Search

This article captures a collective reflection on the successes and challenges we experienced when teaching physiology laboratories online during the COVID-19 pandemic. Physiology instructors from six institutions discussed their own efforts to redesign meaningful physiology laboratories that could be taught remotely, as the nation scrambled to respond to the sudden shift out of the classroom. Despite the complexity of this task, clear themes emerged as our courses transitioned to an online format in spring 2020 and were solidified in the fall of 2020. This article reflects on the history, features, benefits, and challenges of current laboratory teaching when applying a scientific teaching approach to facilitate the redesign process. We believe online networks like ours can facilitate information sharing, promote innovations, and provide support for instructors. The insights we gained through this collaboration will influence our thinking about the future of the physiology lab, whether online or in person.

Proliferation of pancreatic β-cells has long been known to reach its peak in the neonatal stages and decline during adulthood. However, β-cell proliferation has been studied under the assumption that all β-cells constitute a single, homogenous population. It is unknown whether a subpopulation of β-cells retains the capacity to proliferate at a higher rate and thus contributes disproportionately to the maintenance of mature β-cell mass in adults. We therefore assessed the proliferative capacity and turnover potential of virgin β-cells, a novel population of immature β-cells found at the islet periphery. We demonstrate that virgin β-cells can proliferate but do so at rates similar to those of mature β-cells from the same islet under normal and challenged conditions. Virgin β-cell proliferation rates also conform to the age-dependent decline previously reported for β-cells at large. We further show that virgin β-cells represent a long-lived, stable subpopulation of β-cells with low turnover into mature β-cells under healthy conditions. Our observations indicate that virgin β-cells at the islet periphery can divide but do not contribute disproportionately to the maintenance of adult β-cell mass.

Background High throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene expression signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. However, chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity. Results Here, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we not only confirm and visualize previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin, but identify novel regions as well. We compiled our chromatin accessibility data in a freely accessible database of common endocrine- and cell specific-enhancer regions that can be navigated with minimal bioinformatics expertise. Conclusions Both alpha and delta cells appear poised, but repressed, from becoming beta cells in murine pancreatic islets. These data broadly support earlier findings on the plasticity in identity of non-beta cells under certain circumstances. Furthermore, differential chromatin accessibility shows preferentially enriched distal-intergenic regions in beta cells, when compared to either alpha or delta cells.

There is great interest in generating functionally mature beta cells from stem cells, as loss of functional beta cell mass contributes to the pathophysiology of diabetes. Identifying markers of beta cell maturity is therefore very helpful for distinguishing stem cells that have been successfully differentiated into fully mature beta cells from stem cells that did not. Urocortin 3 (UCN3) is a peptide hormone whose expression is associated with the acquisition of functional maturity in beta cells. The onset of its expression occurs after other beta cell maturity markers are already expressed and its loss marks the beginning of beta cell dedifferentiation. Its expression pattern is therefore tightly correlated with beta cell maturity. While this makes UCN3 an excellent marker of beta cell maturity, it is not established whether UCN3 is required for beta cell maturation. Here, we compared gene expression and function of beta cells from Ucn3-null mice relative to WT mice to determine whether beta cells are functionally mature in the absence of UCN3. Our results show that genetic deletion of Ucn3 does not cause a loss of beta cell maturity or an increase in beta cell dedifferentiation. Furthermore, virgin beta cells, first identified as insulin-expressing, UCN3-negative beta cells, can still be detected at the islet periphery in Ucn3-null mice. Beta cells from Ucn3-null mice also exhibit normal calcium response when exposed to high glucose. Collectively, these observations indicate that UCN3 is an excellent mature beta cell marker that is nevertheless not necessary for beta cell maturation.

The role of beta and α-cells to glucose control are established, but the physiological role of δ-cells is poorly understood. Delta-cells are ideally positioned within pancreatic islets to modulate insulin and glucagon secretion at their source. We review the evidence for a negative feedback loop between delta and β-cells that determines the blood glucose set point and suggest that local δ-cell-mediated feedback stabilizes glycemic control.

ShcA is a cytoplasmic signaling protein that supports signal transduction by receptor protein-tyrosine kinases by providing auxiliary tyrosine phosphorylation sites that engage additional signaling proteins. The principal binding partner for tyrosine phosphorylation sites on ShcA is Grb2. In the current study, we have used phosphotyrosine-containing peptides to isolate and identify STS-1 as a novel ShcA-binding protein. Our results further show that the interaction between STS-1 and ShcA is regulated in response to EGF receptor activation.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

We recently discovered a novel subset of beta cells that resemble immature beta cells during pancreas development. We named these "virgin" beta cells as they do not stem from existing mature beta cells. Virgin beta cells are found exclusively at the islet periphery in areas that we therefore designated as the "neogenic niche." As beta cells are our only source of insulin, their loss leads to diabetes. Islets also contain glucagon-producing alpha cells and somatostatin-producing delta cells, that are important for glucose homeostasis and form a mantle surrounding the beta cell core. This 3D architecture is important and determines access to blood flow and innervation. We propose that the distinctive islet architecture may also play an important, but hitherto unappreciated role in generation of new endocrine cells, including beta cells. We discuss several predictions to further test the contribution of the neogenic niche to beta cell regeneration.

© 2015 Nature America, Inc. All rights reserved. The peptide hormone urocortin3 (Ucn3) is abundantly expressed by mature beta cells, yet its physiological role is unknown. Here we demonstrate that Ucn3 is stored and co-released with insulin and potentiates glucose-stimulated somatostatin secretion via cognate receptors on delta cells. Further, we found that islets lacking endogenous Ucn3 have fewer delta cells, reduced somatostatin content, impaired somatostatin secretion, and exaggerated insulin release, and that these defects are rectified by treatment with synthetic Ucn3 in vitro. Our observations indicate that the paracrine actions of Ucn3 activate a negative feedback loop that promotes somatostatin release to ensure the timely reduction of insulin secretion upon normalization of plasma glucose. Moreover, Ucn3 is markedly depleted from beta cells in mouse and macaque models of diabetes and in human diabetic islets. This suggests that Ucn3 is a key contributor to stable glycemic control, whose reduction during diabetes aggravates glycemic volatility and contributes to the pathophysiology of this disease.

Pancreatic alpha cells retain considerable plasticity and can – under the right circumstances – transdifferentiate into functionally mature beta cells. In search of a targetable mechanistic basis, a recent paper suggested that the widely used antimalarial drug artemether suppresses the alpha cell transcription factor Arx to promote transdifferentiation into beta cells. However, key initial experiments in this paper were carried out in islet cell lines and most subsequent validation experiments implied transdifferentiation without direct demonstration of alpha to beta cell conversion. Indeed, we find no evidence that artemether promotes transdifferentiation of primary alpha cells into beta cells. Moreover, artemether reduces Ins2 expression in primary beta cells >100-fold, suppresses glucose uptake, and abrogates beta cell calcium responses and insulin secretion in response to glucose. Our observations suggest that artemether induces general islet endocrine cell dedifferentiation and call into question the utility of artemisinins to promote alpha to beta cell transdifferentiation in treating diabetes.

Type 1 diabetes (T1D) is a devastating disease precipitated by an autoimmune response directed at the insulin-producing beta-cells of the pancreas for which no cure exists. Stem cell-derived beta-cells show great promise for a cure as they have the potential to supply unlimited numbers of cells that could be derived from a patient's own cells, thus eliminating the need for immunosuppression. Current in vitro protocols for the differentiation of stem cell-derived beta-cells can successfully generate pancreatic endoderm cells. In diabetic rodents, such cells can differentiate further along the beta-cell lineage until they are eventually capable of restoring normoglycemia. While these observations demonstrate that stem cell-derived pancreatic endoderm has the potential to differentiate into mature, glucose-responsive beta-cells, the signals that direct differentiation and maturation from pancreatic endoderm onwards remain poorly understood. In this review, we analyze the sequence of events that culminates in the formation of beta-cells during embryonic development. and summarize how current protocols to generate beta-cells have sought to capitalize on this ontogenic template. We place particular emphasis on the current challenges and opportunities which occur in the later stages of beta-cell differentiation and maturation of transplantable stem cell-derived beta-cells. Another focus is on the question how the use of recently identified maturation markers such as urocortin 3 can be instrumental in guiding these efforts.

SUMMARYThe fast muscle fibres in the anterior trunk of teleost fish are primarily responsible for large amplitude undulatory swimming motions. Previous theoretical studies suggested that the near-helical arrangement of these fibres results in a (fairly) uniform distribution of fibre strain and work output during swimming. However, the underlying simplifications of these studies precluded unequivocal support for this hypothesis. We studied the fast muscle-fibre reorientation and the concomitant myotomal strain variance in a body segment near the anus during larval and juvenile development in the zebrafish. From 2 to 4 days post fertilization (d.p.f.), the measured angles between the muscle fibres and the longitudinal axis of the zebrafish were small. Yet, onset of a near-helical muscle-fibre arrangement was recognized. Juveniles of 51 d.p.f. have larger mean fibre angles and already possess the near-helical pattern of adult teleosts. We present a model that computes the distribution of the strain along the muscle fibres from measured muscle-fibre orientations, body curvature and prescribed tissue deformations. We selected the most extreme body curvatures, which only occur during fast starts and turning manoeuvres. Using the model, we identified the (non-linear) tissue deformations that yield the least variance in the muscle-fibre strain. We show that simple beam theory cannot reliably predict the strain distribution: it results in very small strains and negligible work output of the most medial fibres. In our model, we avoided these functional limitations by adding a shear deformation to the simple beam deformation. At 2 d.p.f., the predicted variance in the muscle-fibre strain for the shear deformation optimized for strain uniformity is fairly small, due to the small variation in the fibre distances to the medial plane that is caused by the relatively large spinal cord and notochord. The predicted minimal strain variance increases sharply from 2 d.p.f. to 3 d.p.f., remains relatively large at 4 d.p.f., but decreases again considerably at 15 and 39 d.p.f. The 51 d.p.f. stage exhibits the smallest variance in the fibre strains (for the identified optimal deformation), in spite of the widely varying muscle-fibre distances to the medial plane. The non-linear nature of the body deformations with the least strain variance implies an interesting optimization constraint: the juvenile muscle-fibre arrangement results in small predicted spatial strain variations at large-amplitude body curvatures, at the (modest) expense of a large coefficient of variation for small curvatures. We conclude that larval fish rapidly change their muscle-fibre orientations (probably in response to mechanical signals). Within the theoretically examined plausible range of deformations, the closest correspondence to a uniform strain field was found for the juvenile stage.

Three novel CXC chemokines were identified in common carp (Cyprinus carpio L.) through homology cloning. Phylogenetic analyses show that one of the three CXC chemokines is an unambiguous orthologue of CXCL14, whereas both others are orthologues of CXCL12, and were named CXCL12a and CXCL12b. Percentages of amino acid identity between each of these carp chemokines and their human and mouse orthologues are markedly higher than those reported previously for other carp CXC chemokines, suggestive of involvement in vital processes, which have allowed for relatively few structural changes. Furthermore, all three novel carp CXC chemokines are expressed during early development, in contrast to established immune CXC chemokines. In noninfected adult carp, CXCL12b and CXCL14 are predominantly expressed in the brain. CXCL12a is highly expressed in kidney and anterior kidney, but its expression is still more abundant in brain than any other carp CXC chemokine. Clearly, these chemokines must play key roles in the patterning and maintenance of the (developing) vertebrate central nervous system.

Postnatal maintenance or regeneration of pancreatic beta cells is considered to occur exclusively via the replication of existing beta cells, but clinically meaningful restoration of human beta cell mass by proliferation has never been achieved. We discovered a population of immature beta cells that is present throughout life and forms from non-beta precursors at a specialized micro-environment or "neogenic niche" at the islet periphery. These cells express insulin, but lack other key beta cell markers, and are transcriptionally immature, incapable of sensing glucose, and unable to support calcium influx. They constitute an intermediate stage in the transdifferentiation of alpha cells to cells that are functionally indistinguishable from conventional beta cells. We thus identified a lifelong source of new beta cells at a specialized site within healthy islets. By comparing co-existing immature and mature beta cells within healthy islets, we stand to learn how to mature insulin-expressing cells into functional beta cells.

Peptide hormones are key physiological regulators, and many would make terrific drugs; however, the therapeutic use of peptides is limited by poor metabolism including rapid proteolysis. To develop novel proteolysis-resistant peptide hormone analogs, we utilize a strategy that relies on data from simple mass spectrometry experiments to guide the chemical synthesis of proteolysis-resistant analogs (i.e., data-driven synthesis). Application of this strategy to oxyntomodulin (OXM), a peptide hormone that stimulates insulin secretion from islets and lowers blood glucose in vivo, defined the OXM cleavage site in serum, and this information was used to synthesize a proteolysis-resistant OXM analog (prOXM). prOXM and OXM have similar activity in binding and glucose stimulated-insulin secretion assays. Furthermore, prOXM is also active in vivo. prOXM reduces basal glucose levels and improves glucose tolerance in mice. The discovery of prOXM suggests that proteolysis-resistant variants of other important peptide hormones can also be found using this strategy to increase the number of candidate therapeutic peptides.

Grb2 and ShcA are two phosphotyrosine-binding proteins that link receptor protein-tyrosine kinases to activation of the Ras-Erk pathway. While some receptors bind Grb2 directly, others bind ShcA, which provides a binding site for Grb2. In order to compare signal transduction through a Grb2-binding site with signal transduction through a ShcA-binding site, we replaced the ShcA-binding site in the NGF receptor with a Grb2-binding site. Our results show that the Grb2- and ShcA-binding sites have similar abilities to activate the Ras-Erk and PI 3-kinase-Akt pathways. In contrast, they displayed dramatic differences in their ability to activate DNA synthesis.

Contains fulltext : 32903.pdf (Publisher’s version ) (Closed access) The generation of lymphoid cells during carp development was studied by analyzing expression of the recombination activating genes (rag) using in situ hybridization and real time quantitative PCR. These data were combined with immunohistochemistry using the mAb's WCL9 (cortical thymocytes) and WCI12 (B cells). Carp rag-1 and rag-2 showed 90 and 89% amino acid identity, respectively, to the corresponding zebrafish sequences. Rag-1 was first expressed in the thymus at 4 days post-fertilization (dpf), while both rag-1+/WCL9+ and rag-1-/WCL9- areas were distinguished from 1 week post-fertilization (wpf), suggesting early cortex/medulla differentiation. From 6 dpf, rag-1+ cells were also present cranio-lateral of the head kidney. From 1 wpf, rag-1/rag-2 was expressed in kidney (together with immunoglobulin heavy chain expression) but not in spleen, while WCI12+ cells appeared 1 week later in both organs, suggesting B cell recombination in kidney but not in spleen. Rag-1 expression exceeded rag-2 levels in thymus and in head- and trunk-kidney of juveniles, but this ratio was reversed in head- and trunk-kidney from approximately 16 wpf onwards. Rag-1/rag-2 expression was detected in thymi of animals over 1-year-old, but in kidney only at low levels, indicating life-long new formation of putative T cells but severely reduced formation of B cells in older fish.

Three novel CXC chemokines were identified in common carp (Cyprinus carpio L.) through homology cloning. Phylogenetic analyses show that one of the three CXC chemokines is an unambiguous orthologue of CXCL14, whereas both others are orthologues of CXCL12, and were named CXCL12a and CXCL12b. Percentages of amino acid identity between each of these carp chemokines and their human and mouse orthologues are markedly higher than those reported previously for other carp CXC chemokines, suggestive of involvement in vital processes, which have allowed for relatively few structural changes. Furthermore, all three novel carp CXC chemokines are expressed during early development, in contrast to established immune CXC chemokines. In noninfected adult carp, CXCL12b and CXCL14 are predominantly expressed in the brain. CXCL12a is highly expressed in kidney and anterior kidney, but its expression is still more abundant in brain than any other carp CXC chemokine. Clearly, these chemokines must play key roles in the patterning and maintenance of the (developing) vertebrate central nervous system.

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3(+) beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3(+) hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3(+) alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies.