Retinal ganglion cells (RGCs) convey the major output of information collected from the eye to the brain. Thirty subtypes of RGCs have been identified to date. Here, we analyze 6225 RGCs (average of 5000 genes per cell) from right and left eyes by single-cell RNA-seq and classify them into 40 subtypes using clustering algorithms. We identify additional subtypes and markers, as well as transcription factors predicted to cooperate in specifying RGC subtypes. Zic1, a marker of the right eye-enriched subtype, is validated by immunostaining in situ. Runx1 and Fst, the markers of other subtypes, are validated in purified RGCs by fluorescent in situ hybridization (FISH) and immunostaining. We show the extent of gene expression variability needed for subtype segregation, and we show a hierarchy in diversification from a cell-type population to subtypes. Finally, we present a website for comparing the gene expression of RGC subtypes.

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Throughout evolution, secretion has played an essential role in the ability of organisms and single cells to survive in the face of a changing environment. Peptidylglycine α-amidating monooxygenase (PAM) is an integral membrane monooxygenase, first identified for its role in the biosynthesis of neuroendocrine peptides released by the regulated secretory pathway. PAM was subsequently identified in Chlamydomonas reinhardtii, a unicellular green alga, where it plays an essential role in constitutive secretion and in ciliogenesis. Reduced expression of C. reinhardtii PAM resulted in significant changes in secretion and ciliogenesis. Hence, a screen was performed for transcripts and proteins whose expression responded to changes in PAM levels in a mammalian corticotrope tumor cell line. The goal was to identify genes not previously known to play a role in secretion. The screen identified transcription factors, peptidyl prolyl isomerases, endosomal/lysosomal proteins, and proteins involved in tissue-specific responses to glucose and amino acid availability that had not previously been recognized as relevant to the secretory pathway. Perhaps reflecting the dependence of PAM on molecular oxygen, many PAM-responsive genes are known to be hypoxia responsive. The data highlight the extent to which the performance of the secretory pathway may be integrated into a wide diversity of signaling pathways.

Amino acids and other soluble α-amino compounds are liberated into a leaching medium from tissues of black locust bark, alfalfa, and wheat that are frozen to temperatures which are injurious to the tissues. The amounts liberated increase with lowering in freezing temperature and are proportional to the loss in vital capacity of the tissue. Insignificant amounts of amino acids are released by leaching of non-frozen tissue while a maximum is reached at freezing temperatures which are completely lethal. The amino acids liberated from frozen and thawed tissues must originate from the destruction of living cells by the freezing process. The determination of the concentration of amino acids in the medium in which the plant tissues are leached after freezing can be used therefore as a quantitative method for the estimation of the injury sustained in the freezing.The use of the ninhydrin reaction for the purpose of this determination is described and its application to the estimation of freezing injury and resistance in a variety of hardy and non-hardy tissues of alfalfa, wheat, and black locust tree bark is shown. The extension of this procedure to the determination of injury produced by toxic and other detrimental agents is indicated.

The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal Schwann cells (nm‐cSCs). This anatomical organization is believed to be important for central vision. Here we employed single‐cell RNA sequencing (scRNA‐seq), microscopy, and transgenics to characterize these nm‐cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA‐seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm‐cSCs. To confirm protein expression of cSC genes, we performed cross‐species validation, employing corneal whole‐mount immunostaining with confocal microscopy in mouse corneas. The expression of several representative proteins of nm‐cSCs were validated. As the proteolipid protein 1 (PLP1) gene was also expressed in nm‐cSCs, we explored the Plp1‐eGFP transgenic reporter mouse line to visualize cSCs. Specific and efficient eGFP expression was observed in cSCs in adult mice of different ages. Of several putative cornea‐specific SC genes identified, Dickkopf‐related protein 1 was shown to be present in nm‐cSCs. Taken together, our findings, for the first time, identify important insights and tools toward the study nm‐cSCs in isolated tissue and adult animals. We expect that our results will advance the future study of nm‐cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function.