The SkillsCenter: Creating scalable research opportunities for STEM students.

Undergraduate students generally need laboratory skills and experience to be accepted into a position within an academic lab or a company. However, those settings are traditionally where students would develop that necessary expertise. We developed a laboratory course paradigm to equip students with the skills they need to access future opportunities.

Making mechanistic sense: are we teaching students what they need to know?

Evaluating learning outcomes depends upon objective and actionable measures of what students know - that is, what can they do with what they have learned. In the context of a developmental biology course, a capstone of many molecular biology degree programs, I asked students to predict the behaviors of temporal and spatial signaling gradients. Their responses led me to consider an alternative to conventional assessments, namely a process in which students are asked to build and apply plausible explanatory mechanistic models ("PEMMs"). A salient point is not whether students' models are correct, but whether they "work" in a manner consistent with underlying scientific principles. Analyzing such models can reveal the extent to which students recognize and accurately apply relevant ideas. An emphasis on model building, analysis and revision, an authentic scientific practice, can be expected to have transformative effects on course and curricular design as well as on student engagement and learning outcomes.

Identifying domains of EFHC1 involved in ciliary localization, ciliogenesis, and the regulation of Wnt signaling.

EFHC1 encodes a ciliary protein that has been linked to Juvenile Myoclonic Epilepsy. In ectodermal explants, derived from Xenopus laevis embryos, the morpholino-mediated down-regulation of EFHC1b inhibited multiciliated cell formation. In those ciliated cells that did form, axoneme but not basal body formation was inhibited. EFHC1b morphant embryos displayed defects in central nervous system (CNS) and neural crest patterning that were rescued by a EFHC1b-GFP chimera. EFHC1b-GFP localized to ciliary axonemes in epidermal, gastrocoele roof plate, and neural tube cells. In X. laevis there is a link between Wnt signaling and multiciliated cell formation. While down-regulation of EFHC1b led to a ~2-fold increase in the activity of the ß-catenin/Wnt-responsive TOPFLASH reporter, EFHC1b-GFP did not inhibit ß-catenin activation of TOPFLASH. Wnt8a RNA levels were increased in EFHC1b morphant ectodermal explants and intact embryos, analyzed prior to the on-set of ciliogenesis. Rescue of the EFHC1b MO's ciliary axonemal phenotypes required the entire protein; in contrast, the EFHC1b morpholino's Wnt8a, CNS, and neural crest phenotypes were rescued by a truncated form of EFHC1b. The EFHC1b morpholino's Wnt8a phenotype was also rescued by the injection of RNAs encoding secreted Wnt inhibitors, suggesting that these phenotypes are due to effects on Wnt signaling, rather than the loss of cilia, an observation of potential relevance to understanding EFHC1's role in human neural development.

Chibby functions in Xenopus ciliary assembly, embryonic development, and the regulation of gene expression.

Wnt signaling and ciliogenesis are core features of embryonic development in a range of metazoans. Chibby (Cby), a basal-body associated protein, regulates ß-catenin-mediated Wnt signaling in the mouse but not Drosophila. Here we present an analysis of Cby's embryonic expression and morphant phenotypes in Xenopus laevis. Cby RNA is supplied maternally, negatively regulated by Snail2 but not Twist1, preferentially expressed in the neuroectoderm, and regulates ß-catenin-mediated gene expression. Reducing Cby levels reduced the density of multiciliated cells, the number of basal bodies per multiciliated cell, and the numbers of neural tube primary cilia; it also led to abnormal development of the neural crest, central nervous system, and pronephros, all defects that were rescued by a Cby-GFP chimera. Reduction of Cby led to an increase in Wnt8a and decreases in Gli2, Gli3, and Shh RNA levels. Many, but not all, morphant phenotypes were significantly reversed by the Wnt inhibitor SFRP2. These observations extend our understanding of Cby's role in mediating the network of interactions between ciliogenesis, signaling systems and tissue patterning.

Snail2 controls mesodermal BMP/Wnt induction of neural crest.

The neural crest is an induced tissue that is unique to vertebrates. In the clawed frog Xenopus laevis, neural crest induction depends on signals secreted from the prospective dorsolateral mesodermal zone during gastrulation. The transcription factors Snail2 (Slug), Snail1 and Twist1 are expressed in this region. It is known that Snail2 and Twist1 are required for both mesoderm formation and neural crest induction. Using targeted blastomere injection, morpholino-based loss of function and explant studies, we show that: (1) Snail1 is also required for mesoderm and neural crest formation; (2) loss of snail1, snail2 or twist1 function in the C2/C3 lineage of 32-cell embryos blocks mesoderm formation, but neural crest is lost only in the case of snail2 loss of function; (3) snail2 mutant loss of neural crest involves mesoderm-derived secreted factors and can be rescued synergistically by bmp4 and wnt8 RNAs; and (4) loss of snail2 activity leads to changes in the RNA levels of a number of BMP and Wnt agonists and antagonists. Taken together, these results identify Snail2 as a key regulator of the signals involved in mesodermal induction of neural crest.

Rethinking (again) Hardy-Weinberg and genetic drift in undergraduate biology.

Designing effective curricula is challenging. Content decisions can impact both learning outcomes and student engagement. As an example consider the place of Hardy-Weinberg equilibria (HWE) and genetic drift calculations in introductory biology courses, as discussed by Masel (2012). Given that population genetics, "a fairly arcane speciality", can be difficult to grasp, there is little justification for introducing introductory students to HWE calculations. It is more useful to introduce them to the behavior of alleles in terms of basic features of biological systems, and that in the absence of selection recessive alleles are no "weaker" or preferentially lost from a population than are dominant alleles. On the other hand, stochastic behaviors, such as genetic drift, are ubiquitous in biological systems and often play functionally significant roles; they can be introduced to introductory students in mechanistic and probabilistic terms. Specifically, genetic drift emerges from the stochastic processes involved in meiotic chromosome segregation and recombination. A focus on stochastic processes may help counteract naive bio-deterministic thinking and can reinforce, for students, the value of thinking quantitatively about biological processes.

How Do Instructors Explain The Mechanism by which ATP Drives Unfavorable Processes?

Concerns regarding students' difficulties with the concept of energy date back to the 1970s. They become particularly apparent for systems involving adenosine triphosphate (ATP), which plays a central role in maintaining the nonequilibrium state of biological systems and in driving energetically unfavorable processes. One of the most well-documented misconceptions related to ATP is the idea that breaking bonds releases energy, when the opposite is true. This misconception is often attributed to language used in biology referring to the "high-energy bonds" in ATP. We interviewed chemistry, biology, and biochemistry instructors to learn how they think about and teach the mechanism(s) by which ATP is used as an energy source in biological systems. Across 15 interviews, we found that instructors relied primarily on two mechanisms to explain the role of ATP: 1) energy release, focused on ATP hydrolysis and bond energies; and/or 2) energy transfer, focused on phosphorylation and common intermediates. Many instructors shared negative and uncomfortable experiences related to teaching ATP and energy release. Based on these findings, we suggest instructional strategies that: 1) aim to ease the concerns expressed by introductory biology instructors, and 2) emphasize the role of ATP so as to support students' understanding of molecular mechanisms.

A maternally established SoxB1/SoxF axis is a conserved feature of chordate germ layer patterning.

Despite deep evolutionary roots in the metazoa, the gene regulatory network driving germ layer specification is surprisingly labile both between and within phyla. In Xenopus laevis, SoxB1- and SoxF-type transcription factors are intimately involved in germ-layer specification, in part through their regulation of Nodal signaling. However, it is unclear if X. laevis is representative of the ancestral vertebrate condition, as the precise roles of SoxF and SoxB1 in germ-layer specification vary among vertebrates, and there is no evidence that SoxF mediates germ-layer specification in any invertebrate. To better understand the evolution of germ-layer specification in the vertebrate lineage, we analyzed the expression of soxB1 and soxF genes in embryos and larvae of the basal vertebrate lamprey, and the basal chordate amphioxus. We find that both species maternally deposit soxB1 mRNA in the animal pole, soxF mRNA in the vegetal hemisphere, and zygotically express soxB1 and soxF throughout nascent ectoderm and mesendoderm, respectively. We also find that soxF is excluded from the vegetalmost blastomeres in lamprey and that, in contrast to vertebrates, amphioxus does not express soxF in the oral epithelium. In the context of recent work, our results suggest that a maternally established animal/vegetal Sox axis is a deeply conserved feature of chordate development that predates the role of Nodal in vertebrate germ-layer specification. Furthermore, exclusion of this axis from the vegetal pole in lamprey is consistent with the presence of an extraembryonic yolk mass, as has been previously proposed. Finally, conserved expression of SoxF in the forming mouth across the vertebrates, but not in amphioxus, lends support to the idea that the larval amphioxus mouth is nonhomologous to the vertebrate mouth.

Unexpected functional redundancy between Twist and Slug (Snail2) and their feedback regulation of NF-kappaB via Nodal and Cerberus.

A NF-kappaB-Twist-Snail network controls axis and mesoderm formation in Drosophila. Using translation-blocking morpholinos and hormone-regulated proteins, we demonstrate the presence of an analogous network in the early Xenopus embryo. Loss of twist (twist1) function leads to a reduction of mesoderm and neural crest markers, an increase in apoptosis, and a decrease in snail1 (snail) and snail2 (slug) mRNA levels. Injection of snail2 mRNA rescues twist's loss of function phenotypes and visa versa. In the early embryo NF-kappaB/RelA regulates twist, snail2, and snail1 mRNA levels; similarly Nodal/Smad2 regulate twist, snail2, snail1, and relA RNA levels. Both Twist and Snail2 negatively regulate levels of cerberus RNA, which encodes a Nodal, bone morphogenic protein (BMP), and Wnt inhibitor. Cerberus's anti-Nodal activity inhibits NF-kappaB activity and decreases relA RNA levels. These results reveal both conserved and unexpected regulatory interactions at the core of a vertebrate's mesodermal specification network.

Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe.

Epithelial-mesenchymal transition (EMT) describes a series of rapid changes in cellular phenotype. During EMT, epithelial cells down-modulate cell-cell adhesion structures, alter their polarity, reorganize their cytoskeleton, and become isolated, motile, and resistant to anoikis. The term EMT is often applied to distinct biological events as if it were a single conserved process, but in fact EMT-related processes can vary in intensity from a transient loss of cell polarity to the total cellular reprogramming, as found by transcriptional analysis. Based on clinical observations, it is more appropriate in most cases to describe the emergence of an EMT-like phenotype during tumor progression. Although EMT implies complete trans-differentiation, EMT-like emphasizes the intermediary phenotype associated with tumor cell renewal and adaptation to specific microenvironments. Here, we categorize the various EMT-like phenotypes found in human carcinomas that, depending on the tumor type, may or not represent analogous stages in tumor progression. We based these categories on the global tumor phenotype. The tumor microenvironment, which is associated with stromal reactions, hypoxia, paucity of nutrients, impaired differentiation, and activation of various EMT-associated pathways, modulates overall tumor phenotype and leads to tumor heterogeneity.

Eya1 and Six1 promote neurogenesis in the cranial placodes in a SoxB1-dependent fashion.

Genes of the Eya family and of the Six1/2 subfamily are expressed throughout development of vertebrate cranial placodes and are required for their differentiation into ganglia and sense organs. How they regulate placodal neurogenesis, however, remains unclear. Through loss of function studies in Xenopus we show that Eya1 and Six1 are required for neuronal differentiation in all neurogenic placodes. The effects of overexpression of Eya1 or Six1 are dose dependent. At higher levels, Eya1 and Six1 expand the expression of SoxB1 genes (Sox2, Sox3), maintain cells in a proliferative state and block expression of neuronal determination and differentiation genes. At lower levels, Eya1 and Six1 promote neuronal differentiation, acting downstream of and/or parallel to Ngnr1. Our findings suggest that Eya1 and Six1 are required for both the regulation of placodal neuronal progenitor proliferation, through their effects on SoxB1 expression, and subsequent neuronal differentiation.

Filaments and phenotypes: cellular roles and orphan effects associated with mutations in cytoplasmic intermediate filament proteins.

Cytoplasmic intermediate filaments (IFs) surround the nucleus and are often anchored at membrane sites to form effectively transcellular networks. Mutations in IF proteins (IFps) have revealed mechanical roles in epidermis, muscle, liver, and neurons. At the same time, there have been phenotypic surprises, illustrated by the ability to generate viable and fertile mice null for a number of IFp-encoding genes, including vimentin. Yet in humans, the vimentin ( VIM) gene displays a high probability of intolerance to loss-of-function mutations, indicating an essential role. A number of subtle and not so subtle IF-associated phenotypes have been identified, often linked to mechanical or metabolic stresses, some of which have been found to be ameliorated by the over-expression of molecular chaperones, suggesting that such phenotypes arise from what might be termed "orphan" effects as opposed to the absence of the IF network per se, an idea originally suggested by Toivola et al. and Pekny and Lane.

Whole-Mount Immunocytochemistry in Xenopus.

To visualize the effects of experimental perturbations on normal cellular behavior, morphology, and intracellular organization, we use a simple whole-mount immunocytochemical method with Xenopus oocytes, explants, or embryos. This method is applicable to a wide range of systems, including human-induced pluripotent stem cell-derived organoids, and can be used with both chromogenic (horseradish peroxidase/diaminobenzidine) and fluorescent imaging.

Nuclear roles for cilia-associated proteins.

Cilia appear to be derived, evolutionarily, from structures present in the ancestral (pre-ciliary) eukaryote, such as microtubule-based vesicle trafficking and chromosome segregation systems. Experimental observations suggest that the ciliary gate, the molecular complex that mediates the selective molecular movement between cytoplasmic and ciliary compartments, shares features with nuclear pores. Our hypothesis is that this shared transport machinery is at least partially responsible for the observation that a number of ciliary and ciliogenesis-associated proteins are found within nuclei where they play roles in the regulation of gene expression, DNA repair, and nuclear import and export. Recognizing the potential for such nuclear roles is critical when considering the phenotypic effects that arise from the mutational modification of ciliary proteins.

Diagnostic of students' misconceptions using the Biological Concepts Instrument (BCI): A method for conducting an educational needs assessment.

Concept inventories, constructed based on an analysis of students' thinking and their explanations of scientific situations, serve as diagnostics for identifying misconceptions and logical inconsistencies and provide data that can help direct curricular reforms. In the current project, we distributed the Biological Concepts Instrument (BCI) to 17-18-year-old students attending the highest track of the Swiss school system (Gymnasium). Students' performances on many questions related to evolution, genetics, molecular properties and functions were diverse. Important common misunderstandings were identified in the areas of evolutionary processes, molecular properties and an appreciation of stochastic processes in biological systems. Our observations provide further evidence that the BCI is efficient in identifying specific areas where targeted instruction is required. Based on these observations we have initiated changes at several levels to reconsider how biological systems are presented to university biology studies with the goal of improving student's foundational understanding.

TSPAN12 Is a Norrin Co-receptor that Amplifies Frizzled4 Ligand Selectivity and Signaling.

Accessory proteins in Frizzled (FZD) receptor complexes are thought to determine ligand selectivity and signaling amplitude. Genetic evidence indicates that specific combinations of accessory proteins and ligands mediate vascular ß-catenin signaling in different CNS structures. In the retina, the tetraspanin TSPAN12 and the ligand norrin (NDP) mediate angiogenesis, and both genes are linked to familial exudative vitreoretinopathy (FEVR), yet the molecular function of TSPAN12 remains poorly understood. Here, we report that TSPAN12 is an essential component of the NDP receptor complex and interacts with FZD4 and NDP via its extracellular loops, consistent with an action as co-receptor that enhances FZD4 ligand selectivity for NDP. FEVR-linked mutations in TSPAN12 prevent the incorporation of TSPAN12 into the NDP receptor complex. In vitro and in Xenopus embryos, TSPAN12 alleviates defects of FZD4 M105V, a mutation that destabilizes the NDP/FZD4 interaction. This study sheds light on the poorly understood function of accessory proteins in FZD signaling.

Sox3 expression identifies neural progenitors in persistent neonatal and adult mouse forebrain germinative zones.

Neural precursors persist throughout life in the rodent forebrain subventricular zone (SVZ) and hippocampal dentate gyrus. The regulation of persistent neural stem cells is poorly understood, in part because of the lack of neural progenitor markers. The Sox B1 subfamily of HMG-box transcription factors (Sox1-3) is expressed by precursors in the embryonic nervous system, where these factors maintain neural progenitors in an undifferentiated state while suppressing neuronal differentiation. Sox2 expression persists in germinative zones of the adult rodent brain, but Sox3 expression in the postnatal brain remains largely unexplored. Here we examine Sox3 expression in the neonatal and adult mouse brain to gain insight into its potential involvement in regulating persistent neural stem cells and neurogenesis. We also investigate Sox3 expression during expansion and neural differentiation of postnatal mouse SVZ neural stem cell and human embryonic stem cell (hESC) cultures. We find that Sox3 is expressed transiently by proliferating and differentiating neural progenitors in the SVZ-olfactory bulb pathway and dentate gyrus. Sox3 immunoreactivity also persists in specific postmitotic neuronal populations. In vitro, high Sox3 protein expression levels in undifferentiated, SVZ-derived neurospheres decline markedly with differentiation. Sox3 immunoreactivity in hESCs appears upon differentiation to neural progenitors and then decreases as cells differentiate further into neurons. These findings suggest that Sox3 labels specific stages of hESC-derived and murine neonatal and adult neural progenitors and are consistent with a role for Sox3 in neural stem cell maintenance. Persistent Sox3 expression in some mature neuronal populations suggests additional undefined roles for Sox3 in neuronal function.

The Design and Transformation of Biofundamentals: A Nonsurvey Introductory Evolutionary and Molecular Biology Course.

Many introductory biology courses amount to superficial surveys of disconnected topics. Often, foundational observations and the concepts derived from them and students' ability to use these ideas appropriately are overlooked, leading to unrealistic expectations and unrecognized learning obstacles. The result can be a focus on memorization at the expense of the development of a meaningful framework within which to consider biological phenomena. About a decade ago, we began a reconsideration of what an introductory course should present to students and the skills they need to master. The original Web-based course's design presaged many of the recommendations of the Vision and Change report; in particular, a focus on social evolutionary mechanisms, stochastic (evolutionary and molecular) processes, and core ideas (cellular continuity, evolutionary homology, molecular interactions, coupled chemical reactions, and molecular machines). Inspired by insights from the Chemistry, Life, the Universe & Everything general chemistry project, we transformed the original Web version into a (freely available) book with a more unified narrative flow and a set of formative assessments delivered through the beSocratic system. We outline how student responses to course materials are guiding future course modifications, in particular a more concerted effort at helping students to construct logical, empirically based arguments, explanations, and models.

Centrin-2 (Cetn2) mediated regulation of FGF/FGFR gene expression in Xenopus.

Centrins (Cetns) are highly conserved, widely expressed, and multifunctional Ca(2+)-binding eukaryotic signature proteins best known for their roles in ciliogenesis and as critical components of the global genome nucleotide excision repair system. Two distinct Cetn subtypes, Cetn2-like and Cetn3-like, have been recognized and implicated in a range of cellular processes. In the course of morpholino-based loss of function studies in Xenopus laevis, we have identified a previously unreported Cetn2-specific function, namely in fibroblast growth factor (FGF) mediated signaling, specifically through the regulation of FGF and FGF receptor RNA levels. Cetn2 was found associated with the RNA polymerase II binding sites of the Cetn2-regulated FGF8 and FGFR1a genes, but not at the promoter of a gene (BMP4) whose expression was altered indirectly in Cent2 morphant embryos. These observations point to a previously unexpected role of Cetn2 in the regulation of gene expression and embryonic development.