Otx2 and Onecut1 Promote the Fates of Cone Photoreceptors and Horizontal Cells and Repress Rod Photoreceptors

Cone photoreceptors carry out phototransduction in daylight conditions and provide the critical first step in color vision. Despite their importance, little is known about the developmental mechanisms involved in their generation, particularly how they are determined relative to rod photoreceptors, the cells that initiate vision in dim light. Here, we report the identification of a cisregulatory module (CRM) for the thyroid hormone receptor beta (Thrb) gene, an early cone marker. We found that ThrbCRM1 is active in progenitor cells biased to the production of cones and an interneuronal cell type, the horizontal cell (HC). Molecular analysis of ThrbCRM1 revealed that it is combinatorially regulated by the Otx2 and Onecut1 transcription factors. Onecut1 is sufficient to induce cells with the earliest markers of cones and HCs. Conversely, interference with Onecut1 transcriptional activity leads to precocious rod development, suggesting that Onecut1 is critically important in defining cone versus rod fates

Quantitative analysis of the ThrbCRM1-centered gene regulatory network

Enhancer activity is determined by both the activity and occupancy of transcription factors as well as the specific sequences they bind. Experimental investigation of this dynamic requires the ability to manipulate components of the system, ideally in as close to an in vivo context as possible. Here we use electroporation of plasmid reporters to define critical parameters of a specific cis-regulatory element, ThrbCRM1, during retinal development. ThrbCRM1 is associated with cone photoreceptor genesis and activated in a subset of developing retinal cells that co-express the Otx2 and Onecut1 (OC1) transcription factors. Variation of reporter plasmid concentration was used to generate dose response curves and revealed an effect of binding site availability on the number and strength of cells with reporter activity. Critical sequence elements of the ThrbCRM1 element were defined using both mutagenesis and misexpression of the Otx2 and OC1 transcription factors in the developing retina. Additionally, these experiments suggest that the ThrbCRM1 element is co-regulated by Otx2 and OC1 even under conditions of sub-optimal binding of OC1

Doctor of Philosophy

dissertationDuring development, all seven of the major retinal cell types are produced in a distinct yet overlapping order from a single pool of multipotent retinal progenitor cells (RPCs). In order to accomplish this task, it is thought that each individual RPC proceeds irreversibly through a series of intrinsically defined competence states, capable of producing only a subset of these cell types at any given time. To ensure that all early- and late-born cell types are produced in the correct number, RPCs must not only proceed through these competence states in a timely fashion, but also limit their rate of differentiation in order to prevent premature depletion. Lhx2 is a LIM-homeobox transcription factor expressed in many different tissues during development, known to regulate both proliferation and fate choice. It is also expressed in most if not all RPCs, and we assessed its contribution to their various properties by performing conditional inactivation at multiple time points during retinal neurogenesis. We find that Lhx2 is required within a limited temporal window to ensure the balanced production of earlyborn cell types, as retinal ganglion cells (RGCs) are selectively overproduced in the Lhx2 conditional knock-out (CKO) retina. LHX2 is also necessary for the normal cessation of RGC genesis, suggesting that in its absence, RPCs fail to undergo a normal transition in competence. We show that sustained Notch signaling, dependent on the transcription factor Rbpj, is likely responsible for maintaining a low level of both proliferation and neurogenesis in the absence of Lhx2, based on their successive requirements at distinct stages in the lineage progression of RPCs. We find further that LHX2 is required for the iv normal response of RPCs to Sonic Hedgehog (SHH), a negative-feedback signal secreted by RGCs and known to limit their further generation. In addition, we show that both LHX2 and SHH promote the expression of Ascl1, a gene expressed in RPCs and implicated as molecular readout of competence progression. Together, the results presented here demonstrate how intrinsic factors such as LHX2 may perform distinct functions at distinct phases of RPC lineage progression to orchestrate the process of retinal neurogenesis

Evidence of Müller Glia Conversion Into Retina Ganglion Cells Using Neurogenin2

Degenerative retinopathies are the leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Müller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the rodent adult injured retina but contribute weakly to tissue repair when compared to zebrafish retina. However, postnatal and adult mouse MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram postnatal mouse MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1 and both TFs induce the generation of functionally active iNs. Treatment of MGC cultures with EGF and FGF2 prior to Neurog2 or Ascl1 expression enhances reprogramming efficiencies, what can be at least partially explained by an increase in the frequency of MGCs expressing sex determining region Y (SRY)-box 2 (SOX2). Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. Moreover, in vivo electroporation of Neurog2 in late progenitors from the neonatal rat retina, which are transcriptionally similar to MGCs, also induced a shift in the generation of retinal cell subtypes, favoring neuronal differentiation at the expense of MGCs and resuming the generation of RGCs. Altogether, our data indicate that NEUROG2 induces lineage conversion of postnatal rodent MGCs into RGC-like iNs in vitro and resumes the generation of this neuronal type from late progenitors of the retina in vivo

Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures

Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na+ and K+ currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140208/1/scd.2015.0144.pd

Proneural bHLH: cell population dynamics in mouse retina

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2011The simultaneous production of several cell-types during embryonic development of the retina was previously shown to be regulated by the Notch-signaling pathway at multiple levels, through the sequential expression of two Notch ligands, Dll1 and Dll4. While Dll1 activity has been shown to be responsible for progenitor maintenance, Dll4 function has not been addressed until now, although a role in the regulation of cell-fate acquisition and celldiversity had been suggested. To address the role of Dll4 in the developing retina, we first characterized the intrinsic differentiation potential of Dll4-expressing cells during the first wave of retinal differentiation, at E13.5, by comparing its expression with that of three bHLH-encoding genes (Ngn2, Math5 and NeuroD), whose combined expression identify different retinal progenitors. Taking advantage of a Dll4 conditional knock-out (Dll4 cKO) mouse strain, we have used the expression of the same set of genes to evaluate the effects caused by Dll4 deletion on the profile of the differentiating population of cells. Our analysis of Dll4-expressing cells has revealed that these are still multipotent, although heavily biased towards the photoreceptor fate. We have also found that Dll4 absence leads to an increase in the production of photoreceptor and amacrine cells. The present work has allowed us to propose that during the first wave of differentiation Dll4-mediated Notchsignaling inhibits photoreceptor and amacrine cell production through inhibition of Ngn2 and NeuroD in the surrounding cells

The identification of effectors of retinal cell fate determination through single cell transcriptomics

The vertebrate retina is an exquisite model for neurogenesis, with its common pool of retinal progenitors differentiating into six neuronal cell types and one glial variety, each of which interconnects to form a simply organized yet complexly functioning sensory tissue. Our long-term aim is to determine the cell-autonomous genetic programs that drive retinogenesis, but identifying these signals is complicated by many factors. First, retinal progenitor cells generate the various retinal cell types at distinct but overlapping time points, potentially obscuring the progression of cell-intrinsic signals over time. Second, signals that drive the development of rare, but functionally critical, neuronal subtypes can be drowned out by the genes present in more common and transcriptomically homogeneous cellular populations. For these reasons, we have opted to study the transcriptomes of retinal progenitors at the resolution of the individual cell. Using single-cell transcriptomics, we have isolated genes that are present in subsets of developing retinal progenitors, including the Onecut family of transcription factors and a Polo-like kinase (Plk3), and studied the roles these genes play in the production of specific retinal neurons. We have also expanded our studies to include another vertebrate model system, the chicken, to better identify developmentally relevant genes that have been conserved throughout vertebrate evolution. Through the use of these transcriptomic techniques, my studies detailed in this thesis have brought us closer to elucidating the complex process of retinogenesis

Molecular Mechanisms Underlying Cell Fate Choice Within Specific Retinal Lineages

During development, retinal progenitor cells (RPCs) divide to form all of the cell types that make up the retina. Multipotent RPCs are competent to generate all retinal cell types, while restricted RPCs form specific lineages of cells. In particular, one genetically-defined RPC type preferentially gives rise to cone photoreceptors and horizontal cells. Many of the mechanisms that are responsible for directing cell fate choice within this lineage are unknown. This thesis largely focuses on examining the development of specific cell types and subtypes from restricted RPCs and on investigating the gene regulatory events that underlie cone photoreceptor and horizontal cell development in the retina. Short-term assays have concluded that ThrbCRM1 RPCs preferentially generate cone photoreceptors and horizontal cells. However, developmental timing has precluded an extensive cell type characterization of their progeny. The first part of this thesis describes the development and validation of a recombinase-based lineage tracing system for the chicken embryo to further characterize the lineage of these cells. ThrbCRM1 RPCs were found to preferentially form photoreceptors and horizontal cells, as well as a small number of retinal ganglion cells. The photoreceptor cell progeny are exclusively cone photoreceptors and not rod photoreceptors, confirming that ThrbCRM1 RPCs are restricted from the rod fate. In addition, specific subtypes of horizontal cells and retinal ganglion cells were overrepresented, suggesting that ThrbCRM1 RPCs are not only restricted for cell type, but for cell subtype as well. The second part of this thesis utilizes the identification of novel cis-regulatory elements as a method to examine the gene regulatory networks that direct the development of horizontal cells. The OC1ECR22 and Tfap2aACR5 elements were shown to be enhancers for Onecut1 (OC1) and Tfap2a, respectively, and to be specifically active in developing horizontal cells. The OC1ECR22 element is activated by Ptf1a and Rbpj, which translates to regulation of OC1 expression and suggests that Ptf1a is a direct activator of OC1 expression in developing horizontal cells. The region within the Tfap2aACR5 element that is responsible for its activation was determined to be a 100 bp sequence named Motif 4. Both OC1ECR22 and Tfap2aACR5 are negatively regulated by the nuclear receptors Thrb and Rxrg, as is the expression of OC1 and Tfap2a, suggesting that nuclear receptors may have a role in the negative regulation of horizontal cell development

Gene Regulation and Cell Fate Choice in the Developing Vertebrate Retina

The diverse neuronal cell types in the vertebrate retina all originate from multipotent retinal progenitor cells (RPCs). These undergo a series of molecular changes driven by developmental gene regulatory networks (GRNs) as they divide to generate RPCs which are more restricted in their potential fates. It is crucial to understand these GRNs and changes to gene expression in order to understand how cell identity is established during retinal development. In particular, the GRN that promotes the development of cone photoreceptors and horizontal cells is not well-defined. This work focuses on two approaches to further elucidate the components of this regulatory network and how gene expression is controlled in developing cones and horizontal cells. The first part of this work is largely focused on a fate-restricted RPC that can be defined by the activity of the cis-regulatory element ThrbCRM1 and preferentially generates cone photoreceptors and horizontal cells. This element is bound by two transcription factors: Otx2 and Onecut1. To identify upstream factors that contribute to Onecut1 expression in this population, this work used a large-scale enhancer screen which yielded two regulatory elements: Onecut1 ECR65 and Onecut1 ECR9. These enhancers are both active in ThrbCRM1-positive cells and mark populations that are biased to the cone photoreceptor and horizontal cell fates. Through a combination of bioinformatic tools for predictive binding and functional experiments, it was shown that both of these elements respond to bHLH family transcription factors. Theseexperiments provide evidence that bHLH expression is involved in restricting RPCs to the cone and horizontal cell fates. The second part of this work describes a comparative study between cone-dominant and rod-dominant mammalian retinae, using the thirteen-lined ground squirrel and mouse. This work is the first to describe retinal development in the thirteen-lined ground squirrel retina and demonstrates differences in the timing of particular events in cone and horizontal cell development. This work also used ATAC-seq to compare developmental chromatin accessibility between mouse and ground squirrel retinae. This approach led to the identification of several differentially accessible chromatin regions, some near known cone photoreceptor and horizontal cell genes. This work provides the tools needed to further understand the gene regulatory differences between the developing mouse and ground squirrel retinae. This comparative approach can elucidate how the ground squirrel retina promotes the cone and horizontal cell fates