Generation and Staging of Human Retinal Organoids Based on Self-Formed Ectodermal Autonomous Multi-Zone System

Methods for stem cell-derived, three-dimensional retinal organoids induction have been established and shown great potential for retinal development modeling and drug screening. Herein, we reported an exogenous-factors-free and robust method to generate retinal organoids based on “self-formed ectodermal autonomous multi-zone” (SEAM) system, a two-dimensional induction scheme that can synchronously generate multiple ocular cell lineages. Characterized by distinct morphological changes, the differentiation of the obtained retinal organoids could be staged into the early and late differentiation phases. During the early differentiation stage, retinal ganglion cells, cone photoreceptor cells (PRs), amacrine cells, and horizontal cells developed; whereas rod PRs, bipolar cells, and Müller glial cells were generated in the late differentiation phase, resembling early-phase and late-phase retinogenesis in vivo. Additionally, we modified the maintenance strategy for the retinal organoids and successfully promoted their long-term survival. Using 3D immunofluorescence image reconstruction and transmission electron microscopy, the substantial mature PRs with outer segment, inner segment and ribbon synapse were demonstrated. Besides, the retinal pigment epithelium (RPE) was induced with distinct boundary and the formation of ciliary margin was observed by co-suspending retina organoids with the zone containing RPE. The obtained RPE could be expanded and displayed similar marker expression, ultrastructural feature and functional phagocytosis to native RPE. Thus, this research described a simple and robust system which enabled generation of retina organoids with substantial mature PRs, RPE and the ciliary margin without the need of exogenous factors, providing a new platform for research of retinogenesis and retinal translational application.

Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis

The single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins (NPs) and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a folded RNA and a deformed vRNP associated with a looped RNA. Interestingly, the looped RNA was a double-stranded RNA, which likely comprises a nascent RNA and the template RNA detached from NPs of the vRNP. These results suggest that while some vRNPs keep their helical structures during RNA synthesis, for the repeated cycle of RNA synthesis, others accidentally become structurally deformed, which likely results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural feature of vRNPs during RNA synthesis.

Structural and Ultrastructural Morphological Evaluation of Giant Anteater (Myrmecophaga tridactyla) Prostate Gland

The giant anteater (Myrmecophaga tridactyla) is a vulnerable species from Central and South America, and is considered possibly extinct in Belize, Guatemala, El Salvador, and Uruguay. Due to the species’ conservation and reproductive importance, this research aimed to characterize the morphology, histochemical, immunohistochemical, and ultrastructural feature of the giant anteater prostate gland. For this, we collected 11 giant anteater prostate glands and performed macroscopic, morphological, histochemical, immunohistochemical, and ultrastructural analysis. Nine prostate glands from an adult subject and two from young subjects were studied. Grossly, the adult giant anteater prostate gland is divided in two distinct zones; the central zones (composed mainly of ducts) and the peripheral zones (of acini formed by secretory cells). The secretory cells showed positive periodic acid–Schiff staining. Furthermore, the immunohistochemical characterization revealed a similar human prostate pattern, with p63 staining basal cells, uroplakin III (UPIII) superficial cells of prostatic urethra, androgen receptor (AR) expressing nucleus of secretory and stromal cells, and prostatic specific antigen (PSA) staining prostatic epithelial cells. Overall, our research provided an in-depth morphological description of the giant anteater’s prostate gland, providing valuable information for futures studies focused on giant anteater conservation.

Structural and Ultrastructural Morphological Evaluation of Giant Anteater (Myrmecophaga Tridactyla) Prostate Gland

The giant anteater (Myrmecophaga tridactyla) is a vulnerable species from Central and South Americas, that is considered possibly extinct in Belize, Guatemala, El Salvador, and Uruguay. Due to the species conservations and reproduction’s importance, this research aimed to characterize the morphology, histochemical, immunohistochemical, and ultrastructural feature of the giant anteater prostate gland. For this, we collected 11 giant anteater prostate glands and performed macroscopic, morphological, histochemical, immunohistochemical, and ultrastructural analysis. Nine-prostate glands from adult and two from young subjects were studied. Grossly, the adult giant anteater prostate gland is divided in two distinct zones; the central zones composed mainly of ducts and the peripheral zones of acini formed by secretory cells. The secretory cells showed positive periodic acid–Schiff staining. Furthermore, the immunohistochemical characterization revealed a similar human prostate pattern, with p63 staining basal cells, UPIII superficial cells of prostatic urethra, AR expressing nucleus of secretory and stromal cells, and PSA staining prostatic epithelial cells. Overall, our research provided an indepth morphological description of the giant anteater prostate gland, providing a valuable information for futures studies focused on giant anteater conservation.

Differential enrichment of TTF-I and Tip5 in the T-like promoter structures of the rDNA contribute to the epigenetic response of Cyprinus carpio during environmental adaptation

To ensure homeostasis, ectothermic organisms adapt to environmental variations through molecular mechanisms. We previously reported that during the seasonal acclimatization of the common carp Cyprinus carpio, molecular and cellular functions are reprogrammed, resulting in distinctive traits. Importantly, the carp undergoes a drastic rearrangement of nucleolar components during adaptation. This ultrastructural feature reflects a fine modulation of rRNA gene transcription. Specifically, we identified the involvement of the transcription termination factor I (TTF-I) and Tip-5 (member of nucleolar remodeling complex, NoRC) in the control of rRNA transcription. Our results suggest that differential Tip5 enrichment is essential for silencing carp ribosomal genes and that the T0 element is key for regulating the ribosomal gene during the acclimatization process. Interestingly, the expression and content of Tip5 were significantly higher in winter than in summer. Since carp ribosomal gene expression is lower in the winter than in summer, and considering that expression concomitantly occurs with nucleolar ultrastructural changes of the acclimatization process, these results indicate that Tip5 importantly contributes to silencing the ribosomal genes. In conclusion, the current study provides novel evidence on the contributions of TTF-I and NoRC in the environmental reprogramming of ribosomal genes during the seasonal adaptation process in carp.

Unusual ultrastructural findings in dendrites of pyramidal neurons in the cerebral cortex of rabies-infected mice

Previous studies using the Golgi technique have demonstrated alterations in the dendritic morphology of pyramidal neurons of the cerebral cortex of mice inoculated with the rabies virus. However, knowledge about the fine structure of dendrites in rabies infection is scarce. This work had the aim of studying the ultrastructure of dendrites in cortical pyramidal neurons of rabies-infected mice. Mice were inoculated intramuscularly with a street rabies virus of canine origin. The animals that showed an advanced stage of disease were fixed by perfusion with glutaraldehyde and paraformaldehyde. Brains were removed and cut on a vibratome to obtain coronal slices of 200 micrometers of thickness. Vibratome slices were subjected to the following treatment: postfixation, dehydration, embedding in epoxy resin and polymerization between glass slides. Ultrathin sections of oriented tissue fragments from the cerebral cortex were obtained and observed under electron microscope. The most significant ultrastructural findings were located within distal dendrites of cortical pyramidal neurons: loss of mitochondria, disorganization and loss of microtubules, formation of vacuoles interrupting the continuity of the cytoplasm and formation of myelin-like figures. These strange myelin figures, which apparently had not been reported in previous studies of rabies, were the most noticeable ultrastructural feature. They also differ from the best known myelin figures formed by concentric lamellae. The possible origin of these myelin figures as result of mitochondrial degeneration is discussed.

Unusual ultrastructural findings in dendrites of pyramidal neurons in the cerebral cortex of rabies-infected mice

Previous studies using the Golgi technique have demonstrated alterations in the dendritic morphology of pyramidal neurons of the cerebral cortex of mice inoculated with the rabies virus. However, knowledge about the fine structure of dendrites in rabies infection is scarce. This work had the aim of studying the ultrastructure of dendrites in cortical pyramidal neurons of rabies-infected mice. Mice were inoculated intramuscularly with a street rabies virus of canine origin. The animals that showed an advanced stage of disease were fixed by perfusion with glutaraldehyde and paraformaldehyde. Brains were removed and cut on a vibratome to obtain coronal slices of 200 micrometers of thickness. Vibratome slices were subjected to the following treatment: postfixation, dehydration, embedding in epoxy resin and polymerization between glass slides. Ultrathin sections of oriented tissue fragments from the cerebral cortex were obtained and observed under electron microscope. The most significant ultrastructural findings were located within distal dendrites of cortical pyramidal neurons: loss of mitochondria, disorganization and loss of microtubules, formation of vacuoles interrupting the continuity of the cytoplasm and formation of myelin-like figures. These strange myelin figures, which apparently had not been reported in previous studies of rabies, were the most noticeable ultrastructural feature. They also differ from the best known myelin figures formed by concentric lamellae. The possible origin of these myelin figures as result of mitochondrial degeneration is discussed.