The retina is the neural tissue situated at the back of the eyes that samples the visual scene and sends the processed information to the brain. Millions of people worldwide suffer from retinal diseases that affect mainly the light sensing photoreceptors or retinal ganglion cells, the output neurons projecting to the brain. Despite promising attempts in the fields of gene therapy and cell transplantation, a definitive cure for retinal diseases is still missing.
Research on highly regenerative organisms like zebrafish (Danio rerio) offers an attractive perspective to inform gene as well as cell transplantation-based therapies to treat retinal pathologies. Indeed, the zebrafish retina has the same structure and function as the human retina, including the presence of all retinal neurons as well as Müller glia, glial cells that provide structural and metabolic support.
Remarkably, and differently from mammalian species, zebrafish Müller glia behave additionally as stem cells upon tissue damage. In this context, Müller glia re-enter the cell cycle and generate retinal progenitors that eventually differentiate to all retinal neurons.
In the last twenty years, there has been a considerable effort to understand the molecular mechanisms underlying zebrafish Müller glia reprogramming to pro-regenerative stem cells and retinal progenitor production. However, a comprehensive study of the molecular identity of Müller glia, Müller glia-derived retinal progenitors as well as regenerated progeny in uninjured and lesioned conditions is still missing. Furthermore, although retinal progenitors regenerate all retinal neurons, independently of the specific retinal cell type that has been mostly affected by the tissue damage, it is not known whether all regenerated progeny integrate and rewire into the existing circuitry.
The present study had two aims:
• First, it aimed to provide a comprehensive description of Müller glia, Müller glia-derived progenitors as well as regenerated progeny in uninjured and light-lesioned retina at 44 hours as well as at 4 and 6 days post-lesion.
• Second, it aimed to establish a CreERT2 recombinase-based strategy to allow genetic access to follow and manipulate Müller glia-derived progenitors and their progeny during regeneration.
To achieve the first aim, a short-term lineage tracing strategy was devised using the two fluorescent reporters Tg(gfap:mCherry) and Tg(pcna:EGFP) labelling Müller glia and proliferating cells, respectively. Double transgenic animals were employed to sort for Müller glia, Müller glia-derived progenitors as well as regenerated progeny from the uninjured and light-lesioned retina. Subsequently, 10x Genomics, single cell RNA sequencing was performed to characterize their transcriptome and to deduct their differentiation trajectories during retina regeneration.
The sequencing experiment showed the presence of a glial and a neurogenic trajectory in the regenerating retina up to 6 days post-lesion. The glial trajectory starts with non-reactive Müller glia, characterized by canonical glial markers, and continues with injury-reactive Müller glia at 44 hours post-lesion, which upregulate genes associated with glia reprogramming and inflammation as well as proliferation. These early reactive Müller glia divide and generate cells belonging to a population with a hybrid identity that becomes eminent at 4 days post-lesion and is characterized by marker genes of both Müller glia and progenitors. A glial self-renewal and a neurogenic trajectory depart from the hybrid cell population. While the glial self-renewal trajectory feeds back to the non-reactive Müller glia cell population, the neurogenic trajectory continues with neurogenic progenitors, which progressively express markers of restricted fate competence and eventually regenerate several retinal neurons. The birthdate order of the regenerated progeny recapitulates the order observed during retinal development to a great extent. Indeed, retinal ganglion cells and red cone photoreceptors are born at 4 days post-lesion, followed by blue cones, amacrine and bipolar cells at 6 day post-lesion. Regenerated rod photoreceptors as well as horizontal cells were not detected among the sorted progeny, despite detection of their committed precursors.
To achieve the second aim, genetic access to Müller glia-derived cells was established using the TgBAC(mmp9:CreETt2,cryaa:EGFP);Tg(Olactb:loxP-DsRed2-loxP-EGFP) double transgenic line. The injury-induced promoter mmp9 is expressed in reactive Müller glia and drives the expression of CreERT2. Upon administration of the metabolite 4-hydroxytamoxifen, CreERT2 catalyses recombination in the Cre-dependent reporter Tg(Olactb:loxP-DsRed2-loxP-EGFP), resulting in the expression of EGFP under the control of the broadly expressed Olactb promoter. Subsequently, recombined cells, which include progenitors and progeny, express EGFP permanently. Two time points of 4-hydroxytamoxifen intraperitoneal injection were tested to achieve efficient recombination: 6 hours post-lesion, corresponding to 2 hours prior to onset of mmp9 in reactive Müller glia, and 24 hours post-lesion. In both cases, a substantial number of EGFP-positive, Müller glia-derived cells was observed in the regenerating retina at 4 days post-lesion. The majority of the EGFP-positive cells co-localized with PCNA-positive nuclei and corresponded most likely to progenitors. Importantly, EGFP-positive cells were neither observed in the light-lesioned, ethanol injected controls nor in the uninjured, 4-hydroxytamoxifen-injected controls, indicating tight control of CreERT2.
In conclusion, the current PhD thesis provides a comprehensive description of the transcriptome of Müller glia, Müller glia-derived retinal progenitors and regenerated progeny. Moreover, it establishes a CreERT2-based approach to study the composition as well as long-term integration of Müller glia-derived cells in the regenerated retina and allow their genetic manipulations in future studies.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:86228 |
| Date | 28 June 2023 |
| Creators | Celotto, Laura |
| Contributors | Brand, Michael, Englert, Christoph, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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