A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Neuronal migration prevents spatial competition in retinal morphogenesis.

The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1-3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning4, can be involved in coordinating morphogenesis.

Circuit mechanisms underlying embryonic retinal waves.

Spontaneous activity is a hallmark of developing neural systems. In the retina, spontaneous activity comes in the form of retinal waves, comprised of three stages persisting from embryonic day 16 (E16) to eye opening at postnatal day 14 (P14). Though postnatal retinal waves have been well characterized, little is known about the spatiotemporal properties or the mechanisms mediating embryonic retinal waves, designated stage 1 waves. Using a custom-built macroscope to record spontaneous calcium transients from whole embryonic retinas, we show that stage 1 waves are initiated at several locations across the retina and propagate across a broad range of areas. Blocking gap junctions reduced the frequency and size of stage 1 waves, nearly abolishing them. Global blockade of nAChRs similarly nearly abolished stage 1 waves. Thus, stage 1 waves are mediated by a complex circuitry involving subtypes of nAChRs and gap junctions. Stage 1 waves in mice lacking the ß2 subunit of the nAChRs (ß2-nAChR-KO) persisted with altered propagation properties and were abolished by a gap junction blocker. To assay the impact of stage 1 waves on retinal development, we compared the spatial distribution of a subtype of retinal ganglion cells, intrinsically photosensitive retinal ganglion cells (ipRGCs), which undergo a significant amount of cell death, in WT and ß2-nAChR-KO mice. We found that the developmental decrease in ipRGC density is preserved between WT and ß2-nAChR-KO mice, indicating that processes regulating ipRGC numbers and distributions are not influenced by spontaneous activity.

The Rx transcription factor is required for determination of the retinal lineage and regulates the timing of neuronal differentiation.

Understanding the molecular mechanisms leading to retinal development is of great interest for both basic scientific and clinical applications. Several signaling molecules and transcription factors involved in retinal development have been isolated and analyzed; however, determining the direct impact of the loss of a specific molecule is problematic, due to difficulties in identifying the corresponding cellular lineages in different individuals. Here, we conducted genome-wide expression analysis with embryonic stem (ES) cells devoid of the Rx gene, which encodes one of several homeobox transcription factors essential for retinal development. We performed three-dimensional differentiation of wild-type and mutant cells and compared their gene-expression profiles. The mutant tissue failed to differentiate into the retinal lineage and exhibited precocious expression of genes characteristic of neuronal cells. Together, these results suggest that Rx expression is an important biomarker of the retinal lineage and that it helps regulates appropriate differentiation stages.

Topographic map formation and the effects of NMDA receptor blockade in the developing visual system.

The development of functional topography in the developing brain follows a progression from initially coarse to more precisely organized maps. To examine the emergence of topographically organized maps in the retinotectal system, we performed longitudinal visual receptive field mapping by calcium imaging in the optic tectum of GCaMP6-expressing transgenic Xenopus laevis tadpoles. At stage 42, just 1 d after retinal axons arrived in the optic tectum, a clear retinotopic azimuth map was evident. Animals were imaged over the following week at stages 45 and 48, over which time the tectal neuropil nearly doubled in length and exhibited more precise retinotopic organization. By microinjecting GCaMP6s messenger ribonucleic acid (mRNA) into one blastomere of two-cell stage embryos, we acquired bilateral mosaic tadpoles with GCaMP6s expression in postsynaptic tectal neurons on one side of the animal and in retinal ganglion cell axons crossing to the tectum on the opposite side. Longitudinal observation of retinotopic map emergence revealed the presence of orderly representations of azimuth and elevation as early as stage 42, although presynaptic inputs exhibited relatively less topographic organization than the postsynaptic component for the azimuth axis. Retinotopic gradients in the tectum became smoother between stages 42 and 45. Blocking N-methyl-D-aspartate (NMDA) receptor conductance by rearing tadpoles in MK-801 did not prevent the emergence of retinotopic maps, but it produced more discontinuous topographic gradients and altered receptive field characteristics. These results provide evidence that current through NMDA receptors is dispensable for coarse topographic ordering of retinotectal inputs but does contribute to the fine-scale organization of the retinotectal projection.

The influence of spontaneous and visual activity on the development of direction selectivity maps in mouse retina.

In mice, retinal direction selectivity is organized in a map that aligns to the body and gravitational axes of optic flow, and little is known about how this map develops. We find direction selectivity maps are largely present at eye opening and develop normally in the absence of visual experience. Remarkably, in mice lacking the beta2 subunit of neuronal nicotinic acetylcholine receptors (ß2-nAChR-KO), which exhibit drastically reduced cholinergic retinal waves in the first postnatal week, selectivity to horizontal motion is absent while selectivity to vertical motion remains. We tested several possible mechanisms that could explain the loss of horizontal direction selectivity in ß2-nAChR-KO mice (wave propagation bias, FRMD7 expression, starburst amacrine cell morphology), but all were found to be intact when compared with WT mice. This work establishes a role for retinal waves in the development of asymmetric circuitry that mediates retinal direction selectivity via an unknown mechanism.

Spatial and temporal immunoreaction of nestin, CD44, collagen IX and GFAP in human retinal Müller cells in the developing fetal eye.

The purpose of this study was to investigate Müller cells during the fetal development of the human eye. Müller cells in eyes of 39 human fetuses (11-38 weeks of gestation, WOG) and 6 infants (5 died of abusive head trauma, AHT, aged 1-9 months) were immunohistochemically stained and investigated for spatial and temporal immunoreaction of nestin, CD44, collagen IX and GFAP, which are stem cell markers or markers of intermediate filaments, respectively, in one of the hitherto largest cohorts of fetal eyes. Müller cells could be detected immunohistochemically as early as 12 WOG by immunohistochemical staining with nestin. Nestin was more strongly expressed in Müller cells of the peripheral retina and a centroperipheral gradient of immunoreaction over time was observed. CD44 was predominantly expressed in fetal eyes of the late second and early third trimester between (23 and 27 WOG) and significantly stronger in the infant eyes. Collagen IX labeling in the central retina was significantly stronger than in more peripheral sectors and increased with fetal age. GFAP staining in Müller cells was seen in the eye of a fetus of 38 WOG who died postnatally and in the infant eyes with and without history of AHT. Additionally, GFAP staining was present in the astrocytes of fetal and infant eyes. All examined markers were expressed by Müller cells at different developmental stages highlighting the plasticity of Müller cells during the development of the human eye. GFAP should be cautiously used as a marker for AHT as it was also expressed in fetal astrocytes and Müller cells in eyes without history of AHT.

Single-cell ATAC-seq of fetal human retina and stem-cell-derived retinal organoids shows changing chromatin landscapes during cell fate acquisition.

We previously used single-cell transcriptomic analysis to characterize human fetal retinal development and assessed the degree to which retinal organoids recapitulate normal development. We now extend the transcriptomic analyses to incorporate single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), a powerful method used to characterize potential gene regulatory networks through the changes in accessible chromatin that accompany cell-state changes. The combination of scATAC-seq and single-cell RNA sequencing (scRNA-seq) provides a view of developing human retina at an unprecedented resolution. We identify key transcription factors relevant to specific fates and the order of the transcription factor cascades that define each of the major retinal cell types. The changing chromatin landscape is largely recapitulated in retinal organoids; however, there are differences in Notch signaling and amacrine cell gene regulation. The datasets we generated constitute an excellent resource for the continued improvement of retinal organoid technology and have the potential to inform and accelerate regenerative medicine approaches to retinal diseases.

Impact of GAD65 and/or GAD67 deficiency on perinatal development in rats.

GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform-deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)-deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1-/- ; Gad2-/- rats died at postnatal day 0 and showed little alveolar space in their lungs, suggesting that the cause of their death was respiratory failure. All Gad1-/- ; Gad2-/- rats and 18% of Gad1-/- ; Gad2+/- rats showed cleft palate. In contrast, none of the Gad mutant rats including Gad1-/- ; Gad2-/- rats, showed omphalocele. These results suggest that both rat GAD65 and GAD67 are involved in palate formation, while neither isoform is critical for abdominal wall formation. The GABA content in Gad1-/- ; Gad2-/- rat forebrains and retinas at embryonic day 20 was extremely low, indicating that almost all GABA was synthesized from glutamate by GADs in the perinatal period. The present study shows that Gad mutant rats are a good model for further defining the role of GABA during development.

Development and diversification of bipolar interneurons in the mammalian retina.

The bipolar interneurons of the mammalian retina have evolved as a diverse set of cells with distinct subtype characteristics, which reflect specialized contributions to visual circuitry. Fifteen subtypes of bipolar interneurons have been identified in the mouse retina, each with characteristic gene expression, morphology, and light responses. This review provides an overview of the developmental events that underlie the generation of the diverse bipolar cell class, summarizing the current knowledge of genetic programs that establish and maintain bipolar subtype fates, as well as the events that shape the final distribution of bipolar subtypes. With much left to be discovered, bipolar interneurons present an ideal model system for studying the interplay between cell-autonomous and non-cell-autonomous mechanisms that influence neuronal subtype development within the central nervous system.

Gene regulatory networks controlling temporal patterning, neurogenesis, and cell-fate specification in mammalian retina.

Gene regulatory networks (GRNs), consisting of transcription factors and their target sites, control neurogenesis and cell-fate specification in the developing central nervous system. In this study, we use integrated single-cell RNA and single-cell ATAC sequencing (scATAC-seq) analysis in developing mouse and human retina to identify multiple interconnected, evolutionarily conserved GRNs composed of cell-type-specific transcription factors that both activate genes within their own network and inhibit genes in other networks. These GRNs control temporal patterning in primary progenitors, regulate transition from primary to neurogenic progenitors, and drive specification of each major retinal cell type. We confirm that NFI transcription factors selectively activate expression of genes promoting late-stage temporal identity in primary retinal progenitors and identify other transcription factors that regulate rod photoreceptor specification in postnatal retina. This study inventories cis- and trans-acting factors that control retinal development and can guide cell-based therapies aimed at replacing retinal neurons lost to disease.

Widespread translational control regulates retinal development in mouse.

Retinal development is tightly regulated to ensure the generation of appropriate cell types and the assembly of functional neuronal circuitry. Despite remarkable advances have been made in understanding regulation of gene expression during retinal development, how translational regulation guides retinogenesis is less understood. Here, we conduct a comprehensive translatome and transcriptome survey to the mouse retinogenesis from the embryonic to the adult stages. We discover thousands of genes that have dynamic changes at the translational level and pervasive translational regulation in a developmental stage-specific manner with specific biological functions. We further identify genes whose translational efficiencies are frequently controlled by changing usage in upstream open reading frame during retinal development. These genes are enriched for biological functions highly important to neurons, such as neuron projection organization and microtubule-based protein transport. Surprisingly, we discover hundreds of previously uncharacterized micropeptides, translated from putative long non-coding RNAs and circular RNAs. We validate their protein products in vitro and in vivo and demonstrate their potentials in regulating retinal development. Together, our study presents a rich and complex landscape of translational regulation and provides novel insights into their roles during retinogenesis.

Regulatory Mechanisms of Retinal Photoreceptors Development at Single Cell Resolution.

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electric signals. With the discovery of the intrinsically photosensitive retinal ganglion cells, which regulate non-image-forming visual processes, our knowledge of the photosensitive cell family in the retina has deepened. Photoreceptor development is regulated by specific genes and proteins and involves a series of molecular processes including DNA transcription, post-transcriptional modification, protein translation, and post-translational modification. Single-cell sequencing is a promising technology for the study of photoreceptor development. This review presents an overview of the types of human photoreceptors, summarizes recent discoveries in the regulatory mechanisms underlying their development at single-cell resolution, and outlines the prospects in this field.

In utero intraocular AAV injection for early gene expression in the developing rodent retina.

The visual system is the best system to study activity-dependent sensory circuit development. The connections from the retina to the dorsal lateral geniculate nucleus, the retinogeniculate connections, undergo extensive remodeling during early postnatal life. Thus, techniques that allow the expression of transgenes early in the developing retina are essential to study visual system development. Here, we describe a protocol to express genes-of-interest in the developing mouse retina via in utero intraocular adeno-associated virus injections. For complete details on the use and execution of this protocol, please refer to Yasuda et al. (2021).

The Role of Small Molecules and Their Effect on the Molecular Mechanisms of Early Retinal Organoid Development.

Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers aim to manipulate these pathways to achieve better human representative models for retinal development and disease. To achieve this, a plethora of different small molecules and signaling factors have been used at various time points and concentrations in retinal organoid differentiations, with varying success. Additions differ from protocol to protocol, but their usefulness or efficiency has not yet been systematically reviewed. Interestingly, many of these small molecules affect the same and/or multiple pathways, leading to reduced reproducibility and high variability between studies. In this review, we make an inventory of the key signaling pathways involved in early retinogenesis and their effect on the development of the early retina in vitro. Further, we provide a comprehensive overview of the small molecules and signaling factors that are added to retinal organoid differentiation protocols, documenting the molecular and functional effects of these additions. Lastly, we comparatively evaluate several of these factors using our established retinal organoid methodology.

Seeing stars: Development and function of retinal astrocytes.

Throughout the central nervous system, astrocytes adopt precisely ordered spatial arrangements of their somata and arbors, which facilitate their many important functions. Astrocyte pattern formation is particularly important in the retina, where astrocytes serve as a template that dictates the pattern of developing retinal vasculature. Thus, if astrocyte patterning is disturbed, there are severe consequences for retinal angiogenesis and ultimately for vision - as seen in diseases such as retinopathy of prematurity. Here we discuss key steps in development of the retinal astrocyte population. We describe how fundamental developmental forces - their birth, migration, proliferation, and death - sculpt astrocytes into a template that guides angiogenesis. We further address the radical changes in the cellular and molecular composition of the astrocyte network that occur upon completion of angiogenesis, paving the way for their adult functions in support of retinal ganglion cell axons. Understanding development of retinal astrocytes may elucidate pattern formation mechanisms that are deployed broadly by other axon-associated astrocyte populations.

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.

A single-cell guide to retinal development: Cell fate decisions of multipotent retinal progenitors in scRNA-seq.

Recent advances in high throughput single-cell RNA sequencing (scRNA-seq) technology have enabled the simultaneous transcriptomic profiling of thousands of individual cells in a single experiment. To investigate the intrinsic process of retinal development, researchers have leveraged this technology to quantify gene expression in retinal cells across development, in multiple species, and from numerous important models of human disease. In this review, we summarize recent applications of scRNA-seq and discuss how these datasets have complemented and advanced our understanding of retinal progenitor cell competence, cell fate specification, and differentiation. Finally, we also highlight the outstanding questions in the field that advances in single-cell data generation and analysis will soon be able to answer.

Human neural organoids: Models for developmental neurobiology and disease.

Human organoids stand at the forefront of basic and translational research, providing experimentally tractable systems to study human development and disease. These stem cell-derived, in vitro cultures can generate a multitude of tissue and organ types, including distinct brain regions and sensory systems. Neural organoid systems have provided fundamental insights into molecular mechanisms governing cell fate specification and neural circuit assembly and serve as promising tools for drug discovery and understanding disease pathogenesis. In this review, we discuss several human neural organoid systems, how they are generated, advances in 3D imaging and bioengineering, and the impact of organoid studies on our understanding of the human nervous system.

Distribution of planar cell polarity proteins in the developing avian retina.

Planar cell polarity (PCP) is evolutionary conserved and play a critical role in proper tissue development and function. During central nervous system development, PCP proteins exhibit specific patterns of distribution and are indispensable for axonal growth, dendritogenesis, neuronal migration, and neuronal differentiation. The retina constitutes an excellent model in which to study molecular mechanisms involved in neural development. The analysis of the spatiotemporal expression of PCP proteins in this model constitutes an useful histological approach in order to identify possible roles of these proteins in retinogenesis. Immunohistochemical techniques revealed that Frz6, Celsr1, Vangl1, Pk1, Pk3, and Fat1 were present in emerging axons from recently differentiated ganglion cells in the chicken retina. Except for Vangl1, they were also asymmetrically distributed in differentiated amacrine cells. Pk1 and Pk3 were restricted in the outer nuclear layer to the outer segment of photoreceptors. Vangl1 was also located in the cell somata of Müller glia. Given these findings together, the distribution of PCP proteins in the developing chicken retina suggest essential roles in axonal guidance during early retinogenesis and a possible involvement in the establishment of cell asymmetry and maintenance of retinal cell phenotypes.