10.1242/dev.134254
  4. Koenig, K. M., & Gross, J. M. (2020). Evolution and development of complex eyes: a celebration of diversity. Development, 147(19). https://doi.org/10.1242/dev.182923

 

Tags: cephalopod, live cell imaging, neurogenesis, retina

Posted on: 6th December 2021

doi: https://doi.org/10.1242/prelights.31114

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