Reduced mTORC1-signaling in progenitor cells leads to retinal lamination deficits.

BACKGROUND: Neuronal lamination is a hallmark of the mammalian central nervous system (CNS) and underlies connectivity and function. Initial formation of this tissue architecture involves the integration of various signaling pathways that regulate the differentiation and migration of neural progenitor cells. RESULTS: Here, we demonstrate that mTORC1 mediates critical roles during neuronal lamination using the mouse retina as a model system. Down-regulation of mTORC1-signaling in retinal progenitor cells by conditional deletion of Rptor led to decreases in proliferation and increased apoptosis during embryogenesis. These developmental deficits preceded aberrant lamination in adult animals which was best exemplified by the fusion of the outer and inner nuclear layer and the absence of an outer plexiform layer. Moreover, ganglion cell axons originating from each Rptor-ablated retina appeared to segregate to an equal degree at the optic chiasm with both contralateral and ipsilateral projections displaying overlapping termination topographies within several retinorecipient nuclei. In combination, these visual pathway defects led to visually mediated behavioral deficits. CONCLUSIONS: This study establishes a critical role for mTORC1-signaling during retinal lamination and demonstrates that this pathway regulates diverse developmental mechanisms involved in driving the stratified arrangement of neurons during CNS development.

Insights into the etiology and physiopathology of MODY5/HNF1B pancreatic phenotype with a mouse model of the human disease.

Maturity-onset diabetes of the young type 5 (MODY5) is due to heterozygous mutations or deletion of HNF1B. No mouse models are currently available to recapitulate the human MODY5 disease. Here, we investigate the pancreatic phenotype of a unique MODY5 mouse model generated by heterozygous insertion of a human HNF1B splicing mutation at the intron-2 splice donor site in the mouse genome. This Hnf1bsp2/+ model generated with targeted mutation of Hnf1b mimicking the c.544+1G>T (T) mutation identified in humans, results in alternative transcripts and a 38% decrease of native Hnf1b transcript levels. As a clinical feature of MODY5 patients, the hypomorphic mouse model Hnf1bsp2/+ displays glucose intolerance. Whereas Hnf1bsp2/+ isolated islets showed no altered insulin secretion, we found a 65% decrease in pancreatic insulin content associated with a 30% decrease in total large islet volume and a 20% decrease in total ß-cell volume. These defects were associated with a 30% decrease in expression of the pro-endocrine gene Neurog3 that we previously identified as a direct target of Hnf1b, showing a developmental etiology. As another clinical feature of MODY5 patients, the Hnf1bsp2/+ pancreases display exocrine dysfunction with hypoplasia. We observed chronic pancreatitis with loss of acinar cells, acinar-to-ductal metaplasia, and lipomatosis, with upregulation of signaling pathways and impaired acinar cell regeneration. This was associated with ductal cell deficiency characterized by shortened primary cilia. Importantly, the Hnf1bsp2/+ mouse model reproduces the pancreatic features of the human MODY5/HNF1B disease, providing a unique in vivo tool for molecular studies of the endocrine and exocrine defects and to advance basic and translational research. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Sleeve gastrectomy, but not duodenojejunostomy, preserves total beta-cell mass in Goto-Kakizaki rats evaluated by three-dimensional optical projection tomography.

BACKGROUND: In type 2 diabetes mellitus, there is a progressive loss of beta-cell mass. Bariatric surgery has in recent investigations showed promising results in terms of diabetes remission, but little is established regarding the effect of surgery on the survival or regeneration of pancreatic beta-cells. In this study, we aim to explore how bariatric surgery with its subsequent hormonal alterations affects the islets of Langerhans. METHODS: Twenty-four Goto-Kakizaki rats were operated with duodenojejunostomy (DJ), sleeve gastrectomy (SG) or sham operation. From the 38th week after surgery, body weight, fasting blood glucose, glycosylated hemoglobin, mixed meal tolerance with repeated measures of insulin, glucagon-like peptide 1, gastrin and total ghrelin were evaluated. Forty-six weeks after surgery, the animals were euthanized and the total beta-cell mass in all animals was examined by three-dimensional volume quantification by optical projection tomography based on the signal from insulin-specific antibody staining. RESULTS: Body weight did not differ between groups (P(g) = 0.37). SG showed lower fasting blood glucose compared to DJ and sham (P(g) = 0.037); HbA1c levels in SG were lower compared to DJ only (p < 0.05). GLP-1 levels were elevated for DJ compared to SG and sham (P(g) = 0.001), whereas gastrin levels were higher in SG compared to the two other groups (P(g) = 0.002). Beta-cell mass was significantly greater in animals operated with SG compared to both DJ and sham (p = 0.036). CONCLUSION: Sleeve gastrectomy is superior to duodenojejunostomy and sham operation when comparing the preservation of beta-cell mass 46 weeks after surgery in Goto-Kakizaki rats. This could be related to both the increased gastrin levels and the long-term improvement in glycemic parameters observed after this procedure.

Improving signal detection in emission optical projection tomography via single source multi-exposure image fusion.

We demonstrate a technique to improve structural data obtained from Optical Projection Tomography (OPT) using Image Fusion (IF) and contrast normalization. This enables the visualization of molecular expression patterns in biological specimens with highly variable contrast values. In the approach, termed IF-OPT, different exposures are fused by assigning weighted contrasts to each. When applied to projection images from mouse organs and digital phantoms our results demonstrate the capability of IF-OPT to reveal high and low signal intensity details in challenging specimens. We further provide measurements to highlight the benefits of the new algorithm in comparison to other similar methods.

Quantitative 3D OPT and LSFM datasets of pancreata from mice with streptozotocin-induced diabetes.

Mouse models for streptozotocin (STZ) induced diabetes probably represent the most widely used systems for preclinical diabetes research, owing to the compound's toxic effect on pancreatic ß-cells. However, a comprehensive view of pancreatic ß-cell mass distribution subject to STZ administration is lacking. Previous assessments have largely relied on the extrapolation of stereological sections, which provide limited 3D-spatial and quantitative information. This data descriptor presents multiple ex vivo tomographic optical image datasets of the full ß-cell mass distribution in mice subject to single high and multiple low doses of STZ administration, and in glycaemia recovered mice. The data further include information about structural features, such as individual islet ß-cell volumes, spatial coordinates, and shape as well as signal intensities for both insulin and GLUT2. Together, they provide the most comprehensive anatomical record of the effects of STZ administration on the islet of Langerhans in mice. As such, this data descriptor may serve as reference material to facilitate the planning, use and (re)interpretation of this widely used disease model.

3D imaging of human organs with micrometer resolution - applied to the endocrine pancreas.

The possibility to quantitatively study specific molecular/cellular features of complete human organs with preserved spatial 3D context would have widespread implications for pre-clinical and clinical medicine. Whereas optical 3D imaging approaches have experienced a formidable revolution, they have remained limited due to current incapacities in obtaining specific labelling within large tissue volumes. We present a simple approach enabling reconstruction of antibody labeled cells within entire human organs with preserved organ context. We demonstrate the utility of the approach by providing volumetric data and 3D distribution of hundreds of thousands of islets of Langerhans within the human pancreas. By assessments of pancreata from non-diabetic and type 2 diabetic individuals, we display previously unrecognized features of the human islet mass distribution and pathology. As such, this method may contribute not only in unraveling new information of the pancreatic anatomy/pathophysiology, but it may be translated to essentially any antibody marker or organ system.

Distinct Opsin 3 (Opn3) Expression in the Developing Nervous System during Mammalian Embryogenesis.

Opsin 3 (Opn3) is highly expressed in the adult brain, however, information for spatial and temporal expression patterns during embryogenesis is significantly lacking. Here, an Opn3-eGFP reporter mouse line was used to monitor cell body expression and axonal projections during embryonic and early postnatal to adult stages. By applying 2D and 3D fluorescence imaging techniques, we have identified the onset of Opn3 expression, which predominantly occurred during embryonic stages, in various structures during brain/head development. In addition, this study defines over twenty Opn3-eGFP-positive neural structures never reported before. Opn3-eGFP was first observed at E9.5 in neural regions, including the ganglia that will ultimately form the trigeminal, facial and vestibulocochlear cranial nerves (CNs). As development proceeds, expanded Opn3-eGFP expression coincided with the formation and maturation of critical components of the central and peripheral nervous systems (CNS, PNS), including various motor-sensory tracts, such as the dorsal column-medial lemniscus (DCML) sensory tract, and olfactory, acoustic, and optic tracts. The widespread, yet distinct, detection of Opn3-eGFP already at early embryonic stages suggests that Opn3 might play important functional roles in the developing brain and spinal cord to regulate multiple motor and sensory circuitry systems, including proprioception, nociception, ocular movement, and olfaction, as well as memory, mood, and emotion. This study presents a crucial blueprint from which to investigate autonomic and cognitive opsin-dependent neural development and resultant behaviors under physiological and pathophysiological conditions.

Mesoscopic 3D imaging of pancreatic cancer and Langerhans islets based on tissue autofluorescence.

The possibility to assess pancreatic anatomy with microscopic resolution in three dimensions (3D) would significantly add to pathological analyses of disease processes. Pancreatic ductal adenocarcinoma (PDAC) has a bleak prognosis with over 90% of the patients dying within 5 years after diagnosis. Cure can be achieved by surgical resection, but the efficiency remains drearily low. Here we demonstrate a method that without prior immunohistochemical labelling provides insight into the 3D microenvironment and spread of PDAC and premalignant cysts in intact surgical biopsies. The method is based solely on the autofluorescent properties of the investigated tissues using optical projection tomography and/or light-sheet fluorescence microscopy. It does not interfere with subsequent histopathological analysis and may facilitate identification of tumor-free resection margins within hours. We further demonstrate how the developed approach can be used to assess individual volumes and numbers of the islets of Langerhans in unprecedently large biopsies of human pancreatic tissue, thus providing a new means by which remaining islet mass may be assessed in settings of diabetes. Generally, the method may provide a fast approach to provide new anatomical insight into pancreatic pathophysiology.

Topologically selective islet vulnerability and self-sustained downregulation of markers for ß-cell maturity in streptozotocin-induced diabetes.

Mouse models of Streptozotocin (STZ) induced diabetes represent the most widely used preclinical diabetes research systems. We applied state of the art optical imaging schemes, spanning from single islet resolution to the whole organ, providing a first longitudinal, 3D-spatial and quantitative account of ß-cell mass (BCM) dynamics and islet longevity in STZ-treated mice. We demonstrate that STZ-induced ß-cell destruction predominantly affects large islets in the pancreatic core. Further, we show that hyperglycemic STZ-treated mice still harbor a large pool of remaining ß-cells but display pancreas-wide downregulation of glucose transporter type 2 (GLUT2). Islet gene expression studies confirmed this downregulation and revealed impaired ß-cell maturity. Reversing hyperglycemia by islet transplantation partially restored the expression of markers for islet function, but not BCM. Jointly our results indicate that STZ-induced hyperglycemia results from ß-cell dysfunction rather than ß-cell ablation and that hyperglycemia in itself sustains a negative feedback loop restraining islet function recovery.

Retraction Note: Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.

This article has been retracted; see accompanying Retraction Note, which can be accessed via a link at the top of the paper.

Spatial and quantitative datasets of the pancreatic ß-cell mass distribution in lean and obese mice.

A detailed understanding of pancreatic ß-cell mass distribution is a key element to fully appreciate the pathophysiology of models of diabetes and metabolic stress. Commonly, such assessments have been performed by stereological approaches that rely on the extrapolation of two-dimensional data and provide very limited topological information. We present ex vivo optical tomographic data sets of the full ß-cell mass distribution in cohorts of obese ob/ob mice and their lean controls, together with information about individual islet ß-cell volumes, their three-dimensional coordinates and shape throughout the volume of the pancreas between 4 and 52 weeks of age. These data sets offer the currently most comprehensive public record of the ß-cell mass distribution in the mouse. As such, they may serve as a quantitative and topological reference for the planning of a variety of in vivo or ex vivo experiments including computational modelling and statistical analyses. By shedding light on intra- and inter-lobular variations in ß-cell mass distribution, they further provide a powerful tool for the planning of stereological sampling assessments.

Biochemical profiling of diabetes disease progression by multivariate vibrational microspectroscopy of the pancreas.

Despite the dramatic increase in the prevalence of diabetes, techniques for in situ studies of the underlying pancreatic biochemistry are lacking. Such methods would facilitate obtaining mechanistic understanding of diabetes pathophysiology and aid in prognostic and/or diagnostic assessments. In this report we demonstrate how a multivariate imaging approach (orthogonal projections to latent structures - discriminant analysis) can be applied to generate full vibrational microspectroscopic profiles of pancreatic tissues. These profiles enable extraction of known and previously unrecorded biochemical alterations in models of diabetes, and allow for classification of the investigated tissue with regards to tissue type, strain and stage of disease progression. Most significantly, the approach provided evidence for dramatic alterations of the pancreatic biochemistry at the initial onset of immune-infiltration in the Non Obese Diabetic model for type 1 diabetes. Further, it enabled detection of a previously undocumented accumulation of collagen fibrils in the leptin deficient ob/ob mouse islets. By generating high quality spectral profiles through the tissue capsule of hydrated human pancreata and by in vivo Raman imaging of pancreatic islets transplanted to the anterior chamber of the eye, we provide critical feasibility studies for the translation of this technique to diagnostic assessments of pancreatic biochemistry in vivo.

Intra-islet lesions and lobular variations in ß-cell mass expansion in ob/ob mice revealed by 3D imaging of intact pancreas.

The leptin deficient ob/ob mouse is a widely used model for studies on initial aspects of metabolic disturbances leading to type 2 diabetes, including insulin resistance and obesity. Although it is generally accepted that ob/ob mice display a dramatic increase in ß-cell mass to compensate for increased insulin demand, the spatial and quantitative dynamics of ß-cell mass distribution in this model has not been assessed by modern optical 3D imaging techniques. We applied optical projection tomography and ultramicroscopy imaging to extract information about individual islet ß-cell volumes throughout the volume of ob/ob pancreas between 4 and 52 weeks of age. Our data show that cystic lesions constitute a significant volume of the hyperplastic ob/ob islets. We propose that these lesions are formed by a mechanism involving extravasation of red blood cells/plasma due to increased islet vessel blood flow and vessel instability. Further, our data indicate that the primary lobular compartments of the ob/ob pancreas have different potentials for expanding their ß-cell population. Unawareness of the characteristics of ß-cell expansion in ob/ob mice presented in this report may significantly influence ex vivo and in vivo assessments of this model in studies of ß-cell adaptation and function.

A novel mouse model of tuberous sclerosis complex (TSC): eye-specific Tsc1-ablation disrupts visual-pathway development.

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome that is best characterised by neurodevelopmental deficits and the presence of benign tumours (called hamartomas) in affected organs. This multi-organ disorder results from inactivating point mutations in either the TSC1 or the TSC2 genes and consequent activation of the canonical mammalian target of rapamycin complex 1 signalling (mTORC1) pathway. Because lesions to the eye are central to TSC diagnosis, we report here the generation and characterisation of the first eye-specific TSC mouse model. We demonstrate that conditional ablation of Tsc1 in eye-committed progenitor cells leads to the accelerated differentiation and subsequent ectopic radial migration of retinal ganglion cells. This results in an increase in retinal ganglion cell apoptosis and consequent regionalised axonal loss within the optic nerve and topographical changes to the contra- and ipsilateral input within the dorsal lateral geniculate nucleus. Eyes from adult mice exhibit aberrant retinal architecture and display all the classic neuropathological hallmarks of TSC, including an increase in organ and cell size, ring heterotopias, hamartomas with retinal detachment, and lamination defects. Our results provide the first major insight into the molecular etiology of TSC within the developing eye and demonstrate a pivotal role for Tsc1 in regulating various aspects of visual-pathway development. Our novel mouse model therefore provides a valuable resource for future studies concerning the molecular mechanisms underlying TSC and also as a platform to evaluate new therapeutic approaches for the treatment of this multi-organ disorder.

Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells.

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives.

Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism.

Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis.

Differential regulation of myosin heavy chains defines new muscle domains in zebrafish.

Numerous muscle lineages are formed during myogenesis within both slow- and fast-specific cell groups. In this study, we show that six fast muscle-specific myosin heavy chain genes have unique expression patterns in the zebrafish embryo. The expression of tail-specific myosin heavy chain (fmyhc2.1) requires wnt signaling and is essential for fast muscle organization within the tail. Retinoic acid treatment results in reduced wnt signaling, which leads to loss of the fmyhc2.1 domain. Retinoic acid treatment also results in a shift of muscle identity within two trunk domains defined by expression of fmyhc1.2 and fmyhc1.3 in favor of the anteriormost myosin isoform, fmyhc1.2. In summary, we identify new muscle domains along the anteroposterior axis in the zebrafish that are defined by individual nonoverlapping, differentially regulated expression of myosin heavy chain isoforms.

Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.

Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.