Essential functions of MLL1 and MLL2 in retinal development and cone cell maintenance

MLL1 (KMT2A) and MLL2 (KMT2B) are homologous members of the mixed-lineage leukemia (MLL) family of histone methyltransferases involved in epigenomic transcriptional regulation. Their sequence variants have been associated with neurological and psychological disorders, but little is known about their roles and mechanism of action in CNS development. Using mouse retina as a model, we previously reported the roles of MLL1 in retinal neurogenesis and horizontal cell maintenance. Here we determine roles of MLL2 and MLL1/MLL2 together in retinal development using conditional knockout (CKO) mice. Deleting Mll2 from Chx10+ retinal progenitors resulted in a similar phenotype as Mll1 CKO, but removal of both alleles produced much more severe deficits than each single CKO: 1-month double CKO mutants displayed null light responses in electroretinogram; thin retinal layers, including shorter photoreceptor outer segments with impaired phototransduction gene expression; and reduced numbers of M-cones, horizontal and amacrine neurons, followed by fast retinal degeneration. Despite moderately reduced progenitor cell proliferation at P0, the neurogenic capacity was largely maintained in double CKO mutants. However, upregulated apoptosis and reactive gliosis were detected during postnatal retinal development. Finally, the removal of both MLLs in fated rods produced a normal phenotype, but the CKO in M-cones impaired M-cone function and survival, indicating both cell non-autonomous and autonomous mechanisms. Altogether, our results suggest that MLL1/MLL2 play redundant roles in maintaining specific retinal neurons after cell fate specification and are essential for establishing functional neural networks.

Amoeboid-like neuronal migration ensures correct horizontal cell layer formation in the developing vertebrate retina

As neurons are often born at positions different than where they ultimately function, neuronal migration is key to ensure successful nervous system development. Radial migration during which neurons featuring unipolar and bipolar morphology, employ pre-existing processes or underlying cells for directional guidance, is the most well explored neuronal migration mode. However, how neurons that display multipolar morphology, without such processes, move through highly crowded tissue environments towards their final positions remains elusive. To understand this, we here investigated multipolar migration of horizontal cells in the zebrafish retina. We found that horizontal cells tailor their movements to the environmental spatial constraints of the crowded retina, by featuring several characteristics of amoeboid migration. These include cell and nucleus shape changes, and persistent rearward polarization of stable F-actin, which enable horizontal cells to successfully move through the crowded retina. Interference with the organization of the developing retina by changing nuclear properties or overall tissue architecture, hampers efficient horizontal cell migration and layer formation. Thus, cell-tissue interplay is crucial for efficient migration of horizontal cells in the retina. In view of high proportion of multipolar neurons, the here uncovered ameboid-like neuronal migration mode might also be crucial in other areas of the developing brain.

Synaptic Scaffolds, Ion Channels and Polyamines in Mouse Photoreceptor Synapses: Anatomy of a Signaling Complex

Synaptic signaling complexes are held together by scaffold proteins, each of which is selectively capable of interacting with a number of other proteins. In previous studies of rabbit retina, we found Synapse-Associated Protein-102 (SAP102) and Channel Associated Protein of Synapse-110 (Chapsyn110) selectively localized in the tips of horizontal cell processes at contacts with rod and cone photoreceptors, along with several interacting ion channels. We have examined the equivalent suites of proteins in mouse retina and found similarities and differences. In the mouse retina we identified Chapsyn110 as the scaffold selectively localized in the tips of horizontal cells contacting photoreceptors, with Sap102 more diffusely present. As in rabbit, the inward rectifier potassium channel Kir2.1 was present with Chapsyn110 on the tips of horizontal cell dendrites within photoreceptor invaginations, where it could provide a hyperpolarization-activated current that could contribute to ephaptic signaling in the photoreceptor synapses. Pannexin 1 and Pannexin 2, thought to play a role in ephaptic and/or pH mediated signaling, were present in the outer plexiform layer, but likely not in the horizontal cells. Polyamines regulate many ion channels and control the degree of rectification of Kir2.1 by imposing a voltage-dependent block. During the day polyamine immunolabeling was unexpectedly high in photoreceptor terminals compared to other areas of the retina. This content was significantly lower at night, when polyamine content was predominantly in Müller glia, indicating daily rhythms of polyamine content. Both rod and cone terminals displayed the same rhythm. While polyamine content was not prominent in horizontal cells, if polyamines are released, they may regulate the activity of Kir2.1 channels located in the tips of HCs. The rhythmic change in polyamine content of photoreceptor terminals suggests that a daily rhythm tunes the behavior of suites of ion channels within the photoreceptor synapses.

Comparison of Modern In Vitro Permeability Methods with the Aim of Investigation Nasal Dosage Forms

Nowadays, the intranasal route has become a reliable alternative route for drug administration to the systemic circulation or central nervous system. However, there are no official in vitro diffusion and dissolution tests especially for the investigation of nasal formulations. Our main goal was to study and compare a well-known and a lesser-known in vitro permeability investigation method, in order to ascertain which was suitable for the determination of drug permeability through the nasal mucosa from different formulations. The vertical diffusion cell (Franz cell) was compared with the horizontal diffusion model (Side-Bi-Side). Raw and nanonized meloxicam containing nasal dosage forms (spray, gel and powder) were tested and compared. It was found that the Side-Bi-Side cell was suitable for the investigation of spray and powder forms. In contrast, the gel was not measurable on the Side-Bi-Side cell; due to its high viscosity, a uniform distribution of the active substance could not be ensured in the donor phase. The Franz cell, designed for the analysis of semi-solid formulations, was desirable for the investigation of nasal gels. It can be concluded that the application of a horizontal cell is recommended for liquid and solid nasal preparations, while the vertical one should be used for semi-solid formulations.

Synaptic Remodeling in the Cone Pathway After Early Postnatal Horizontal Cell Ablation

The first synapse of the visual pathway is formed by photoreceptors, horizontal cells and bipolar cells. While ON bipolar cells invaginate into the photoreceptor terminal and form synaptic triads together with invaginating horizontal cell processes, OFF bipolar cells make flat contacts at the base of the terminal. When horizontal cells are ablated during retina development, no invaginating synapses are formed in rod photoreceptors. However, how cone photoreceptors and their synaptic connections with bipolar cells react to this insult, is unclear so far. To answer this question, we specifically ablated horizontal cells from the developing mouse retina. Following ablation around postnatal day 4 (P4)/P5, cones initially exhibited a normal morphology and formed flat contacts with OFF bipolar cells, but only few invaginating contacts with ON bipolar cells. From P15 on, synaptic remodeling became obvious with clustering of cone terminals and mislocalized cone somata in the OPL. Adult cones (P56) finally displayed highly branched axons with numerous terminals which contained ribbons and vesicular glutamate transporters. Furthermore, type 3a, 3b, and 4 OFF bipolar cell dendrites sprouted into the outer nuclear layer and even expressed glutamate receptors at the base of newly formed cone terminals. These results indicate that cones may be able to form new synapses with OFF bipolar cells in adult mice. In contrast, cone terminals lost their invaginating contacts with ON bipolar cells, highlighting the importance of horizontal cells for synapse maintenance. Taken together, our data demonstrate that early postnatal horizontal cell ablation leads to differential remodeling in the cone pathway: whereas synapses between cones and ON bipolar cells were lost, new putative synapses were established between cones and OFF bipolar cells. These results suggest that synapse formation and maintenance are regulated very differently between flat and invaginating contacts at cone terminals.

Cis-regulatory dissection of cone development reveals a broad role for Otx2 and Oc transcription factors

ABSTRACT The vertebrate retina is generated by retinal progenitor cells (RPCs), which produce >100 cell types. Although some RPCs produce many cell types, other RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). We used genome-wide assays of chromatin structure to compare the profiles of a restricted cone/HC RPC and those of other RPCs in chicks. These data nominated regions of regulatory activity, which were tested in tissue, leading to the identification of many cis-regulatory modules (CRMs) active in cone/HC RPCs and developing cones. Two transcription factors, Otx2 and Oc1, were found to bind to many of these CRMs, including those near genes important for cone development and function, and their binding sites were required for activity. We also found that Otx2 has a predicted autoregulatory CRM. These results suggest that Otx2, Oc1 and possibly other Onecut proteins have a broad role in coordinating cone development and function. The many newly discovered CRMs for cones are potentially useful reagents for gene therapy of cone diseases.

Function of cone and cone-related pathways in CaV1.4 IT mice

CaV1.4 L-type calcium channels are predominantly expressed in photoreceptor terminals playing a crucial role for synaptic transmission and, consequently, for vision. Human mutations in the encoding gene are associated with congenital stationary night blindness type-2. Besides rod-driven scotopic vision also cone-driven photopic responses are severely affected in patients. The present study therefore examined functional and morphological changes in cones and cone-related pathways in mice carrying the CaV1.4 gain-of function mutation I756T (CaV1.4-IT) using multielectrode array, patch-clamp and immunohistochemical analyses. CaV1.4-IT ganglion cell responses to photopic stimuli were seen only in a small fraction of cells indicative of a major impairment in the cone pathway. Though cone photoreceptors underwent morphological rearrangements, they retained their ability to release glutamate. Our functional data suggested a postsynaptic cone bipolar cell defect, supported by the fact that the majority of cone bipolar cells showed sprouting, while horizontal cells maintained contacts with cones and cone-to-horizontal cell input was preserved. Furthermore a reduction of basal Ca2+ influx by a calcium channel blocker was not sufficient to rescue synaptic transmission deficits caused by the CaV1.4-IT mutation. Long term treatments with low-dose Ca2+ channel blockers might however be beneficial reducing Ca2+ toxicity without major effects on ganglion cells responses.

A cell atlas of the chick retina based on single cell transcriptomics

Retinal structure and function have been studied in many vertebrate orders, but molecular characterization has been largely confined to mammals. We used single-cell RNA sequencing (scRNA-seq) to generate a cell atlas of the chick retina. We identified 136 cell types plus 14 positional or developmental intermediates distributed among the six classes conserved across vertebrates – photoreceptor, horizontal, bipolar, amacrine, retinal ganglion and glial cells. To assess morphology of molecularly defined types, we adapted a method for CRISPR-based integration of reporters into selectively expressed genes. For Müller glia, we found that transcriptionally distinct cells were regionally localized along the anterior-posterior, dorsal-ventral and central-peripheral retinal axes. We also identified immature photoreceptor, horizontal cell and oligodendrocyte types that persist into late embryonic stages. Finally, we analyzed relationships among chick, mouse and primate retinal cell classes and types. Our results provide a foundation for anatomical, physiological, evolutionary, and developmental studies of the avian visual system.