The formation of boundaries between or within tissues is a fundamental aspect of animal development. In the developing vertebrate hindbrain, boundaries separate molecularly and neuroanatomically distinct segments called rhombomeres. Transplantation studies have suggested that rhombomere boundaries form by the local sorting out of cells with different segmental identities. This sorting-out process has been shown to involve repulsive interactions between cells expressing an Eph receptor tyrosine kinase, EphA4, and cells expressing its ephrinB ligands. Although a model for rhombomere-boundary formation based on repulsive Eph-ephrin signaling is well established in the literature, the predictions of this model have not been tested in loss-of-function experiments. Here, we eliminate EphA4 and ephrinB2a proteins in zebrafish with antisense morpholinos (MO) and find that rhombomere boundaries are disrupted in EphA4MO embryos, consistent with a requirement for Eph-ephrin signaling in boundary formation. However, in mosaic embryos, we observe that EphA4MO cells and EphA4-expressing cells sort from one another, an observation that is not predicted by the Eph-ephrin repulsion model but instead suggests that EphA4 promotes cell adhesion within the rhombomeres in which it is expressed. Differential cell adhesion is known to be an effective mechanism for cell sorting. We therefore propose that the well-known EphA4-dependent repulsion between rhombomeres operates in parallel with the EphA4-dependent adhesion within rhombomeres described here to drive the cell sorting that underlies rhombomere-boundary formation.

In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G 1 arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.

During vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell-cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps. ProtocolA step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage. One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps. To proteolytically dechorionate embryos, incubate at the 1-cell stage for~1-5' in a 0.5 mg/ml pronase solution as described in the Zebrafish Book 1. Moniter dechorionation closely and submerge embryos in Embryo Medium (EM) with Pen/Strep 1 as soon as the chorions begin to visibly collapse. Once released from their chorions, blastula and gastrula stage embryos are fragile and will stick to plastic or disintegrate if exposed to air. Therefore keep dechorionated embryos submerged in Pen/Strep EM, transfer between dishes using a wide-bore fire-polished glass pipet, and maintain in either agar-coated plastic dishes (1.2% agarose in Pen/Strep EM) or autoclaved glass petri dishes. Injecting dechorionated embryos with lineage marker.Prepare an injection dish by inserting a glass slide at a 45 degree angle across the widest part of a Petri dish three-quarters full of 1.2% agarose made in Pen/Strep EM. ...

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans. These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.

The midbrain-hindbrain boundary (MHB) is a well-known organizing center during vertebrate brain development. The MHB forms at the expression boundary of Otx2 and Gbx2, mutually repressive homeodomain transcription factors expressed in the midbrain/forebrain and anterior hindbrain, respectively. The genetic hierarchy of gene expression at the MHB is complex, involving multiple positive and negative feedback loops that result in the establishment of non-overlapping domains of Wnt1 and Fgf8 on either side of the boundary and the consequent specification of the cerebellum. The cerebellum derives from the dorsal part of the anterior-most hindbrain segment, rhombomere 1 (r1), which undergoes a distinctive morphogenesis to give rise to the cerebellar primordium within which the various cerebellar neuron types are specified. Previous studies in the mouse have shown that Gbx2 is essential for cerebellar development. Using zebrafish mutants we show here that in the zebrafish gbx1 and gbx2 are required redundantly for morphogenesis of the cerebellar primordium and subsequent cerebellar differentiation, but that this requirement is alleviated by knocking down Otx. Expression of fgf8, wnt1 and the entire MHB genetic program is progressively lost in gbx1-;gbx2- double mutants but is rescued by Otx knock-down. This rescue of the MHB genetic program depends on rescued Fgf signaling, however the rescue of cerebellar primordium morphogenesis is independent of both Gbx and Fgf. Based on our findings we propose a revised model for the role of Gbx in cerebellar development.

In budding yeast, Cla4 and Ste20, two p21-activated kinases, contribute to numerous morphogenetic processes. Loss of Ste20 or Cla4 individually confers distinct phenotypes, implying that they regulate different processes. However, loss of both proteins is lethal, suggesting some functional overlap. To explore the role(s) of Cla4, we and others have sought mutations that are lethal in a cla4⌬ strain. These mutations define Ͼ60 genes. Recently, both Ste20 and Cla4 have been implicated in mitotic exit. Here, we identify a genetic interaction between PHO85, which encodes a cyclin-dependent kinase, and CLA4. We further show that the Pho85-coupled G 1 cyclins Pcl1 and Pcl2 contribute to this Pho85 role. We performed a twohybrid screen with Pcl1. Three Pcl1-interacting proteins were identified: Ncp1, Hms1, and a novel ATPase dubbed Epa1. Each of these proteins interacts with Pcl1 in GST pull-down experiments and is specifically phosphorylated by Pcl1•Pho85 complexes. NCP1, HMS1, and EPA1 also genetically interact with CLA4. Like Cla4, the proteins Hms1, Ncp1, and Pho85 appear to affect mitotic exit, a conclusion that follows from the mislocalization of Cdc14, a key mitotic regulator, in strains lacking these proteins. We propose a model in which the G 1 Pcl1•Pho85 complex regulates mitotic exit machinery.

Background: Outdoor education (OE) should be understood in place, time, and culture because it is not a universal teaching approach. We currently know little about what constitutes the “Canadian ways” of doing OE or what students gain from the experience. Purpose: Our goal was to (a) identify the underlying factors guiding OE programs in Canada and (b) describe the learning outcomes and psychosocial benefits of engaging in OE from the students’ perspective. Methodology/Approach: We conducted a systematic review of qualitative studies that examined OE in Canada. We searched published studies from electronic databases (1980-2018). We used meta-ethnography to synthesize the findings. Findings/Conclusions: We reviewed 21 studies reporting on the experiences of 508 students. Using thematic analysis, we identified eight themes highlighting process, goals, and learning outcomes. We developed a model that describes the common teaching components, learning process, and short-term outcomes for OE in Canada. Implications: Our results represent the first study to synthesize OE published research in Canada, which help form a unified voice and a distinctive national identity for OE in Canada. Our results serve as a catalyst for educators to share ideas, practices, and learning goals.