Reports Step by Step

Project: Development and Function of High Acuity Central Vision (EyeCenter)

Contract: PCE 54/2022

Cod Depunere: PN-III-P4-PCE-2021-0333

Project Director: Tudor C. Badea

The project uses experimental models in genetically modified mice to study the development, function and pathogenesis of high visual acuity in the central retina.

During this year, we have established the research infrastructure (experimental animal facility), acquired further necessary equipment and recruited the project team, consisting of one postdoctoral researcher and three graduate students (two Phd and one MS). In addition we discovered and characterized several Retinal Ganglion Cell populations (RGCs) that belong to the high spatial resolution group, and may contribute to high resolution central vision. Amongst these we studied transient suppressed by contrast cells (tSBCs), RGCs expressing Tusc5, and Brn3c⁺Brn3b⁺ RGCs. Members of the group also helped describe new mechanisms for setting up early retinal topographic layout, which implicitly affects the topography of central retina. Furthermore, we contributed to studies on the mechanisms of autoimmune diseases that also target the visual system, especially the optic nerve. These results were published in three peer reviewed papers (IF between 4 and 8) and presented at two international and two national conferences.

Published papers:

• Boobalan E, Thompson AH, Alur RP, McGaughey DM, Dong L, Shih G, Vieta-Ferrer ER, Onojafe IF, Kalaskar VK, Arno G, Lotery AJ, Guan B, Bender C, Memon O, Brinster L, Soleilhavoup C, Panman L, Badea TC, Minella A, Lopez AJ, Thomasy SM, Moshiri A, Blain D, Hufnagel RB, Cogliati T, Bharti K, Brooks BP. (2022) Zfp503/Nlz2 Is Required for RPE Differentiation and Optic Fissure Closure. Invest Ophthalmol Vis Sci. 2022 Nov 1;63(12):5. doi: 10.1167/iovs.63.12.5. & BioRxiv doi: 10.1101/2022.03.28.486078 IF: 5 Q1 https://doi.org/10.1167/iovs.63.12
• Tatomir A, Cuevas J, Badea TC, Muresanu DF, Rus V, Rus H (2022) Role of RGC-32 in multiple sclerosis and neuroinflammation – few answers and many questions, Frontiers in Immunology IF: 5.7 Q1 https://doi.org/10.3389/fimmu.2022.979414
• Tien NW, Vitale C, Badea TC (co-corresponding author), Kerschensteiner D, (2022) Layer-specific developmentally precise axon targeting of transient suppressed-by-contrast retinal ganglion cells bioRxiv doi: 10.1101/2021.11.26.470118 & J. of Neuroscience doi: 10.1523/JNEUROSCI.2332-21.2022 IF: 4.4 Q1 https://doi.org/10.1523/JNEUROSCI.2332-21.2022

Project: Development and Function of High Acuity Central Vision (EyeCenter)

Contract: PCE 54/2022

Cod Depunere: PN-III-P4-PCE-2021-0333

Project Director: Tudor C. Badea

The project aims to elucidate the mechanisms of development, function and pathogenesis of high visual acuity in the center of the retina, using experimental models on genetically modified animals.

1) We acclimatized the genetically modified lines transferred from NIH, by adjusting the temperature conditions, controlled climate, nutrition and animal care. After several iterations and personnel changes, we optimized the care conditions for the lines, and currently all lines imported from NIH are fertile and properly propagated.

2) During this period, we generated the combined Brn3c^Cre; Brn3b^CKOAP lines, and we determined with high precision the position of ArCe in retinas from these animals.

3) To study the role of Brn3b and Brn3c in ArCe formation, we initiated crosses to generate the parental lines: Brn3c^Cre/WT; Brn3b^KO/WT and Brn3c^KO/WT; Brn3b^CKOAP/CKOAP.

4) To study the role of Brn3a in ArCe formation, we generated the parental lines Brn3a^CKOAP/CKOAP, and Rax:Cre; Brn3a^KO/WT.

5) We have acquired the necessary equipment to complete the behavioral and electrophysiology analysis lines required for objective 3. The assembly of these lines is ongoing.

6) We have identified and recruited two female researchers (two doctoral students) to the team for the vacant positions, who are coming to replace a doctoral student and a master's student who withdrew from the project.

The following results were obtained:

1) The transcription factors Brn3a and Tbr1 cooperate in the formation of RGC OFF - DS ("Jam2 RGCs)

Together with Dr. Mao, we demonstrated that the Brn3a protein binds directly to the regulatory regions of Tbr1 and Jam2, activating their transcription, and thus contributing to the specification of OFF-DS-RGC cells. These results are relevant to Objective 2 (development of high visual acuity RGCs from the ArCe), since the formation of retinal topographic domains (and implicitly the ArCe), is under the control of the transcription factors mentioned above. The specific contribution of the project director was the establishment of the transcriptional dependence of Jam2 and Tbr1 on Brn3a.

2) The relationship between transcription factors and signaling molecules in the initial stages of RGC cell formation.

RGC cell specification is found in a balance between autonomous mechanisms, dictated by transcription factors (see previous paragraph) and adhesion and signaling molecules, which mediate the interaction between surrounding tissues and RGC neurites (dendrites and axons), both during development and during neuronal function and pathology. In order to understand the initial stages of the development of RGC axons exiting the eye, we need to know the adhesion molecules and transcription factors necessary for these stages. Together with Raluca Pascalau MD, PhD student member of the team, we reviewed the knowledge in the field related to this topic, and identified some candidate molecules for the mechanisms of RGC axon formation at the exit from the optic cup.

These results are relevant to Aims 1 and 2, as the formation of topographic domains of the retina (and implicitly the ArCe), is under the control of the transcription factors mentioned above.

3) Restoring sight to patients with optic nerve pathologies: the RReSTORe consortium

RGCs are the target of many blinding diseases. Unfortunately, diseases leading to loss of RGCs (glaucoma, optic nerve hypoplasia, neurodegenerative diseases with damage to the optic nerve) do not benefit from effective therapies in saving RGCs and halting the progression to blindness. That is why we are interested in therapies to reconstruct sight by replacing RGCs, bio-electronic vision prosthesis, or regenerative approaches through cellular reprogramming. For all these purposes, we need to be able to understand in depth the organization of the human visual system, in particular the RGC types of the human retina, their development and the developmental mechanisms of RGC axons en route to the brain. During the last two years, Dr. Badea participated as a senior member in the consortium of researchers including virtually all world experts studying RGCs. RRestore organized a series of face-to-face and online meetings to discuss strategies for vas restoration, resulting in a policy document setting out the research agenda for vas restoration through replacement and regenerative therapies.

This study is relevant to the general objective of the project, which is the use of animal models in exploring the development, function and pathogenetic mechanisms of visual disorders affecting central vision, and identifying therapeutic and restorative strategies.

4) Characterization of high visual acuity RGCs expressing Brn3c and Brn3b.

In these experiments, we infected the retinas of Brn3cCre animals with AAV-FLEX-eGFP viruses, and then stained these retinas with anti-Brn3b and anti-eGFP antibodies. The rare, well-spaced infections allowed us to isolate and characterize the dendritic trees of Brn3cCre cells, and among them we could identify those that also express Brn3b by staining with the antibody. Thus we found that at least three cell types expressed both Brn3b and Brn3c, two OFF-type RGCs (2aw and 1wt) and one RGC with dense arbors and restricted area (5to). At this stage we completed the analysis of all Brn3b+Brn3c+ and Brn3b-Brn3c+ cells from the previously collected data set by measuring dendritic trees and 3D reconstructions (adjacent images).

5) The influence of albinism on ArCe

In humans, albinism leads to hypoplasia of the fovea. To investigate the role of albinism in ArCe formation, we generated triple transgenic mice: Tyr^Cj/Cj; Brn3c^Cre/WT; Brn3b^CKOAP/CKOAP

In collaboration with the group Dr. Brian Brooks of the National Eye Institute. The Tyr^Cj/Cj line has a mutation in the tyrosinase gene, resulting in albinism. Our experiments showed that ArCe position and dimensions are unchanged in albino mice (Tyr^Cj/Cj; Brn3c^Cre/WT; Brn3b^CKOAP/WT) compared to control animals (Tyr^Cj/WT; Brn3c^Cre/WT; Brn3b^CKOAP/WT or Brn3c^Cre/WT) ; Brn3b^CKOAP/WT) suggesting that this form of albinism does not affect formation ArCe.

Published papers:

• Nishida K, Matsumura S, Uchida H, Abe M, Sakimura K, Badea TC, Kobayashi T. (2023) Brn3a controls the soma localization and axonal extension patterns of developing spinal dorsal horn neurons. PLoS One. 2023 Sep 21;18(9):e0285295. doi: 10.1371/journal.pone.0285295. IF: 2.9 Q1 https://doi.org/10.1371/journal.pone.0285295
• Paşcalău R, Badea TC (2023) Signaling – transcription interactions in mouse retinal ganglion cells early axon pathfinding –a literature review, Front. Ophthalmol., 2023 May 17; doi: 10.3389/fopht.2023.1180142 (Scopus) https://doi.org/10.3389/fopht.2023.1180142
• Kiyama T, Altay HY, Badea TC, Mao CA (2023) Pou4f1-Tbr1 transcriptional cascade controls the formation of Jam2-expressing retinal ganglion cells. Front. Ophthalmol., 2023 May 18; doi: 10.3389/fopht.2023.1175568 Scopus) https://doi.org/10.3389/fopht.2023.1175568

Project: Development and Function of High Acuity Central Vision (EyeCenter)

Contract: PCE 54/2022

Cod Depunere: PN-III-P4-PCE-2021-0333

Project Director: Tudor C. Badea

In this final year, we have made advances in several of the goals we have set, and completed a majority of the proposed objectives.

Characterization of high visual acuity RGCs expressing Brn3c and Brn3b.

We completed the classification of Brn3b⁺Brn3c⁺ and Brn3b^-Brn3c⁺ RGCs by measuring dendritic trees and 3D reconstructions. In total we quantified 99 monostratisfied RGCs, of which 17 were Brn3b⁺, and 67 bistratified RGCs, of which 3 were Brn3b⁺. Because AAV infection is random and virus activation occurs only in Brn3c^Cre cells, which express Cre recombinase, Brn3b^-Brn3c⁺ labeled cells covered the retinal surface relatively homogeneously (due to Brn3c expression), but most Brn3b⁺Brn3c⁺ cells found in ArCe (area of increased density of Brn3b and Brn3c). In conclusion, Brn3c⁺Brn3b⁺ cells populating the ArCe include two OFF-type RGCs (2aw-OFFvOS and 1wt-OFF sustained alpha), one RGC with dense arbors and restricted area (5to, ON transient), and a minor population of bilayered cells.

Embryonic/postnatal time interval in which ArCe is formed in Brn3c^Cre/WT; Brn3b^CKOAP/WT.

To identify the stages of ArCe formation, we crossed Brn3c^Cre/WT males with Brn3b^CKOAP/CKOAP females, and we collected embryos at embryonic ages E12 and E15 and postnatal pups at P0, P7, and P15. Whole-mount stainings of E12 and E15 embryos show the absence of RGCs in the eye at E12 and the presence of RGCs and axons at E15. Sections through the brain of E15 embryos show a dorsal-dense, ventral-sparse gradient of RGC cells, and the progression of axons beyond the optic chiasm to the lateral aspect of the thalamus. At P0 the RGC distribution is relatively homogeneous on the retinal surface. At P7, RGC density in the dorsal lobe begins to decrease, and the ArCe begins to define. At P15, the ArCe assumes its nearly mature appearance, with a naso-temporally oriented demarcation line on the dorsal side, and a dorso-ventral gradient on the ventral side. Overall, Brn3b⁺Brn3c⁺ RGCs have a much lower density in the dorsal lobe of the retina, and their density decreases progressively towards the ventral lobe, with a "belt" of maximum density on the naso-temporal axis. These results suggest that ArCe formation is largely a postnatal phenomenon, possibly through activity-dependent mechanisms.

Role of Brn3b in ArCe development

To specifically remove Brn3b from Brn3c⁺ cells, we generated Brn3c^Cre/WT animals; Brn3b^CKOAP/KO and Brn3c^Cre/WT; Brn3b^CKOAP/WT, and we analyzed ArCe at 2 months of age. No difference is observed between the two genotypes in terms of ArCe topography, and only a small difference in overall cell number. From this we conclude that Brn3b does not play an important role in the establishment of ArCe, and the development of Brn3b⁺Brn3c⁺ cells. A limitation is that, by this experiment, Brn3b is removed only after the activation of Brn3c, i.e., at the age of E12–E13, when Brn3c, and thus Brn3cCre, begins to be expressed.

The role of Brn3a in RGC differentiation with ipsi or contralateral projections

An important element of high-resolution vision is the ipsi/contralateral projection of RGC axons. Previous publications have argued that Brn3a/Pou4f1 is a critical factor for the correct development of ipsi and/or contralateral fibers, but using only postnatal (P0) Brn3a^KO/KO pups, as they do not survive beyond the first postnatal day. Using the Cre-dependent conditional allele of Brn3a (Brn3a^CKOAP), we eliminated age-specific Brn3a expression (by crossing with the Rax^Cre allele, which expresses Cre specifically in the optic vesicle starting from embryonic day 9.5), thus avoiding postnatal lethality. We determined the ipsi and contralateral composition of axonal projections originating from the two eyes using the Cholera Toxin tracer A488 (left eye) and A555 (right eye), and quantified the proportion of ipsi and contra fibers in the lateral geniculate nuclei (LGN), pretectal olivary (OPN), supra-chiasmatic (SCN) and superior colliculus (SC), in control animals (Brn3a^CKOAP/WT), heterozygous (Rax^Cre; Brn3a^CKOAP/WT) and knock-out (Rax^Cre; Brn3a^CKOAP/KO). While the contralateral projections are 100% segregated from the ipsilateral ones in the SC, at the level of the SCN they are almost equal. At the LGN and OPN level, the ipsilateral projection represents around 15% of the total projections. Absence of one or both copies of Brn3a in RGC cells produces only statistically insignificant effects on the proportion of ipsilateral axons in the four nuclei. Thus, Brn3a does not seem to play a role in specifying ipsi vs contralateral segregation, even though it is expressed in large amounts in contra-projecting RGCs.

Preprints:

• Rodgers J., Hughes S., Ebrahimi A, Allen AE, Storchi R, Lindner M., Peirson S.N., Badea T.C., Hankins M.W., Lucas R.J. (2024) Enhanced restoration of visual code after targeting on bipolar cells compared to retinal ganglion cells with optogenetic therapy. 07.23.24 bioRxiv 2024.07.22.604613; doi: https://doi.org/10.1101/2024.07.22.604613

  This work is in second round revision in Molecular Therapy: IF: 12.1 Q1

Peer Reviewed Research Articles:

• Soucy JR, Aguzzi EA, Cho J, Gilhooley MJ, Keuthan C, Luo Z, Monavarfeshani A, Saleem MA, Wang XW, Wohlschlegel J; RReSTORe Consortium (consortium author, including Badea TC); Baranov P, Di Polo A, Fortune B, Gokoffski KK, Goldberg JL, Guido W, Kolodkin AL, Mason CA, Ou Y, Reh TA, Ross AG, Samuels BC, Welsbie D, Zack DJ, Johnson TV. (2023) Retinal ganglion cell repopulation for vision restoration in optic neuropathy: a roadmap from the RReSTORe Consortium. Mol Neurodegener. 2023 Sep 21;18(1):64. doi: 10.1186/s13024-023-00655-y. IF: 15.1 Q1 https://doi.org/10.1186/s13024-023-00655-y