Digital workflows for pathological assessment of rat estrous cycle stage using images of uterine horn and vaginal tissue.

Assessment of the estrous cycle of mature female mammals is an important component of verifying the efficacy and safety of drug candidates. The common pathological approach of relying on expert observation has several drawbacks, including laborious work and inter-viewer variability. The recent advent of image recognition technologies using deep learning is expected to bring substantial benefits to such pathological assessments. We herein propose 2 distinct deep learning-based workflows to classify the estrous cycle stage from tissue images of the uterine horn and vagina, respectively. These constructed models were able to classify the estrous cycle stages with accuracy comparable with that of expert pathologists. Our digital workflows allow efficient pathological assessments of the estrous cycle stage in rats and are thus expected to accelerate drug research and development.

A Positive Feedback Loop Comprising LHW-TMO5 and Local Auxin Biosynthesis Regulates Initial Vascular Development in Arabidopsis Roots.

The phytohormone auxin governs various developmental processes in plants including vascular formation. Auxin transport and biosynthesis are important factors in determining auxin distribution in tissues. Although the role of auxin transport in vein pattern formation is widely recognized, that of auxin biosynthesis in vascular development is poorly understood. Heterodimer complexes comprising two basic helix-loop-helix protein families, LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS5 (TMO5)/TMO5-LIKE1 (T5L1), are master transcriptional regulators of the initial process of vascular development. The LHW-TMO5/T5L1 dimers regulate vascular initial cell production, vascular cell proliferation and xylem fate determination in the embryo and root apical meristem (RAM). In this study, we investigated the function of local auxin biosynthesis in initial vascular development in RAM. Results showed that LHW-T5L1 upregulated the expression of YUCCA4 (YUC4), a key auxin biosynthesis gene. The expression of YUC4 was essential for promoting xylem differentiation and vascular cell proliferation in RAM. Conversely, auxin biosynthesis was required for maintaining the expression levels of LHW, TMO5/T5L1 and their targets. Our results suggest that local auxin biosynthesis forms a positive feedback loop for fine-tuning the level of LHW-TMO5/T5L1, which is necessary for initiating vascular development.

A Rho-actin signaling pathway shapes cell wall boundaries in Arabidopsis xylem vessels.

Patterned cell wall deposition is crucial for cell shapes and functions. In Arabidopsis xylem vessels, ROP11 GTPase locally inhibits cell wall deposition through microtubule disassembly, inducing pits in cell walls. Here, we show that an additional ROP signaling pathway promotes cell wall growth at pit boundaries. Two proteins, Boundary of ROP domain1 (BDR1) and Wallin (WAL), localize to pit boundaries and regulate cell wall growth. WAL interacts with F-actin and promotes actin assembly at pit boundaries while BDR1 is a ROP effector. BDR1 interacts with WAL, suggesting that WAL could be recruited to the plasma membrane by a ROP-dependent mechanism. These results demonstrate that BDR1 and WAL mediate a ROP-actin pathway that shapes pit boundaries. The study reveals a distinct machinery in which two closely associated ROP pathways oppositely regulate cell wall deposition patterns for the establishment of tiny but highly specialized cell wall domains.

A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels.

Rho GTPases play crucial roles in cell polarity and pattern formation. ROPs, Rho of plant GTPases, are widely involved in cell wall patterning in plants, yet the molecular mechanism underlying their action remains unknown. Arabidopsis ROP11 is locally activated to form plasma membrane domains, which direct formation of cell wall pits in metaxylem vessel cells through interaction with cortical microtubules. Here, we show that the pattern formation of cell wall pits is governed by ROP activation via a reaction-diffusion mechanism. Genetic analysis and reconstructive assays revealed that ROPGEF4/7 and ROPGAP3/4, which encode ROP activators and inactivators, respectively, regulated the formation of ROP-activated domains; these in turn determined the pattern of cell wall pits. Mathematical modelling showed that ROP-activation cycle generated ROP domains by reaction-diffusion mechanism. The model predicted that a positive feedback and slow diffusion of ROP11-ROPGEF4 complex were required to generate ROP-activated domains. ROPGEF4 formed a dimer that interacted with activated ROP11 in vivo, which could provide positive feedback for ROP activation. ROPGEF4 was highly stable on the plasma membrane and inhibited ROP11 diffusion. Our study indicated that ROP-based reaction-diffusion system self-organizes ROP-activated domains, thereby determines the pit pattern of metaxylem vessels.

Reconstruction of ROP GTPase Domains on the Plasma Membrane in Tobacco Leaves.

Rho-type small GTPases (Rho GTPases) play central roles in various cellular events. Rho GTPases are often activated locally on the plasma membrane, forming plasma membrane domains, which induce downstream signaling. We describe an experimental procedure designed for inducing the production of de novo plasma membrane domains using Arabidopsis ROP11 GTPase. Introduction of ROP11 and its activator and inactivator into the tobacco leaf epidermis leads to formation of ROP11-activated plasma membrane domains on the plasma membrane. Effectors and marker genes can also be introduced alongside ROP11. This reconstruction system allows identifying molecules regulating Rho GTPase polarization.

Novel coiled-coil proteins regulate exocyst association with cortical microtubules in xylem cells via the conserved oligomeric golgi-complex 2 protein.

Xylem vessel cells develop secondary cell walls in distinct patterns. Cortical microtubules are rearranged into distinct patterns and regulate secondary cell wall deposition; however, it is unclear how exocytotic membrane trafficking is linked to cortical microtubules. Here, we show that the novel coiled-coil proteins vesicle tethering 1 (VETH1) and VETH2 recruit EXO70A1, an exocyst subunit essential for correct patterning of secondary cell wall deposition, to cortical microtubules via the conserved oligomeric Golgi complex (COG) 2 protein. VETH1 and VETH2 encode an uncharacterized domain of an unknown function designated DUF869, and were preferentially up-regulated in xylem cells. VETH1-green fluorescent protein (GFP) and VETH2-GFP co-localized at novel vesicle-like small compartments, which exhibited microtubule plus-end-directed and end-tracking dynamics. VETH1 and VETH2 interacted with COG2, and this interaction promoted the association between cortical microtubules and EXO70A1 These results suggest that the VETH-COG2 complex ensures the correct secondary cell wall deposition pattern by recruiting exocyst components to cortical microtubules.

New insights into the dynamics of plant cell nuclei and chromosomes.

The plant lamin-like protein NMCP/AtLINC and orthologues of the SUN-KASH complex across the nuclear envelope (NE) show the universality of nuclear structure in eukaryotes. However, depletion of components in the connection complex of the NE in plants does not induce severe defects, unlike in animals. Appearance of the Rabl configuration is not dependent on genome size in plant species. Topoisomerase II and condensin II are not essential for plant chromosome condensation. Plant endoreduplication shares several common characteristics with animals, including involvement of cyclin-dependent kinases and E2F transcription factors. Recent finding regarding endomitosis regulator GIG1 shed light on the suppression mechanism of endomitosis in plants. The robustness of plants, compared with animals, is reflected in their genome redundancy. Spatiotemporal functional analyses using chromophore-assisted light inactivation, super-resolution microscopy, and 4D (3D plus time) imaging will reveal new insights into plant nuclear and chromosomal dynamics.