Impact of gross anatomy laboratory on student written examination performance: A 3-year study of a large-enrollment undergraduate anatomy course.

The efficacy of the various pedagogies that are used in human anatomy laboratories has been extensively debated. Nevertheless, an important question remains relatively unexamined-how the learning experience in the anatomy laboratory impacts students' mastery and application of anatomical knowledge beyond the laboratory setting. In this study, the effect of a prosection-based anatomy laboratory on overall comprehension and mastery of anatomical knowledge was evaluated in an upper division undergraduate anatomy curriculum that consists of a mandatory lecture course and an optional laboratory course. This flexible curricular structure permitted assessing the merit of laboratory learning on the written examination performance of the lecture course. In 2019 and 2022, the anatomy laboratory was taught in-person using prosections, while in 2021 due to the Covid-19 pandemic related regulations, it was taught remotely with live-streaming of prosections using document cameras. In both in-person and remote instructive formats, written examination scores of the lecture course were compared between two cohorts of students: Those enrolled in lecture only and those enrolled in both lecture and laboratory. Results showed that the cohort enrolled in both lecture and laboratory courses consistently outperformed the lecture-only cohort by one full letter grade. Furthermore, when the degrees of improvement on written examination scores were compared between the two instructive formats, in-person laboratory had a greater increase compared to remote laboratory. Altogether this study demonstrates that the prosection-based anatomy laboratory enhances students' mastery of anatomical knowledge beyond the laboratory setting by promoting comprehension of spatial relationships of anatomical structures.

Molecular evolution of the Thrombospondin superfamily.

Thrombospondins (TSPs) are multidomain, calcium-binding glycoproteins that have wide-ranging roles in vertebrates in cell interactions, extracellular matrix (ECM) organisation, angiogenesis, tissue remodelling, synaptogenesis, and also in musculoskeletal and cardiovascular functions. Land animals encode five TSPs, which assembly co-translationally either as trimers (subgroup A) or pentamers (subgroup B). The vast majority of research has focused on this canonical TSP family, which evolved through the whole-genome duplications that took place early in the vertebrate lineage. With benefit of the growth in genome- and transcriptome-predicted proteomes of a much wider range of animal species, examination of TSPs throughout metazoan phyla has revealed extensive conservation of subgroup B-type TSPs in invertebrates. In addition, these searches established that canonical TSPs are, in fact, one branch within a TSP superfamily that includes other clades designated mega-TSPs, sushi-TSPs and poriferan-TSPs. Despite the apparent simplicity of poriferans and cnidarians as organisms, these phyla encode a greater diversity of TSP superfamily members than vertebrates. We discuss here the molecular characteristics of the TSP superfamily members, current knowledge of their expression profiles and functions in invertebrates, and models for the evolution of this complex ECM superfamily.

Advances on the roles of tenascin-C in cancer.

The roles of the extracellular matrix molecule tenascin-C (TNC) in health and disease have been extensively reviewed since its discovery over 40 years ago. Here, we will describe recent insights into the roles of TNC in tumorigenesis, angiogenesis, immunity and metastasis. In addition to high levels of expression in tumors, and during chronic inflammation, and bacterial and viral infection, TNC is also expressed in lymphoid organs. This supports potential roles for TNC in immunity control. Advances using murine models with engineered TNC levels were instrumental in the discovery of important functions of TNC as a danger-associated molecular pattern (DAMP) molecule in tissue repair and revealed multiple TNC actions in tumor progression. TNC acts through distinct mechanisms on many different cell types with immune cells coming into focus as important targets of TNC in cancer. We will describe how this knowledge could be exploited for cancer disease management, in particular for immune (checkpoint) therapies.

Revisiting the Tenascins: Exploitable as Cancer Targets?

For their full manifestation, tumors require support from the surrounding tumor microenvironment (TME), which includes a specific extracellular matrix (ECM), vasculature, and a variety of non-malignant host cells. Together, these components form a tumor-permissive niche that significantly differs from physiological conditions. While the TME helps to promote tumor progression, its special composition also provides potential targets for anti-cancer therapy. Targeting tumor-specific ECM molecules and stromal cells or disrupting aberrant mesenchyme-cancer communications might normalize the TME and improve cancer treatment outcome. The tenascins are a family of large, multifunctional extracellular glycoproteins consisting of four members. Although each have been described to be expressed in the ECM surrounding cancer cells, tenascin-C and tenascin-W are currently the most promising candidates for exploitability and clinical use as they are highly expressed in various tumor stroma with relatively low abundance in healthy tissues. Here, we review what is known about expression of all four tenascin family members in tumors, followed by a more thorough discussion on tenascin-C and tenascin-W focusing on their oncogenic functions and their potential as diagnostic and/or targetable molecules for anti-cancer treatment purposes.

Did Tenascin-C Co-Evolve With the General Immune System of Vertebrates?

Tenascin-C plays important roles in immunity. Toll-like receptor 4, integrin α9ß1 and chemokines have already been identified as key players in executing the immune regulatory functions of tenascin-C. Tenascin-C is also found in reticular fibers in lymphoid tissues, which are major sites involved in the regulation of adaptive immunity. Did the "tool box" for reading and interpreting the immune-regulating instructions imposed by tenascins and tenascin-C co-evolve? Though the extracellular matrix is ancient, tenascins evolved relatively recently. Tenascin-like genes are first encountered in cephalochordates and urochordates, which are widely accepted as the early branching chordate lineages. Vertebrates lacking jaws like the lamprey have tenascins, but a tenascin gene that clusters in the tenascin-C clade first appears in cartilaginous fish. Adaptive immunity apparently evolved independently in jawless and jawed vertebrates, with the former using variable lymphocyte receptors for antigen recognition, and the latter using immunoglobulins. Thus, while tenascins predate the appearance of adaptive immunity, the first tenascin-C appears to have evolved in the first organisms with immunoglobulin-based adaptive immunity. While a C-X-C chemokine is present in the lamprey, C-C chemokines also appear in the first organisms with immunoglobulin-based adaptive immunity, as does the major histocompatibility complex, T-cell receptors, Toll-like receptor 4 and integrin α9ß1. Given the importance of tenascin-C in inflammatory events, the co-evolution of tenascin-C and key elements of adaptive and innate immunity is suggestive of a fundamental role for this extracellular matrix glycoprotein in the immune response of jawed vertebrates.

Tenascin-W Is a Novel Stromal Marker in Biliary Tract Cancers.

Extrahepatic cancers of the biliary system are typically asymptomatic until after metastasis, which contributes to their poor prognosis. Here we examined intrahepatic cholangiocarcinomas (n = 8), carcinomas of perihilar bile ducts (n = 7), carcinomas of the gallbladder (n = 11) and hepatic metastasis from carcinomas of the gallbladder (n = 4) for the expression of the extracellular matrix glycoproteins tenascin-C and tenascin-W. Anti-tenascin-C and anti-tenascin-W immunoreactivity was found in all biliary tract tumors examined. Unlike tenascin-C, tenascin-W was not detected in normal hepatobiliary tissue. Tenascin-W was also expressed by the cholangiocarcinoma-derived cell line Huh-28. However, co-culture of Huh-28 cells with immortalized bone marrow-derived stromal cells was necessary for the formation and organization of tenascin-W fibrils in vitro. Our results indicate that tenascin-W may be a novel marker of hepatobiliary tumor stroma, and its absence from many normal tissues suggests that it may be a potential target for biotherapies.

Tenascin-W: Discovery, Evolution, and Future Prospects.

Of the four tenascins found in bony fish and tetrapods, tenascin-W is the least understood. It was first discovered in the zebrafish and later in mouse, where it was mistakenly named tenascin-N. Tenascin-W is expressed primarily in developing and mature bone, in a subset of stem cell niches, and in the stroma of many solid tumors. Phylogenetic studies show that it is the most recent tenascin to evolve, appearing first in bony fishes. Its expression in bone and the timing of its evolutionary appearance should direct future studies to its role in bone formation, in stem cell niches, and in the treatment and detection of cancer.

Immunohistochemistry and In Situ Hybridization in the Developing Chicken Brain.

One of the first steps in studies of gene function is the spatiotemporal analysis of patterns of gene expression. Indirect immunohistochemistry is a method that allows the detection of a protein of interest by incubating a histological section with an antibody or antiserum raised against the protein, and then localizing this primary antibody with a tagged secondary antibody. To determine the cellular source of a protein of interest, or if a specific antibody is not available, specific transcripts can be localized using in situ hybridization. A histological section is incubated with a labeled RNA probe that is complementary to the target transcript; after hybridization with the target transcript the labeled RNA probe can be identified with an antibody. Here we describe materials and methods used to perform basic indirect immunohistochemistry and in situ hybridization on frozen sections through the developing chicken brain, emphasizing controls and potential problems that may be encountered.

The Expression and Possible Functions of Tenascin-W During Development and Disease.

Tenascins are a family of multifunctional glycoproteins found in the extracellular matrix of chordates. Two of the tenascins, tenascin-C and tenascin-W, form hexabrachions. In this review, we describe the discovery and domain architecture of tenascin-W, its evolution and patterns of expression during embryogenesis and in tumors, and its effects on cells in culture. In avian and mammalian embryos tenascin-W is primarily expressed at sites of osteogenesis, and in the adult tenascin-W is abundant in certain stem cell niches. In primary cultures of osteoblasts tenascin-W promotes cell migration, the formation of mineralized foci and increases alkaline phosphatase activity. Tenascin-W is also prominent in many solid tumors, yet it is missing from the extracellular matrix of most adult tissues. This makes it a potential candidate for use as a marker of tumor stroma and a target for anti-cancer therapies.

Emergence of a Thrombospondin Superfamily at the Origin of Metazoans.

Extracellular matrix (ECM) is considered central to the evolution of metazoan multicellularity; however, the repertoire of ECM proteins in nonbilaterians remains unclear. Thrombospondins (TSPs) are known to be well conserved from cnidarians to vertebrates, yet to date have been considered a unique family, principally studied for matricellular functions in vertebrates. Through searches utilizing the highly conserved C-terminal region of TSPs, we identify undisclosed new families of TSP-related proteins in metazoans, designated mega-TSP, sushi-TSP, and poriferan-TSP, each with a distinctive phylogenetic distribution. These proteins share the TSP C-terminal region domain architecture, as determined by domain composition and analysis of molecular models against known structures. Mega-TSPs, the only form identified in ctenophores, are typically >2,700 aa and are also characterized by N-terminal leucine-rich repeats and central cadherin/immunoglobulin domains. In cnidarians, which have a well-defined ECM, Mega-TSP was expressed throughout embryogenesis in Nematostella vectensis, with dynamic endodermal expression in larvae and primary polyps and widespread ectodermal expression in adult Nematostella vectensis and Hydra magnipapillata polyps. Hydra Mega-TSP was also expressed during regeneration and siRNA-silencing of Mega-TSP in Hydra caused specific blockade of head regeneration. Molecular phylogenetic analyses based on the conserved TSP C-terminal region identified each of the TSP-related groups to form clades distinct from the canonical TSPs. We discuss models for the evolution of the newly defined TSP superfamily by gene duplications, radiation, and gene losses from a debut in the last metazoan common ancestor. Together, the data provide new insight into the evolution of ECM and tissue organization in metazoans.

The teneurin C-terminal domain possesses nuclease activity and is apoptogenic.

Teneurins are type 2 transmembrane proteins expressed by developing neurons during periods of synaptogenesis and apoptosis. Neurons expressing teneurin-1 synapse with other teneurin-1-expressing neurons, and neurons expressing teneurin-2 synapse with other teneurin-2-expressing neurons. Knockdowns and mutations of teneurins lead to abnormal neuronal connections, but the mechanisms underlying teneurin action remain unknown. Teneurins appear to have evolved via horizontal gene transfer from prokaryotic proteins involved in bacterial self-recognition. The bacterial teneurin-like proteins contain a cytotoxic C-terminal domain that is encapsulated in a tyrosine-aspartic acid repeat barrel. Teneurins are likely to be organized in the same way, but it is unclear if the C-terminal domains of teneurins have cytotoxic properties. Here we show that expression of teneurin C-terminal domains or the addition of purified teneurin C-terminal domains leads to an increase in apoptosis in vitro The C-terminal domains of teneurins are most similar to bacterial nucleases, and purified C-terminal domains of teneurins linearize pcDNA3 and hydrolyze mitochondrial DNA. We hypothesize that yet to be identified stimuli lead to the release of the encapsulated teneurin C-terminal domain into the intersynaptic region, resulting in programmed cell death or the disruption of mitochondrial DNA and the subsequent pruning of inappropriate contacts.

Teneurins: Domain Architecture, Evolutionary Origins, and Patterns of Expression.

Disruption of teneurin expression results in abnormal neural networks, but just how teneurins support the development of the central nervous system remains an area of active research. This review summarizes some of what we know about the functions of the various domains of teneurins, the possible evolution of teneurins from a bacterial toxin, and the intriguing patterns of teneurin expression. Teneurins are a family of type-2 transmembrane proteins. The N-terminal intracellular domain can be processed and localized to the nucleus, but the significance of this nuclear localization is unknown. The extracellular domain of teneurins is largely composed of tyrosine-aspartic acid repeats that fold into a hollow barrel, and the C-terminal domains of teneurins are stuffed, and least partly, into the barrel. A 6-bladed beta-propeller is found at the other end of the barrel. The same arrangement-6-bladed beta-propeller, tyrosine-aspartic acid repeat barrel, and the C-terminal domain inside the barrel-is seen in toxic proteins from bacteria, and there is evidence that teneurins may have evolved from a gene encoding a prokaryotic toxin via horizontal gene transfer into an ancestral choanoflagellate. Patterns of teneurin expression are often, but not always, complementary. In the central nervous system, where teneurins are best studied, interconnected populations of neurons often express the same teneurin. For example, in the chicken embryo neurons forming the tectofugal pathway express teneurin-1, whereas neurons forming the thalamofugal pathway express teneurin-2. In Drosophila melanogaster, Caenorhabditis elegans, zebrafish and mice, misexpression or knocking out teneurin expression leads to abnormal connections in the neural networks that normally express the relevant teneurin. Teneurins are also expressed in non-neuronal tissue during development, and in at least some regions the patterns of non-neuronal expression are also complementary. The function of teneurins outside the nervous system remains unclear.

Tenascin-C at a glance.

Tenascin-C (TNC) is a hexameric, multimodular extracellular matrix protein with several molecular forms that are created through alternative splicing and protein modifications. It is highly conserved amongst vertebrates, and molecular phylogeny indicates that it evolved before fibronectin. Tenascin-C has many extracellular binding partners, including matrix components, soluble factors and pathogens; it also influences cell phenotype directly through interactions with cell surface receptors. Tenascin-C protein synthesis is tightly regulated, with widespread protein distribution in embryonic tissues, but restricted distribution of tenascin-C in adult tissues. Tenascin-C is also expressed de novo during wound healing or in pathological conditions, including chronic inflammation and cancer. First described as a modulator of cell adhesion, tenascin-C also directs a plethora of cell signaling and gene expression programs by shaping mechanical and biochemical cues within the cellular microenvironment. Exploitment of the pathological expression and function of tenascin-C is emerging as a promising strategy to develop new diagnostic, therapeutic and bioengineering tools. In this Cell Science at a Glance article and the accompanying poster we provide a succinct and comprehensive overview of the structural and functional features of tenascin-C and its potential roles in developing embryos and under pathological conditions.

The expression of tenascin-C and tenascin-W in human ossicles.

The ossicles of the middle ear (the malleus, incus and stapes) transmit forces resulting from vibrations of the tympanic membrane to the cochlea where they are coded as sound. Hearing loss can result from diseases such as rheumatoid arthritis (RA) that affect the joints between the ossicles or degenerative processes like otosclerosis that lead to ankylosis of the footplate of the stapes in the oval window of the cochlea. In this study, immunohistochemistry was used to determine if the extracellular matrix glycoproteins tenascin-C or tenascin-W are expressed in the incudomalleolar and incudostapedial joints of ossicles dissected from human cadavers. Tenascin-C, which is expressed during inflammatory conditions including RA, was seen in the articular cartilage of the incudomalleolar joints and the head of the stapes. Tenascin-W, in contrast, was enriched in the annular ligament that anchors the footplate of the stapes into the oval window of the cochlea.

Tenascin-C: Its functions as an integrin ligand.

This review summarizes the experimental evidence of tenascin-C/integrin interactions, emphasizing the identification of integrin binding sites and the effects of specific interactions on cell behavior. At least four integrins appear to bind to the third fibronectin-type 3 domain of tenascin-C: α9ß1, αVß3, α8ß1 and αVß6. The α9ß1 integrin recognizes a highly conserved IDG motif in this domain, while the others recognize an RGD motif. There is also significant evidence that the collagen receptor α2ß1 can bind to tenascin-C, but the interacting site is unknown. Tenascin-C interactions with α9ß1 and αVß3 can promote cell proliferation and interactions with αVß3 can also inhibit apoptosis. Interactions with α7ß1 integrin, which may bind to the alternatively spliced domain of tenascin-C, and α9ß1 integrin are able to influence the differentiation of mesenchymal stem cells into the neuronal lineage. This illustrates the potential for using our knowledge of tenascins and their integrin receptors in stem cell-based therapies.

The evolution of tenascins and fibronectin.

Tenascins are extracellular matrix glycoproteins that act both as integrin ligands and as modifiers of fibronectin-integrin interactions to regulate cell adhesion, migration, proliferation and differentiation. In tetrapods, both tenascins and fibronectin bind to integrins via RGD and LDV-type tripeptide motifs found in exposed loops in their fibronectin-type III domains. We previously showed that tenascins appeared early in the chordate lineage and are represented by single genes in extant cephalochordates and tunicates. Here we have examined the genomes of the coelacanth Latimeria chalumnae, the elephant shark Callorhinchus milii as well as the lampreys Petromyzon marinus and Lethenteron japonicum to learn more about the evolution of the tenascin gene family as well as the timing of the appearance of fibronectin during chordate evolution. The coelacanth has 4 tenascins that are more similar to tetrapod tenascins than are tenascins from ray-finned fishes. In contrast, only 2 tenascins were identified in the elephant shark and the Japanese lamprey L. japonicum. An RGD motif exposed to integrin binding is observed in tenascins from many, but not all, classes of chordates. Tetrapods that lack this RGD motif in tenascin-C have a similar motif in the paralog tenascin-W, suggesting the potential for some overlapping function. A predicted fibronectin with the same domain organization as the fibronectin from tetrapods is found in the sea lamprey P. marinus but not in tunicates, leading us to infer that fibronectin first appeared in vertebrates. The motifs that recognize LDV-type integrin receptors are conserved in fibronectins from a broad spectrum of vertebrates, but the RGD integrin-binding motif may have evolved in gnathostomes.

Tenascin-C is required for normal Wnt/ß-catenin signaling in the whisker follicle stem cell niche.

Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/ß-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed ß-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear ß-catenin in wild-type mice reflecting active Wnt/ß-catenin signaling, whereas ß-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/ß-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted ß-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/ß-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/ß-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.

Tenascins in stem cell niches.

Tenascins are extracellular matrix proteins with distinct spatial and temporal expression during development, tissue homeostasis and disease. Based on their expression patterns and knockout phenotypes an important role of tenascins in tissue formation, cell adhesion modulation, regulation of proliferation and differentiation has been demonstrated. All of these features are of importance in stem cell niches where a precise regulation of growth versus differentiation has to be guaranteed. In this review we summarize the expression and possible functions of tenascins in neural, epithelial and osteogenic stem cell niches during normal development and organ turnover, in the hematopoietic and pro-inflammatory niche as well as in the metastatic niche during cancer progression.