Initiation of Otx2 expression in the developing mouse retina requires a unique enhancer and either Ascl1 or Neurog2 activity.

During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation. How these retinal progenitor cells initially activate Otx2 expression is unclear. To address this, we investigated the cis-regulatory network that controls Otx2 expression in mice. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. CRISPR/Cas9-mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor-binding sites were required for its activity. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Our findings suggest that Ascl1 and Neurog2 act either redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. We observed redundancy or compensation at both the transcriptional and enhancer utilization levels, suggesting that the mechanisms governing Otx2 regulation in the retina are flexible and robust.

Prdm1 overexpression causes a photoreceptor fate-shift in nascent, but not mature, bipolar cells.

The transcription factors Prdm1 (Blimp1) and Vsx2 (Chx10) work downstream of Otx2 to regulate photoreceptor and bipolar cell fates in the developing retina. Mice that lack Vsx2 fail to form bipolar cells while Prdm1 mutants form excess bipolars at the direct expense of photoreceptors. Excess bipolars in Prdm1 mutants appear to derive from rods, suggesting that photoreceptor fate remains mutable for some time after cells become specified. Here we tested whether bipolar cell fate is also plastic during development. To do this, we created a system to conditionally misexpress Prdm1 at different stages of bipolar cell development. We found that Prdm1 blocks bipolar cell formation if expressed before the fate choice decision occurred. When we misexpressed Prdm1 just after the decision to become a bipolar cell was made, some cells were reprogrammed into photoreceptors. In contrast, Prdm1 misexpression in mature bipolar cells did not affect cell fate. We also provide evidence that sustained misexpression of Prdm1 was selectively toxic to photoreceptors. Our data show that bipolar fate is malleable, but only for a short temporal window following fate specification. Prdm1 and Vsx2 act by stabilizing photoreceptor and bipolar fates in developing OTX2+ cells of the retina.

Simultaneous deletion of Prdm1 and Vsx2 enhancers in the retina alters photoreceptor and bipolar cell fate specification, yet differs from deleting both genes.

The transcription factor OTX2 is required for photoreceptor and bipolar cell formation in the retina. It directly activates the transcription factors Prdm1 and Vsx2 through cell type-specific enhancers. PRDM1 and VSX2 work in opposition, such that PRDM1 promotes photoreceptor fate and VSX2 bipolar cell fate. To determine how OTX2+ cell fates are regulated in mice, we deleted Prdm1 and Vsx2 or their cell type-specific enhancers simultaneously using a CRISPR/Cas9 in vivo retina electroporation strategy. Double gene or enhancer targeting effectively removed PRDM1 and VSX2 protein expression. However, double enhancer targeting favored bipolar fate outcomes, whereas double gene targeting favored photoreceptor fate. Both conditions generated excess amacrine cells. Combined, these fate changes suggest that photoreceptors are a default fate outcome in OTX2+ cells and that VSX2 must be present in a narrow temporal window to drive bipolar cell formation. Prdm1 and Vsx2 also appear to redundantly restrict the competence of OTX2+ cells, preventing amacrine cell formation. By taking a combinatorial deletion approach of both coding sequences and enhancers, our work provides new insights into the complex regulatory mechanisms that control cell fate choice.

Balancing Extrasynaptic Excitation and Synaptic Inhibition within Olfactory Bulb Glomeruli.

Glutamatergic transmission in the brain typically occurs at well-defined synaptic connections, but increasing evidence indicates that neural excitation can also occur through activation of "extrasynaptic" glutamate receptors. Here, we investigated the underlying mechanisms and functional properties of extrasynaptic signals that are part of a feedforward path of information flow in the olfactory bulb. This pathway involves glutamatergic interneurons, external tufted cells (eTCs), that are excited by olfactory sensory neurons (OSNs) and in turn excite output mitral cells (MCs) extrasynaptically. Using pair-cell and triple-cell recordings in rat bulb slices (of either sex), combined with ultrastructural approaches, we first present evidence that eTC-to-MC signaling results from "spillover" of glutamate released at eTC synapses onto GABAergic periglomerular (PG) cells in glomeruli. Thus, feedforward excitation is an indirect result of and must cooccur with activation of inhibitory circuitry. Next, to examine the dynamics of the competing signals, we assayed the relationship between the number of spikes in eTCs and excitation of MCs or PG cells in pair-cell recordings. This showed that extrasynaptic excitation in MCs is very weak due to single spikes but rises sharply and supralinearly with increasing spikes, differing from sublinear behavior for synaptic excitation of PG cells. Similar dynamics leading to a preference for extrasynaptic excitation were also observed during recordings of extrasynaptic and inhibitory currents in response to OSN input of increasing magnitude. The observed alterations in the balance between extrasynaptic excitation and inhibition in glomeruli with stimulus strength could underlie an intraglomerular mechanism for olfactory contrast enhancement.

Transcriptional profiling of murine retinas undergoing semi-synchronous cone photoreceptor differentiation.

Uncovering the gene regulatory networks that control cone photoreceptor formation has been hindered because cones only make up a few percent of the retina and form asynchronously during development. To overcome these limitations, we used a γ-secretase inhibitor, DAPT, to disrupt Notch signaling and force proliferating retinal progenitor cells to rapidly adopt neuronal identity. We treated mouse retinal explants at the peak of cone genesis with DAPT and examined tissues at several time-points by histology and bulk RNA-sequencing. We found that this treatment caused supernumerary cone formation in an overwhelmingly synchronized fashion. This analysis revealed several categorical patterns of gene expression changes over time relative to DMSO treated control explants. These were placed in the temporal context of the activation of Otx2, a transcription factor that is expressed at the onset of photoreceptor development and that is required for both rod and cone formation. One group of interest had genes, such as Mybl1, Ascl1, Neurog2, and Olig2, that became upregulated by DAPT treatment before Otx2. Two other groups showed upregulated gene expression shortly after Otx2, either transiently or permanently. This included genes such as Mybl1, Meis2, and Podxl. Our data provide a developmental timeline of the gene expression events that underlie the initial steps of cone genesis and maturation. Applying this strategy to human retinal organoid cultures was also sufficient to induce a massive increase in cone genesis. Taken together, our results provide a temporal framework that can be used to elucidate the gene regulatory logic controlling cone photoreceptor development.

Prdm13 is required for Ebf3+ amacrine cell formation in the retina.

Amacrine interneurons play a critical role in the processing of visual signals within the retina. They are highly diverse, representing 30 or more distinct subtypes. Little is known about how amacrine subtypes acquire their unique gene expression and morphological features. We characterized the gene expression pattern of the zinc-finger transcription factor Prdm13 in the mouse. Consistent with a developmental role, Prdm13 was expressed by Ptf1a+ amacrine and horizontal precursors. Over time, Prdm13 expression diverged from the transiently expressed Ptf1a and marked just a subset of amacrine cells in the adult retina. While heterogeneous, we show that most of these Prdm13+ amacrine cells express the transcription factor Ebf3 and the calcium binding protein calretinin. Loss of Prdm13 did not affect the number of amacrine cells formed during development. However, we observed a modest loss of amacrine cells and increased apoptosis that correlated with the onset timing of Ebf3 expression. Adult Prdm13 loss-of-function mice had 25% fewer amacrine cells, altered calretinin expression, and a lack of Ebf3+ amacrines. Forcing Prdm13 expression in retinal progenitor cells did not significantly increase amacrine cell formation, Ebf3 or calretinin expression, and appeared detrimental to the survival of photoreceptors. Our data show that Prdm13 is not required for amacrine fate as a class, but is essential for the formation of Ebf3+ amacrine cell subtypes. Rather than driving subtype identity, Prdm13 may act by restricting competing fate programs to maintain identity and survival.

5HTR3A-driven GFP labels immature olfactory sensory neurons.

The ionotropic serotonin receptor, 5-HT3 , is expressed by many developing neurons within the central nervous system. Since the olfactory epithelium continues to generate new olfactory sensory neurons (OSNs) throughout life, we investigated the possibility that 5-HT3 is expressed in the adult epithelium. Using a transgenic mouse in which the promoter for the 5-HT3a subunit drives expression of green fluorescent protein (GFP), we assessed the expression of this marker in the olfactory epithelium of adult mice. Both the native 5-HT3a mRNA and GFP are expressed within globose basal cells of the olfactory and vomeronasal epithelium in adult mice. Whereas the 5-HT3a mRNA disappears relatively quickly after final cell division, the GFP label persists for about 5 days, thereby labeling immature OSNs in both the main olfactory system and vomeronasal organ. The GFP-labeled cells include both proliferative globose basal cells as well as immature OSNs exhibiting the hallmarks of ongoing differentiation including GAP43, PGP9.5, but the absence of olfactory marker protein. Some of the GFP-labeled OSNs show characteristics of more mature yet still developing OSNs including the presence of cilia extending from the apical knob and expression of NaV1.5, a component of the transduction cascade. These findings suggest that 5-HT3a is indicative of a proliferative or developmental state, regardless of age, and that the 5-HT3A GFP mice may prove useful for future studies of neurogenesis in the olfactory epithelium. J. Comp. Neurol. 525:1743-1755, 2017. © 2016 Wiley Periodicals, Inc.