Monica Lynn Vetter

Neural diversity depends on both the temporal competence of progenitors to generate particular cell types and their progression into a neurogenic state. How these distinct features of progenitor development are mechanistically coordinated remains poorly understood. Here, we identify Foxp1 as a dual function transcriptional regulator that couples early progenitor identity to neurogenic progression in the developing retina. By defining its direct transcriptional targets, we show that Foxp1 coordinates complementary regulatory programs: it sustains early competence by activating early-stage retinal progenitor genes and repressing late temporal factors, while promoting neurogenic progression through activation of the proneural factors Atoh7 and Neurog2 . We establish a cis-regulatory mechanism linking these functions: Foxp1 activates Atoh7 through its remote shadow enhancer, and deletion of this enhancer prevents Foxp1-dependent induction of Atoh7 and retinal ganglion cell (RGC) production. We further reveal that Neurog2 partially compensates for loss of Atoh7 function. This coordinated early progenitor program is temporally restricted by progressive silencing of Foxp1 , involving sequential deposition of H2AK119ub1 followed by H3K27me3 at a distal conserved silencer essential for its repression. Together, these findings establish a mechanistic link between temporal competence and neurogenic progression, revealing how a shared transcriptional regulator coordinates progenitor identity with neuronal production through distinct cis-regulatory elements. Significance statement: Temporal competence and neurogenic progression are distinct features of neural progenitor development, yet how they are mechanistically coordinated remains poorly understood. We identify Foxp1 as a transcriptional regulator that couples early progenitor identity to neurogenic progression in the developing retina. Foxp1 activates early competence genes, represses late temporal factors, and directly activates proneural regulators. Functional dissection of cis-regulatory elements establishes an enhancer requirement for Foxp1-dependent neuronal production and a silencer requirement for repression of Foxp1 itself. These findings reveal how coordinated transcriptional activation and repression connect progenitor competence to neuronal differentiation, providing a mechanistic framework for understanding the temporal control of neural cell diversity.

Microglia and CNS-associated macrophages (CAMs) originate from yolk sac erythromyeloid progenitors, yet the process that mediates their colonization of the mammalian eye and the developmental cues that promote their diversification remain unclear. Here, we identify the optic fissure as the primary entry site for myeloid cells into the embryonic mouse retina. This entry is spatially and temporally coupled with a localized wave of cell death. This apoptotic niche contains colonizing myeloid cells in a transient CD206+ state, and apoptotic burden directly correlates with CD206 expression across development. We find that disrupting apoptotic recognition pathways, specifically Cx3cr1 and Itgam , promotes enrichment of CD206+ cells; likewise, increasing cell death via genetic loss of Atoh7 results in elevated CD206+ population, indicating that impaired clearance and increased death converge on a common outcome within a constrained developmental window. Both CD206+ and CD206- subsets contribute to phagocytosis during optic fissure closure, indicating that CD206 expression does not mark a specialized phagocytic subset. Intriguingly, unlike parenchymal microglia, CD206+ cells are selectively spared from pharmacological CSF1R inhibition, revealing distinct survival requirements within the fissure niche. Together, these findings indicate that localized apoptotic niches encountered during myeloid cell entry influence the initial states and properties of CNS-resident myeloid cells.

Microglia can selectively phagocytose live neurons during normal development and also in response to stress, injury or disease by recognizing phagocytic cues to target cells for elimination. In the developing retina at embryonic stages we previously found that microglia refine retinal ganglion cell (RGC) numbers by targeting non-apoptotic newborn RGCs for phagocytosis, utilizing complement receptor 3 (CR3) to recognize and eliminate RGCs. Here, we investigate additional phagocytic mechanisms and cues that microglia utilize to clear a subset of viable RGCs. Our findings indicate that both Mer tyrosine kinase (Mertk) and CR3 are required for clearance of a subpopulation of embryonic RGCs. In Mertk/CR3 double knockouts, we show that C1q-tagged RGCs accumulate and excess RGCs persist indicating failure of normal clearance by microglia. We also show that microglia target RGCs that have phosphorylated c-JUN (p-cJUN) expression, suggesting stress pathway activation. RGCs with p-cJUN expression also accumulate in Mertk/CR3 double knockout retinas, but this appears to resolve by P0, suggesting this is a transient stress state exhibited by a subset of RGCs that remain viable. By depleting microglia we establish that microglia are not required for p-cJUN induction in RGCs but show that they are the sole source of complement protein C1q, which marks these cells for elimination. Altogether the data suggests that a subset of stressed RGCs are recognized by local microglia that tag them with opsonins for removal using specific recognition receptors.

Development of the vertebrate retina involves the interaction of multiple signaling pathways and cell types, and there is growing appreciation of the role of innate immune pathways in this process. Resident innate immune cells, particularly microglia, play myriad roles in retinal development, disease, and regeneration. Here we aim to highlight what is known about innate immune cell populations and pathways in retinal cell development and regeneration. Resident innate immune cells are present from the earliest stages of retinal development and regulate developmental cell elimination, synapse refinement, angiogenesis, and recovery from retinal damage. We discuss the signaling pathways mediating immune cell interactions with other cell populations in developing and regenerating retina and highlight species-specific differences in retinal innate immune cell function, which are particularly evident in retinal cell regeneration.

Retinal ganglion cells (RGCs) are the projection neurons of the retina, and their death promotes an irreversible blindness. Several factors were described to control their genesis during retinal development. These include Atoh7, a major orchestrator of the RGC program, and downstream targets of this transcription factor, including Pou4f factors, that in turn regulate key aspects of terminal differentiation. The absence of POU4F family genes results in defects in RGC differentiation, aberrant axonal elaboration and, ultimately, RGC death. This confirms the requirement of POU4F factors for RGC development and survival, with a crucial role in regulating RGC axon outgrowth and pathfinding. Here, we have investigated in vivo whether ectopic Pou4f2 expression in late retinal progenitor cells (late RPCs) is sufficient to induce the generation of cells with RGC properties, including long-range axon projections. We show that Pou4f2 overexpression generates RGC-like cells that share morphological and transcriptional features with RGCs that are normally generated during early development and extend axonal projections up to the brain. In conclusion, these results show that POU4F2 alone is sufficient to promote the crucial properties of projection neurons that arise from retinal progenitors outside their developmental window.

Microglia, the parenchymal macrophage of the central nervous system, serve crucial remodeling functions throughout development. Microglia are transcriptionally heterogenous, suggesting that distinct microglial states confer discrete roles. Currently, little is known about how dynamic these states are, the cues that promote them, or how they impact microglial function. In the developing retina, we previously found a significant proportion of microglia express CD11c (Integrin αX, Itgax, subunit of complement receptor 4) which has also been reported in other developmental and disease contexts. Here, we sought to understand the regulation and function of CD11c+ microglia. We found that CD11c+ microglia track with prominent waves of neuronal apoptosis in postnatal retina. Using genetic fate mapping, we provide evidence that microglia transition out of the CD11c state to return to homeostasis. We show that CD11c+ microglia have elevated lysosomal content and contribute to the clearance of apoptotic neurons, and found that acquisition of CD11c expression is partially dependent upon the TAM receptor AXL. Using selective ablation, we found CD11c+ microglia are not uniquely critical for phagocytic clearance of apoptotic cells. Together, our data suggest that CD11c+ microglia are a transient state induced by developmental apoptosis rather than a specialized subset mediating phagocytic elimination.

Microglia, the parenchymal macrophage of the central nervous system serve crucial remodeling functions throughout development. Microglia are transcriptionally heterogenous, suggesting that distinct microglial states confer discrete roles. Currently, little is known about how dynamic these states are, the cues that promote them, or how they impact microglial function. In the developing retina, we previously found a significant proportion of microglia express CD11c (Integrin αX, complement receptor 4, Itgax ) which has also been reported in other developmental and disease contexts. Here, we sought to understand the regulation and function of CD11c+ microglia. We found that CD11c+ microglia track with prominent waves of neuronal apoptosis in postnatal retina. Using genetic fate mapping, we provide evidence that microglia transition out of the CD11c state to return to homeostasis. We show that CD11c+ microglia have elevated lysosomal content and contribute to the clearance of apoptotic neurons, and found that acquisition of CD11c expression is, in part, dependent upon the TAM receptor Axl. Using selective ablation, we found CD11c+ microglia are not uniquely critical for phagocytic clearance of apoptotic cells. Together, our data suggest CD11c+ microglia are a transient state induced by developmental apoptosis rather than a specialized subset mediating phagocytic elimination.

Retinal ganglion cells (RGCs) are the projection neurons of the retina. In early retinal progenitor cells (RPCs), Atoh7 orchestrates the developmental RGC program and regulates the expression of critical downstream targets, including Pou4f factors. The absence of Pou4f2 or more POU4F family genes results in defects in RGC differentiation, aberrant axonal elaboration and ultimately RGC death, confirming the requirement of POU4F factors for RGC development and survival, with a critical role in regulating RGC axon outgrowth and pathfinding. Here, we investigated in vivo whether ectopic Pou4f2 expression in late retinal progenitor cells (late RPCs) is sufficient to induce the generation of cells with RGC properties, including projecting axons to the brain. Using a strong ubiquitous promoter to induce Pou4f2 overexpression in neonates, we observed a change in targeted cell distribution in the retinal tissue, including the presence of cells in the ganglion cell layer and inner plexiform layer with high density of GFP ⁺ processes along the retina. Similar results on the induction of neuron processes were obtained when we overexpressed Pou4f2 in Atoh7 knockout mice, suggesting that POU4F2 is sufficient to induce them. Single-cell RNA sequencing (scRNA-seq) analysis shows that several RGC-genes (such as Rbpms , Gap - 43 , Hs6st3 , and Foxp2 ) are upregulated after Pou4f2 overexpression. Additionally, gene ontology analysis indicates the induction of genes related to axonogenesis and neuronal differentiation. Imaging throughout the visual pathway revealed high density of axons projecting toward the optic nerve head and extending to brain regions, such as the superior colliculus and geniculate nucleus. Thus, Pou4f2 induced neurons with specific RGC characteristics that share similarities with resident RGCs and notably project axons that reach brain targets. In conclusion, these results showed that POU4F2 alone was sufficient to promote critical properties of projection neurons from retinal progenitors outside their developmental window.