Daigo Inoue

One for All—A Highly Efficient and Versatile Method for Fluorescent Immunostaining in Fish Embryos

Background For the detection and sub-cellular (co)-localization of proteins in the context of the tissue or organism immunostaining in whole mount preparations or on sections is still the best approach. So far, each antibody required its own fixation and antigen retrieval protocol so that optimizing immunostaining turned out to be tedious and time consuming. Methodology/Principal Finding Here we present a novel method to efficiently retrieve the antigen in a widely applicable standard protocol, facilitating fluorescent immunostaining of both cryosections and whole mount preparations in zebrafish (Danio rerio) and medaka (Oryzias latipes). Conclusions/Significance Our method overcomes the loss of sections and damage of tissue and cell morphology, and allows parallel immunostaining in multiple colors, co-immunostaining with fluorescent proteins in transgenic fish lines and in combination with whole mount in situ hybridization.

The centriolar satellite protein SSX2IP promotes centrosome maturation

SSX2IP promotes centrosome maturation and maintenance at the onset of vertebrate development, preserving centrosome integrity and mitosis during rapid cleavage divisions and in somatic cells.

Co-expression of VAL- and TMT-opsins uncovers ancient photosensory interneurons and motorneurons in the vertebrate brain.

Evolutionarily conserved, nonvisual opsins appear to endow specific interneurons and motorneurons of the vertebrate brain with light sensitivity, suggesting that environmental light may be able to modulate information processing.

Emi2 Inhibition of the Anaphase-promoting Complex/Cyclosome Absolutely Requires Emi2 Binding via the C-Terminal RL Tail

Both the D-box and the zinc-binding region (ZBR) of Emi2 are implicated in APC/C inhibition. This article shows that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. The RL tail apparently promotes the inhibitory interactions of the D-box and the ZBR with the APC/C. The RL tail thus serves as a docking site for the APC/C.

Dynamic Regulation of Emi2 by Emi2-Bound Cdk1/Plk1/CK1 and PP2A-B56 in Meiotic Arrest of Xenopus Eggs

In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Mos and Emi2, an inhibitor of the APC/C ubiquitin ligase. In Xenopus, Cdk1 phosphorylates Emi2 and both destabilizes and inactivates it, whereas Mos recruits PP2A phosphatase to antagonize the Cdk1 phosphorylation. However, how Cdk1 phosphorylation inhibits Emi2 is largely unknown. Here we show that multiple N-terminal Cdk1 phosphorylation motifs bind cyclin B1-Cdk1 itself, Plk1, and CK1δ/ε to inhibit Emi2. Plk1, after rebinding to other sites by self-priming phosphorylation, partially destabilizes Emi2. Cdk1 and CK1δ/ε sequentially phosphorylate the C-terminal APC/C-docking site, thereby cooperatively inhibiting Emi2 from binding the APC/C. In the presence of Mos, however, PP2A-B56β/ε bind to Emi2 and keep dephosphorylating it, particularly at the APC/C-docking site. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP2A phosphatase. Our data also suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation.

Emi2 Inhibition of the APC/C Absolutely Requires Emi2 Binding via the C-terminal RL Tail

Both the D-box and the zinc-binding region (ZBR) of Emi2 are implicated in APC/C inhibition. This article shows that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. The RL tail apparently promotes the inhibitory interactions of the D-box and the ZBR with the APC/C. The RL tail thus serves as a docking site for the APC/C.

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis‐regulatory element (pCRE) of the retina‐specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2‐positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans‐regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain‐ and loss‐of‐function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2‐mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina.

Noninvasive In Toto Imaging of the Thymus Reveals Heterogeneous Migratory Behavior of Developing T Cells

The migration of developing T cells (thymocytes) between distinct thymic microenvironments is crucial for their development. Ex vivo studies of thymus tissue explants suggest two distinct migratory behaviors of thymocytes in the thymus. In the cortex, thymocytes exhibit a stochastic migration, whereas medullary thymocytes show confined migratory behavior. Thus far, it has been difficult to follow all thymocytes in an entire thymus and relate their differentiation steps to their migratory dynamics. To understand the spatial organization of the migratory behavior and development of thymocytes in a fully functional thymus, we developed transgenic reporter lines for the chemokine receptors ccr9a and ccr9b, as well as for rag2, and used them for noninvasive live imaging of the entire thymus in medaka (Oryzias latipes). We found that the expression of these two chemokine receptors in the medaka juvenile thymus defined two spatially distinct subpopulations of thymocytes. Landmark events of T cell development including proliferation, somatic recombination, and thymic selection can be mapped to subregions of the thymus. The migratory behavior of thymocytes within each of the subpopulations is equally heterogeneous, and specific migratory behaviors are not associated with particular domains in the thymus. During the period when thymocytes express rag2 their migratory behavior was more homogeneous. Therefore, the migratory behavior of thymocytes is partly correlated with their developmental stage rather than being defined by their spatial localization.

Loss and Rebirth of the Animal Microtubule Organizing Center: How Maternal Expression of Centrosomal Proteins Cooperates with the Sperm Centriole in Zygotic Centrosome Reformation

Centrosomes are the main microtubule organizing centers in animal cells. In particular during embryogenesis, they ensure faithful spindle formation and proper cell divisions. As metazoan centrosomes are eliminated during oogenesis, they have to be reassembled upon fertilization. Most metazoans use the sperm centrioles as templates for new centrosome biogenesis while the eggs cytoplasm re‐prepares all components for on‐going centrosome duplication in rapidly dividing embryonic cells. We discuss our knowledge and the experimental challenges to analyze zygotic centrosome reformation, which requires genetic experiments to enable scrutinizing respective male and female contributions. Male and female knockout animals and mRNA injection to mimic maternal expression of centrosomal proteins could point a way to the systematic molecular dissection of the process. The most recent data suggest that timely expression of centrosome components in oocytes is the key to zygotic centrosome reformation that uses male sperm as coordinators for de novo centrosome production.

Expression of the novel maternal centrosome assembly factor Wdr8 is required for vertebrate embryonic mitoses.

The assembly of the first centrosome occurs upon fertilisation when male centrioles recruit pericentriolar material (PCM) from the egg cytoplasm. The mechanisms underlying the proper assembly of centrosomes during early embryogenesis remain obscure. We identify Wdr8 as a novel maternally essential protein that is required for centrosome assembly during embryonic mitoses of medaka (Oryzias latipes). By CRISPR–Cas9-mediated knockout, maternal/zygotic Wdr8-null (m/zWdr8−/−) blastomeres exhibit severe defects in centrosome structure that lead to asymmetric division, multipolar mitotic spindles and chromosome alignment errors. Via its WD40 domains, Wdr8 interacts with the centriolar satellite protein SSX2IP. Combining targeted gene knockout and in vivo reconstitution of the maternally essential Wdr8–SSX2IP complex reveals an essential link between maternal centrosome proteins and the stability of the zygotic genome for accurate vertebrate embryogenesis. Our approach provides a way of distinguishing maternal from paternal effects in early embryos and should contribute to understanding molecular defects in human infertility.