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RhoU and RhoV are members of the Rho family of small GTPases that comprise their own subfamily. RhoUV GTPases are classified as atypical due to the kinetics of their GTP/GDP binding cycles. They also possess unique N- and C-termini that regulate their subcellular localization and activity. RhoU and RhoV have been linked to cytoskeletal regulation, cell adhesion, and cell migration. They each exhibit distinct expression patterns during embryonic development and diseases such as cancer metastasis, suggesting they have specialized functions. In this review, we will discuss the known functions of RhoU and RhoV, with a focus on their roles in early development, organogenesis, and disease.

Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. In this study, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

AbstractInducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. Here, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

Introduction and Objectives Over the past two decades, advancements in screening programs have led to a decrease in the incidence and mortality rates of colorectal cancer. The recommended benchmark for primary screening colonoscopy adenoma detection rate (ADR) is 25%. However, recent research supports higher ADR benchmarks following positive stool testing. Findings from a Dutch screening program have suggested higher ADRs in fecal immunochemical test positive patients with an inverse relationship with interval cancer development. Our study aims to assess ADRs in a U.S. patient population with positive stool screenings and investigate any correlation to occurrences of interval cancers. Materials and Methods Data from all positive stool-based screening participants who subsequently underwent colonoscopy at a tertiary care center between 2017 and 2021 were collected. A retrospective chart review was performed to determine the ADR and interval colon cancers. Results From a total of 120 patients (32 fecal occult blood test [FOBT] positive patients, 43 fecal immunochemical test [FIT] positive patients, 45 FIT-DNA-positive patients), the average ADR was 35%. Nonadvanced polyps were the most identified adenomas at 78.6%. No interval colorectal cancer cases were identified. There was a clear difference in ADR between stool-testing methods, with FIT-DNA showing higher ADRs than FIT and FOBT. Conclusion Endoscopists should recognize the importance of higher ADR targets in colonoscopies conducted after positive stool-based screening as a means to maintain high-quality colonoscopy standards. [ABSTRACT FROM AUTHOR]

During embryogenesis, the germ layers, including the endoderm, undergo convergence and extension movements to narrow and elongate the body plan. In zebrafish, the dorsal migration of endodermal cells during gastrulation is controlled by chemokine signaling, but little is known about how they migrate during segmentation. Here, we show that glypican 4 (Gpc4), a member of the heparin sulfate proteoglycan family, is required for efficient migration of anterior endodermal cells during early segmentation, regulating Rac activation to maintain polarized actin-rich lamellipodia. An endoderm transplantation assay showed that Gpc4 regulates endoderm migration in a non-cell-autonomous fashion. Further analyses revealed that the impaired endoderm migration in gpc4 mutants results from increases in the expression and assembly of fibronectin and laminin, major components of the extracellular matrix (ECM). Notably, we found that matrix metalloproteinase 14 (Mmp14a/b) is required for the control of ECM expression during endoderm migration, with Gpc4 acting through Mmp14a/b to limit ECM expression. Our results suggest that Gpc4 is crucial for generating the environment required for efficient migration of endodermal cells, uncovering a novel function of Gpc4 during development.

Here, we describe an optogenetic gene expression system optimized for use in zebrafish. This system overcomes the limitations of current inducible expression systems by enabling robust spatial and temporal regulation of gene expression in living organisms. Because existing optogenetic systems show toxicity in zebrafish, we re-engineered the blue-light-activated EL222 system for minimal toxicity while exhibiting a large range of induction, fine spatial precision and rapid kinetics. We validate several strategies to spatially restrict illumination and thus gene induction with our new TAEL (TA4-EL222) system. As a functional example, we show that TAEL is able to induce ectopic endodermal cells in the presumptive ectoderm via targeted sox32 induction. We also demonstrate that TAEL can be used to resolve multiple roles of Nodal signaling at different stages of embryonic development. Finally, we show how inducible gene editing can be achieved by combining the TAEL and CRISPR/Cas9 systems. This toolkit should be a broadly useful resource for the fish community.

Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions.

The author states that raising her twins the Montessori way has made her life easy. Imagine two 1-year-olds eating entire meals on their own, setting their own tables by 20 months, and becoming potty-trained before 2. These are not statistics found in just one household. Children raised the Montessori way can take care of themselves and their environment at surprisingly young ages. The author shares her beliefs and experiences of raising her twins the Montessori way and promoting independence.

One barrier to widespread public access to empirically supported treatments (ESTs) is the limited availability and high cost of professionals trained to deliver them. Our earlier work from 2 clinical trials demonstrated that front-line addiction counselors could be trained to deliver a manualized, group-based cognitive behavioral therapy (GCBT) for depression, a prototypic example of an EST, with a high level of adherence and competence. This follow-up article provides specific recommendations for the selection and initial training of counselors, and for the structure and process of their ongoing clinical supervision. We highlight unique challenges in working with counselors unaccustomed to traditional clinical supervision. The recommendations are based on comprehensive feedback derived from clinician notes taken throughout the clinical trials, a focus group with counselors conducted 1 year following implementation, and interviews with key organization executives and administrators. (Contains 4 tables.)