Molecular codes for cell type specification in Brn3 retinal ganglion cells.

Visual information is conveyed from the eye to the brain by distinct types of retinal ganglion cells (RGCs). It is largely unknown how RGCs acquire their defining morphological and physiological features and connect to upstream and downstream synaptic partners. The three Brn3/Pou4f transcription factors (TFs) participate in a combinatorial code for RGC type specification, but their exact molecular roles are still unclear. We use deep sequencing to define (i) transcriptomes of Brn3a- and/or Brn3b-positive RGCs, (ii) Brn3a- and/or Brn3b-dependent RGC transcripts, and (iii) transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation, and synaptogenesis. We reveal a combinatorial code of TFs, cell surface molecules, and determinants of neuronal morphology that is differentially expressed in specific RGC populations and selectively regulated by Brn3a and/or Brn3b. This comprehensive molecular code provides a basis for understanding neuronal cell type specification in RGCs.

Potential of Gene Editing and Induced Pluripotent Stem Cells (iPSCs) in Treatment of Retinal Diseases.

The advent of gene editing has introduced the ability to make changes to the genome of cells, thus allowing for correction of genetic mutations in patients with monogenic diseases. Retinal diseases are particularly suitable for the application of this new technology because many retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and Leber congenital amaurosis (LCA), are monogenic. Moreover, gene delivery techniques such as the use of adeno-associated virus (AAV) vectors have been optimized for intraocular use, and phase III trials are well underway to treat LCA, a severe form of inherited retinal degeneration, with gene therapy. This review focuses on the use of gene editing techniques and another relatively recent advent, induced pluripotent stem cells (iPSCs), and their potential for the study and treatment of retinal disease. Investment in these technologies, including overcoming challenges such as off-target mutations and low transplanted cell integration, may allow for future treatment of many debilitating inherited retinal diseases.

Interaction patterns of nurturant support exchanged in online health social networking.

BACKGROUND: Expressing emotion in online support communities is an important aspect of enabling e-patients to connect with each other and expand their social resources. Indirectly it increases the amount of support for coping with health issues. Exploring the supportive interaction patterns in online health social networking would help us better understand how technology features impacts user behavior in this context. OBJECTIVE: To build on previous research that identified different types of social support in online support communities by delving into patterns of supportive behavior across multiple computer-mediated communication formats. Each format combines different architectural elements, affecting the resulting social spaces. Our research question compared communication across different formats of text-based computer-mediated communication provided on the MedHelp.org health social networking environment. METHODS: We identified messages with nurturant support (emotional, esteem, and network) across three different computer-mediated communication formats (forums, journals, and notes) of an online support community for alcoholism using content analysis. Our sample consisted of 493 forum messages, 423 journal messages, and 1180 notes. RESULTS: Nurturant support types occurred frequently among messages offering support (forum comments: 276/412 messages, 67.0%; journal posts: 65/88 messages, 74%; journal comments: 275/335 messages, 82.1%; and notes: 1002/1180 messages, 84.92%), but less often among messages requesting support. Of all the nurturing supports, emotional (ie, encouragement) appeared most frequently, with network and esteem support appearing in patterns of varying combinations. Members of the Alcoholism Community appeared to adapt some traditional face-to-face forms of support to their needs in becoming sober, such as provision of encouragement, understanding, and empathy to one another. CONCLUSIONS: The computer-mediated communication format may have the greatest influence on the supportive interactions because of characteristics such as audience reach and access. Other factors include perception of community versus personal space or purpose of communication. These results lead to a need for further research.

Belantamab mafodotin associated corneal microcyst-like epithelial changes.

PURPOSE: To report a case of bilateral corneal microcyst-like epithelial changes associated with belantamab mafodotin (belamaf) therapy. OBSERVATIONS: A 70-year-old man with refractory multiple myeloma was placed on belamaf, a recently FDA-approved treatment for relapsed or refractory multiple myeloma. He developed decreased visual acuity and bilateral corneal microcyst-like peripheral epithelial changes. Belamaf was withheld.Anterior segment OCT showed intra-epithelial opacities at various depths. After resolution of corneal changes and recovery of vision, belamaf was restarted. The patient underwent two additional treatments, each time with recurrence of diffuse microcyst-like corneal epithelial changes. It took a total of 8, 11.5 and 17 weeks after each respective infusion for the microcyst-like epithelial changes to resolve. This suggested a longer recovery time after each subsequent infusion. CONCLUSIONS AND IMPORTANCE: The care for patients on belamaf requires the collaboration of eye care providers and hematologists-oncologists to assess for ocular adverse effects and adjust treatment as necessary. Further study is needed to illustrate the mechanism of corneal microcyst-like epithelial changes and its effects on limbal stem cells.

Identification of retinal ganglion cell types and brain nuclei expressing the transcription factor Brn3c/Pou4f3 using a Cre recombinase knock-in allele.

Members of the POU4F/Brn3 transcription factor family have an established role in the development of retinal ganglion cell (RGCs) types, the main transducers of visual information from the mammalian eye to the brain. Our previous work using sparse random recombination of a conditional knock-in reporter allele expressing alkaline phosphatase (AP) and intersectional genetics had identified three types of Brn3c positive (Brn3c+ ) RGCs. Here, we describe a novel Brn3cCre mouse allele generated by serial Dre to Cre recombination and use it to explore the expression overlap of Brn3c with Brn3a and Brn3b and the dendritic arbor morphologies and visual stimulus response properties of Brn3c+ RGC types. Furthermore, we explore brain nuclei that express Brn3c or receive input from Brn3c+ neurons. Our analysis reveals a much larger number of Brn3c+ RGCs and more diverse set of RGC types than previously reported. Most RGCs expressing Brn3c during development are still Brn3c positive in the adult, and all express Brn3a while only about half express Brn3b. Genetic Brn3c-Brn3b intersection reveals an area of increased RGC density, extending from dorsotemporal to ventrolateral across the retina and overlapping with the mouse binocular field of view. In addition, we report a Brn3c+ RGC projection to the thalamic reticular nucleus, a visual nucleus that was not previously shown to receive retinal input. Furthermore, Brn3c+ neurons highlight a previously unknown subdivision of the deep mesencephalic nucleus. Thus, our newly generated allele provides novel biological insights into RGC type classification, brain connectivity, and cytoarchitectonic.

Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies.

Site-specific recombinases (SSRs) such as Cre are widely used in gene targeting and genetic approaches for cell labeling and manipulation. They mediate DNA strand exchange between two DNA molecules at dedicated recognition sites. Precise understanding of the Cre recombination mechanism, including the role of individual base pairs in its loxP target site, guided the generation of mutant lox sites that specifically recombine with themselves but not with the wild type loxP. This has led to the development of a variety of combinatorial Cre-dependent genetic strategies, such as multicolor reporters, irreversible inversions, or recombination-mediated cassette exchange. Dre, a Cre-related phage integrase that recognizes roxP sites, does not cross-react with the Cre-loxP system, but has similar recombination efficiency. We have previously described intersectional genetic strategies combining Dre and Cre. We now report a mutagenesis screen aimed at identifying roxP base pairs critical for self-recognition. We describe several rox variant sites that are incompatible with roxP, but are able to efficiently recombine with themselves in either purified systems or bacterial and eukaryotic tissue culture systems. These newly identified rox sites are not recognized by Cre, thus enabling potential combinatorial strategies involving Cre, Dre, and target loci including multiple loxP and roxP variants.

Notch activity modulates the responsiveness of neural progenitors to sonic hedgehog signaling.

Throughout the developing nervous system, neural stem and progenitor cells give rise to diverse classes of neurons and glia in a spatially and temporally coordinated manner. In the ventral spinal cord, much of this diversity emerges through the morphogen actions of Sonic hedgehog (Shh). Interpretation of the Shh gradient depends on both the amount of ligand and duration of exposure, but the mechanisms permitting prolonged responses to Shh are not well understood. We demonstrate that Notch signaling plays an essential role in this process, enabling neural progenitors to attain sufficiently high levels of Shh pathway activity needed to direct the ventral-most cell fates. Notch activity regulates subcellular localization of the Shh receptor Patched1, gating the translocation of the key effector Smoothened to primary cilia and its downstream signaling activities. These data reveal an unexpected role for Notch shaping the interpretation of the Shh morphogen gradient and influencing cell fate determination.