Whole-mount Immunohistochemistry Research Articles

Development of branchial ionocytes in embryonic and larval stages of cloudy catshark, Scyliorhinus torazame.

In teleost fish, branchial ionocytes are important sites for osmoregulation and acid-base regulation by maintaining ionic balance in the body fluid. During the early developmental stages before the formation of the gills, teleost ionocytes are localized in the yolk-sac membrane and body skin. By comparing with teleost fish, much less is known about ionocytes in developing embryos of elasmobranch fish. The present study investigated the development of ionocytes in the embryo and larva of cloudy catshark, Scyliorhinus torazame. We first observed ionocyte distribution by immunohistochemical staining with anti-Na+/K+-ATPase (NKA) and anti-vacuolar-type H+-ATPase (V-ATPase) antibodies. The NKA- and V-ATPase-rich ionocytes appeared as single cells in the gill filaments from stage 31, the stage of pre-hatching, while the ionocytes on the body skin and yolk-sac membrane were also observed. From stage 32, in addition to single ionocytes on the gill filaments, some outstanding follicular structures of NKA-immunoreactive cells were developed to fill the inter-filament region of the gill septa. The follicular ionocytes possess NKA in the basolateral membrane and Na+/H+ exchanger 3 in the apical membrane, indicating that they are involved in acid-base regulation like single NKA-rich ionocytes. Three-dimensional analysis and whole-mount immunohistochemistry revealed that the distribution of follicular ionocytes was limited to the rostral side of gill septum. The rostral sides of gill septum might be exposed to faster water flow than caudal side because the gills of sharks gently curved backward. This dissymmetric distribution of follicular ionocytes is considered to facilitate efficient body-fluid homeostasis of catshark embryo.

Effect of valproic acid on the formation and migration of cranial neural crest cells at the early developmental stages in rat embryos.

Cranial neural crest cells (NCCs) are critical for craniofacial development. The administration of valproic acid (VPA) to pregnant females causes craniofacial malformations in offspring. However, the in vivo influence of VPA on mammalian cranial NCCs remains unclear. In this study, we aimed to elucidate the developmental stage-specific effect of VPA on cranial NCCs through the administration of a single dose of VPA to pregnant rat females immediately prior to the formation of the cranial neural crest (NC). We performed whole-mount immunohistochemistry or in situ hybridization to examine localization changes of gene transcripts associated with the epithelial-mesenchymal transition of the cranial NC (i.e., cranial NCC formation) and cranial NCC migration. The results showed that Hoxa2 mRNA was abnormally detected and Sox9 mRNA expression was decreased in the midbrain-rhombomere (R) 1/2 NC, which forms cranial NCCs that migrate to the frontonasal mass (FNM) and branchial arch (BA) 1, through VPA administration, thus reducing the formation of SNAI2-positive NCCs. Hoxa2-positive NCCs were detected normally in BA2 and abnormally in FNM and BA1, which are normally Hox-free, implying VPA-induced abnormal cranial NCC migration. In vitro verification experiments using the whole embryo culture system revealed that midbrain-R4 NCC migration was abnormal. These results indicate that VPA reduces the formation/delamination of the midbrain-R1/2 NCCs in a developmental stage-specific manner and subsequently causes the abnormal migration of R4 NCCs, which suggests that the abnormal formation and migration of cranial NCCs contribute to the inhibition of axonal elongation in the trigeminal nerve and a reduction in head size.

Probiotic Lactobacillus spp. improves Drosophila memory by increasing lactate dehydrogenase levels in the brain mushroom body neurons

ABSTRACT Probiotics are live microorganisms that offer potential benefits to their hosts and can occasionally influence behavioral responses. However, the detailed mechanisms by which probiotics affect the behavior of their hosts and the underlying biogenic effects remain unclear. Lactic acid bacteria, specifically Lactobacillus spp. are known probiotics. Drosophila melanogaster, commonly known as the fruit fly, is a well-established model organism for investigating the interaction between the host and gut microbiota in translational research. Herein, we showed that 5-day administration of Lactobacillus acidophilus (termed GMNL-185) or Lacticaseibacillus rhamnosus (termed GMNL-680) enhances olfactory-associative memory in Drosophila. Moreover, a combined diet of GMNL-185 and GMNL-680 demonstrated synergistic effects on memory functions. Live brain imaging revealed a significant increase in calcium responses to the training odor in the mushroom body β and γ lobes of flies that underwent mixed feeding with GMNL-185 and GMNL-680. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and whole-mount brain immunohistochemistry revealed significant upregulation of lactate dehydrogenase (LDH) expression in the fly brain following the mixed feeding. Notably, the genetic knockdown of Ldh in neurons, specifically in mushroom body, ameliorated the beneficial effects of mixed feeding with GMNL-185 and GMNL-680 on memory improvement. Altogether, our results demonstrate that supplementation with L. acidophilus and L. rhamnosus enhances memory functions in flies by increasing brain LDH levels.

Quantitative dynamic contrast-enhance MRI parameters for rectal carcinoma characterization: correlation with tumor tissue composition

ObjectiveTo investigate the relationship between dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) measurements and the potential composition of rectal carcinoma.MethodsTwenty-four patients provided informed consent for this study. DCE-MRI was performed before total mesorectal excision. Quantitative parameters were calculated based on a modified Tofts model. Whole-mount immunohistochemistry and Masson staining sections were generated and digitized at histological resolution. The percentage of tissue components area was measured. Pearson correlation analysis was used to evaluate the correlations between pathological parameters and DCE-MRI parameters.ResultsOn the World Health Organization (WHO) grading scale, there were significant differences in extracellular extravascular space (Ktrans) (F = 9.890, P = 0.001), mean transit time (MTT) (F = 9.890, P = 0.038), CDX-2 (F = 4.935, P = 0.018), and Ki-67 (F = 4.131, P = 0.031) among G1, G2, and G3. ECV showed significant differences in extramural venous invasion (t = − 2.113, P = 0.046). Ktrans was strongly positively correlated with CD34 (r = 0.708, P = 0.000) and moderately positively correlated with vimentin (r = 0.450, P = 0.027). Interstitial volume (Ve) was moderately positively correlated with Masson’s (r = 0.548, P = 0.006) and vimentin (r = 0.417, P = 0.043). There was a moderate negative correlation between Ve and CDX-2 (r = − 0.441, P = 0.031). The rate constant from extracellular extravascular space to blood plasma (Kep) showed a strong positive correlation with CD34 expression (r = 0.622, P = 0.001). ECV showed a moderate negative correlation with CDX-2 (r = − 0.472, P = 0.020) and a moderate positive correlation with collagen fibers (r = 0.558, P = 0.005).ConclusionThe dynamic contrast-enhanced MRI-derived parameters measured in rectal cancer were significantly correlated with the proportion of histological components. This may serve as an optimal imaging biomarker to identify tumor tissue components.

Disruption of Col9a2 expression leads to defects in osteochondral homeostasis and osteoarthritis-like phenotype in mice

Background/ObjectiveAs one of the branched chains of Type IX collagen (Col9), Collagen IX alpha2 (Col9a2) has been reported to be associated with several orthopedic conditions. However, the relationship between Col9a2 and knee osteoarthritis (KOA) remains to be elucidated. MethodsTo probe the relationship between Col9a2 and KOA, we performed a systematic analysis of Col9a2-deficient (Col9a2−/−) mice using whole-mount skeletal staining, Micro-CT (μCT), biomechanics, histomorphometry, immunohistochemistry (IHC), immunofluorescence (IF) and Elisa. ResultsWe found that the subchondral bone (SCB) in the knee joint of Col9a2−/− mice became sparse and deformed in the early stage, with altered bone morphometric parameters, reduced load-bearing capacity, dysfunctional bone homeostasis (decreased osteogenesis capacity and elevated bone resorption capacity), diminished cartilage proteoglycans and disrupted cartilage extracellular matrix (ECM) anabolism and catabolism compared with the Col9a2+/+ mice. In the late stage, the cartilage degeneration in Col9a2−/− mice were particularly pronounced compared to Col9a2+/+ mice, as evidenced by severe cartilage destruction and a marked reduction in cartilage thickness and area. ConclusionOverall, Col9a2 is essential for maintaining osteochondral homeostasis in the knee joint of mice, and the absence of this gene is accompanied by distinct sclerosis of the SCB and a reduction in load-bearing capacity; in the late stage, in the lack of SCB stress inhibition, excessive load is consistently exerted on the cartilage, ultimately leading to osteoarthritic-like articular cartilage damage.

Taste arbor structural variability analyzed across taste regions.

Taste ganglion neurons are functionally and molecularly diverse, but until recently morphological diversity was completely unexplored. Specifically, taste arbors (the portion of the neuron within the taste bud) vary in structure, but the reason for this variability is unclear. Here, we analyzed structural variability in taste arbors to determine which factors determine their morphological diversity. To characterize arbor morphology and its relationship to taste bud cells capable of transducing taste stimuli (taste-transducing cell) number and type, we utilized sparse cell genetic labeling of taste ganglion neurons in combination with whole-mount immunohistochemistry. Reconstruction of 151 taste arbors revealed variation in arbor size, complexity, and symmetry. Overall, taste arbors exist on a continuum of complexity, cannot be categorized into discrete morphological groups, and do not have stereotyped endings. Arbor size/complexity was not related to the size of the taste bud in which it was located or the type of taste-transducing cell contacted (membranes within 180nm). Instead, arbors could be broadly categorized into three groups: large asymmetrical arbors contacting many taste-transducing cells, small symmetrical arbors contacting one or two taste-transducing cells, and unbranched arbors. Neurons with multiple arbors had arbors in more than one of these categories, indicating that this variability is not an intrinsic feature of neuron type. Instead, we speculate that arbor structure is determined primarily by nerve fiber remodeling in response to cell turnover and that large asymmetrical arbors represent a particularly plastic state.

Isozyme-specific histone deacetylase 1/2 inhibitor K560 attenuates oxidative stress-induced retinal cell death

Oxidative stress-induced damage is an underlying mechanism in the pathogenesis of age-related retinal diseases. Here, we examined the effects of K560, a potential candidate drug for the treatment of these diseases, on oxidative stress-induced retinal cell death. K560 is a novel isozyme-specific inhibitor of histone deacetylase 1 and 2 (HDAC1/2). Histone acetylation in retinal lysates and dissociated retinal cells was detected with a western blot analysis and cell-based enzyme-linked immunosorbent assay (ELISA), respectively. The viability of mouse retinal cells was measured with an alamarBlue assay. We used immunohistochemistry for RNA binding protein with multiple splicing (RBPMS) to visualize the retinal ganglion cells (RGCs) of mice. An ELISA analysis indicated that histone acetylation was enhanced in dissociated mouse retinal cells treated with K560. The cell viability assay indicated that K560 attenuated both exogenous hydrogen peroxide–induced and endogenous oxidative stress–induced cell death in dissociated retinal cells. Western blot analysis indicated that intravitreal K560 administration enhanced the acetylation of histones H3 and H4 in mouse retinal lysates. To examine the effect of K560 on oxidative stress–induced RGC death, we performed whole-mount immunohistochemistry for RBPMS on retinas dissected from eyes treated with K560 or vehicle on day one, and K560 or vehicle and NMDA on day two. Quantification of RBPMS-immunopositive cells indicated that K560 attenuated NMDA-induced RGC death. Taken together, our findings suggest that administration of a HDAC1/2-specific inhibitor K560 may be effective in the treatment of oxidative stress–mediated retinal degeneration and have less cytotoxicity than other known HDAC inhibitors, which are known to target a wide range of HDAC family members.

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

Development of ribs and intercostal muscles in the chicken embryo.

In the thorax of higher vertebrates, ribs and intercostal muscles play a decisive role in stability and respiratory movements of the body wall. They are derivatives of the somites, the ribs originating in the sclerotome and the intercostal muscles originating in the myotome. During thorax development, ribs and intercostal muscles extend into the lateral plate mesoderm and eventually contact the sternum during ventral closure. Here, we give a detailed description of the morphogenesis of ribs and thoracic muscles in the chicken embryo (Gallus gallus). Using Alcian blue staining as well as Sox9 and Desmin whole‐mount immunohistochemistry, we monitor synchronously the development of rib cartilage and intercostal muscle anlagen. We show that the muscle anlagen precede the rib anlagen during ventrolateral extension, which is in line with the inductive role of the myotome in rib differentiation. Our studies furthermore reveal the temporary formation of a previously unknown eighth rib in the chicken embryonic thorax.

Conservation of Kidney Development in the Red‐eared Slider, Trachemys scripta

Mammals, birds, and reptiles all possess a metanephric kidney in adult animals. The process of kidney development, or nephrogenesis, ceases in mammals prior to or immediately after birth. Termination of mammalian nephrogenesis is due to kidney progenitor cell populations halting self‐renewal and undergoing terminal differentiation. Our previous studies have shown in several reptile species the presence of post‐embryonic nephrogenesis accompanying embryonic‐like adult progenitor cells. It is unknown how or why kidney progenitor cell populations are lost during mammalian development but persist in reptiles. To better understand the persistence of kidney progenitor cells in reptiles, we have set out to characterize kidney development to determine if adult progenitor cell populations are a continuation of embryonic processes. We have chosen to use the red‐eared slider turtle, Trachemys scripta, as a model for reptile metanephric kidney development as this species showed the highest number of nephrogenic zones in adults. While the embryonic development of external morphology (such as head, limbs, shell) in this species has been documented, organogenesis has not been studied as thoroughly. We have utilized gross observations of morphology, histology, whole‐mount immunohistochemistry and in situ hybridization to characterize and assess the conservation of kidney development in this species. The embryonic kidney progenitor cell marker, Six2, was detected as early as stage‐15 as a linear group of metanephric mesenchyme. Little to no cytokeratin positive epithelium was detected at this stage of kidney development. As kidney development proceeds, the progenitor mesenchyme cells and adjacent ureteric bud epithelium are found in highly organized rows. Several genes known to play roles within mammalian kidney development displayed conserved expression domains in the developing T. scripta kidney. The embryonically expressed Six2 transcription factor was also detected in progenitor cells of adult kidneys. Future investigation will continue to assess kidney ontogeny in reptiles.

Implementation of tissue clearing and 3‐dimensional volumetric imaging approaches for visualization of anatomical structures in autonomic nuclei

Alterations within central nervous system (CNS) autonomic nuclei contribute to a wide array of diseases. In this context, significant advancements have been made into disease‐related molecular mechanisms in autonomic brain regions. While helpful, it is important to consider that information processing within the CNS occurs through dynamic micro‐architectural structural interactions, such astrocyte‐neuron synaptic connections. However the traditional method for evaluating CNS morphology and synaptic contacts between cells is performed with immunohistochemistry on thin brain sections. An inherent limitation of this approach is not only the narrow 2‐dimensional field of view, but also the lack of optical transparency that minimizes high‐resolution imaging of anatomical structures and cell‐cell interactions. To overcome these limitations, here we sought to optimize tissue clearing approaches combined with 3D volumetric imaging for autonomic CNS nuclei. We focused our initial efforts on the forebrain subfornical organ (SFO), a region lacking a blood‐brain‐barrier, given: 1 Alterations in the SFO have been described to contribute to numerous disease conditions; and 2 The SFO represents a unique technical challenge for 3D imaging due to the complexity of cellular processes and surrounding lateral ventricle space. As a first step, we used a “passive” CLARITY tissue approach in 8‐week‐old C57Bl/6J male mice. Mice were transcardially perfused with an acrylamide‐based hydrogel in standard fixative. Subsequently, a brain section encompassing the entire SFO (~1mm) was obtained, and a series of thermal diffusion steps was performed to place existing protein structures in a “scaffold” and remove the lipid contents ‐ rendering the tissue transparent. Whole‐mount immunohistochemistry for astrocytes was then conducted using GFAP and DAPI as a nuclei marker, followed by 3D volumetric multiphoton imaging to acquire high‐resolution visualization of the entire SFO in three planes (Figure top). In brief, this approach proved to be a significant advancement beyond standard 2D histology, and 3D visualization of intricate astrocyte processes throughout the whole SFO was possible. However, a major limitation was the extensive amount of time required to process a single sample; 2 weeks for tissue clearing and 2 weeks for primary and secondary antibody incubation. Based on this, we adapted our protocol to an “active” CLARITY approach and employed electrophoretic‐based clearing of large brain sections (n=5), which resulted in a reduction in clearing time from 2 weeks to 4 hours. It was also readily apparent that this approach rendered the tissue more transparent than passive clearing, allowing for greater visualization of astrocyte processes (Figure middle). Preliminary analysis with IMARIS software to analyze astrocyte surface area and dendritic processing (Figure bottom) revealed highly complex SFO astrocytic structures with some dendrites extending from soma at the lateral ventricle throughout and surrounding nearly the entire SFO; these processes are not visualized with standard histology. Collectively, these approaches highlight the utility of tissue‐based clearing for 3D visualization of anatomical structures within autonomic nuclei and provide a framework to understand structure‐function relationships within these brain regions in the regulation of physiological and pathophysiological conditions.

A Vegfc-Emilin2a-Cxcl8a Signaling Axis Required for Zebrafish Cardiac Regeneration.

Ischemic heart disease following the obstruction of coronary vessels leads to the death of cardiac tissue and the formation of a fibrotic scar. In contrast to adult mammals, zebrafish can regenerate their heart after injury, enabling the study of the underlying mechanisms. One of the earliest responses following cardiac injury in adult zebrafish is coronary revascularization. Defects in this process lead to impaired cardiomyocyte repopulation and scarring. Hence, identifying and investigating factors that promote coronary revascularization holds great therapeutic potential. We used wholemount imaging, immunohistochemistry and histology to assess various aspects of zebrafish cardiac regeneration. Deep transcriptomic analysis allowed us to identify targets and potential effectors of Vegfc (vascular endothelial growth factor C) signaling. We used newly generated loss- and gain-of-function genetic tools to investigate the role of Emilin2a (elastin microfibril interfacer 2a) and Cxcl8a (chemokine (C-X-C) motif ligand 8a)-Cxcr1 (chemokine (C-X-C) motif receptor 1) signaling in cardiac regeneration. We first show that regenerating coronary endothelial cells upregulate vegfc upon cardiac injury in adult zebrafish and that Vegfc signaling is required for their proliferation during regeneration. Notably, blocking Vegfc signaling also significantly reduces cardiomyocyte dedifferentiation and proliferation. Using transcriptomic analyses, we identified emilin2a as a target of Vegfc signaling and found that manipulation of emilin2a expression can modulate coronary revascularization as well as cardiomyocyte proliferation. Mechanistically, Emilin2a induces the expression of the chemokine gene cxcl8a in epicardium-derived cells, while cxcr1, the Cxcl8a receptor gene, is expressed in coronary endothelial cells. We further show that Cxcl8a-Cxcr1 signaling is also required for coronary endothelial cell proliferation during cardiac regeneration. These data show that after cardiac injury, coronary endothelial cells upregulate vegfc to promote coronary network reestablishment and cardiac regeneration. Mechanistically, Vegfc signaling upregulates epicardial emilin2a and cxcl8a expression to promote cardiac regeneration. These studies aid in understanding the mechanisms underlying coronary revascularization in zebrafish, with potential therapeutic implications to enhance revascularization and regeneration in injured human hearts.

Identification of MrgprD expression in mouse enteric neurons.

Mas-related G protein-coupled receptor D (MrgprD) was first identified in small-diameter sensory neurons of mouse dorsal root ganglion (DRG). The role of MrgprD has been studied in somatosensation, especially in pain and itch response. We recently showed that MrgprD also participated in the modulation of murine intestinal motility. The treatment of MrgprD receptor agonist suppressed the spontaneous contractions in the isolated intestinal rings of mice, indicating the intrinsic expression of MrgprD in the murine gastrointestinal (GI) tract. Although the expression of Mrgprd in GI tract has been previously detected by the way of quantitative real-time PCR, the cell-type-specific expression of MrgprD in GI tract is no yet determined. Herein, we employed Mrgprd-tdTomato reporter mouse line and the whole-mount immunohistochemistry to observe the localization of MrgprD in the smooth muscle layers of ileum and colon. We show that tdTomato-positive cells colocalized with NeuN-immunostaining in the myenteric plexus in the whole-mount preparations of the ileum and the colon. Further immunohistochemistry using the commercially available MrgprD antibody revealed the expression of MrgprD in NeuN-labeled enteric neurons in the myenteric plexus. Our results demonstrate the expression of MrgprD in the enteric neurons in the murine GI tract, highlighting the implications of MrgprD in the physiology and pathophysiology of the GI tract.

Chemical phenotypes of intrinsic cardiac neurons in the newborn pig (Sus scrofa domesticus Erxleben, 1777).

Intrinsic cardiac neurons (ICNs) are crucial cells in the neural regulation of heart rhythm, myocardial contractility, and coronary blood flow. ICNs exhibit diversity in their morphology and neurotransmitters that probably are age-dependent. Therefore, neuroanatomical heart studies have been currently focused on the identification of chemical phenotypes of ICNs to disclose their possible functions in heart neural regulation. Employing whole-mount immunohistochemistry, we examined ICNs from atria of the newborn pigs (Sus scrofa domesticus) as ICNs at this stage of development have never been neurochemically characterized so far. We found that the majority of the examined ICNs (>60%) were of cholinergic phenotype. Biphenotypic neuronal somata (NS), that is, simultaneously positive for two neuronal markers, were also rather common and distributed evenly within the sampled ganglia. Simultaneous positivity for cholinergic and adrenergic neuromarkers was specific in 16.4%, for cholinergic and nitrergic-in 3.5% of the examined NS. Purely either adrenergic or nitrergic ICNs were observed at 13% and 3.1%, correspondingly. Purely adrenergic and nitrergic NS were the most frequent in the ventral left atrial subplexus. Similarly to neuronal phenotype, sizes of NS also varied depending on the atrial region providing insights into their functional implications. Axons, but not NS, positive for classic sensory neuronal markers (vesicular glutamate transporter 2 and calcitonin gene-related peptide) were identified within epicardiac nerves and ganglia. Moreover, a substantial number of ICNs could not be attributed to any phenotype as they were not immunoreactive for antisera used in this study. Numerous dendrites with putative peptidergic and adrenergic contacts on cholinergic NS contributed to neuropil of ganglia. Our observations demonstrate that intrinsic cardiac ganglionated plexus is not fully developed in the newborn pig despite of dense network of neuronal processes and numerous signs of neural contacts within ganglia.

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning.

Dorsoventral (DV) patterning is a key landmark of embryonic development that is primarily regulated by bone morphogenetic protein (BMP) signaling. Disruption of DV patterning can result in downstream effects on cell specification and organogenesis. Zebrafish embryos have been extensively used to understand signaling pathways that regulate DV patterning because zebrafish embryos develop ex utero and, in contrast to mammalian embryos, which develop in utero, can be observed in real time using brightfield and fluorescence microscopy. Embryos with disrupted DV patterning are either dorsalized or ventralized, with lack of development of head or trunk/tail structures, respectively. Although these phenotypes are typically accompanied by effects on BMP signaling, exceptions exist where some drugs or environmental chemicals can disrupt DV patterning in the absence of effects on BMP signaling. Therefore, assessments of DV patterning should be accompanied by BMP signaling-specific readouts to confirm the role of BMP disruption. Here, we describe an exposure paradigm and steps for phenotyping zebrafish embryos for two types of DV defects, dorsalization and ventralization, with a range of severities. In addition, we describe a strategy for whole-mount immunohistochemistry of zebrafish embryos with an antibody specific for phospho-SMAD 1/5/9 (pSMAD 1/5/9), as disruption in pSMAD 1/5/9 localization is indicative of an effect on BMP signaling. Taken together, these protocols describe an initial strategy for evaluating DV patterning defects under various experimental conditions and confirming BMP-mediated DV patterning disruptions, which can be followed by additional studies that aim to uncover mechanisms leading to these adverse phenotypes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Phenotyping for dorsalization and ventralization Basic Protocol 2: Whole-mount immunohistochemistry with antibody to phospho-SMAD 1/5/9.

Type II/III cell composition and NCAM expression in taste buds.

Taste buds are localized in fungiform (FF), foliate (FL), and circumvallate (CV) papillae on the tongue, and taste buds also occur on the soft palate (SP). Mature elongate cells within taste buds are constantly renewed from stem cells and classified into three cell types, Types I, II, and III. These cell types are generally assumed to reside in respective taste buds in a particular ratio corresponding to taste regions. A variety of cell-type markers were used to analyze taste bud cells. NCAM is the first established marker for Type III cells and is still often used. However, NCAM was examined mainly in the CV, but not sufficiently in other regions. Furthermore, our previous data suggested that NCAM may be transiently expressed in the immature stage of Type II cells. To precisely assess NCAM expression as a Type III cell marker, we first examined Type II and III cell-type markers, IP3R3 and CA4, respectively, and then compared NCAM with them using whole-mount immunohistochemistry. IP3R3 and CA4 were segregated from each other, supporting the reliability of these markers. The ratio between Type II and III cells varied widely among taste buds in the respective regions (Pearson's r=0.442 [CV], 0.279 [SP], and -0.011 [FF]), indicating that Type II and III cells are contained rather independently in respective taste buds. NCAM immunohistochemistry showed that a subset of taste bud cells were NCAM(+)CA4(-). While NCAM(+)CA4(-) cells were IP3R3(-) in the CV, the majority of them were IP3R3(+) in the SP and FF.

Whole-mount Immunohistochemistry to Visualize Mouse Embryonic Dermal Lymphatic Vasculature.

Lymphatic vessels are abundant in the skin where they regulate interstitial fluid uptake and immune surveillance. Defects in dermal lymphatic vessels, such as fewer vessels and abnormal lymphatic vessel coverage with mural cells, are frequently associated with lymphedema and other lymphatic disorders. Whole-mount immunohistochemistry allows the visualization of dermal lymphatic vessels and identifies morphogenetic defects. Most dermal lymphatic vessels start growing during embryogenesis from lymph sacs that are located close to the axilla towards the dorsal and ventral midlines. Here, we present an approach that we have developed to permeabilize, immunolabel, clear, and visualize the lymphatic vessels. These simple and inexpensive techniques reproducibly generate images of dermal lymphatic vessels with great clarity.

Whole-mount staining of neuromuscular junctions in adult mouse diaphragms with a sandwich-like apparatus

BackgroundInvestigation of neuromuscular junction (NMJ) morphology by immunochemistry can provide important insights into the physiological and pathological status of neuromuscular disorders. Sectioning and muscle fiber tearing are commonly required to prepare experimentally accessible samples, while muscles that are flat and thin can be investigated with whole-mount immunohistochemistry for a comprehensive overview of the entire innervation pattern. The diaphragm is important for respiratory function and one of the flat muscles frequently used for studying neuromuscular development as well as neuromuscular pathology. Nevertheless, techniques for reliable whole-mount immunolabeling of adult diaphragms are lacking, mainly due to the poor tissue permeability of labeling reagents. An effective approach for researchers to be able to comprehensively visualize and characterize NMJ defects of the adult diaphragm in mouse models is therefore of clear importance. New MethodThis protocol demonstrates that the diaphragm can be thinned and spread out under even pressure using two Perspex boards for better whole-mount immunostaining. ResultsThe expanded mouse diaphragm allows the comprehensive assessment of a number of NMJ phenotypes. Comparison with existing methodsMost peer-reviewed and online protocols can be applied to the embryonic diaphragm but fail to show the entire innervation pattern in the adult diaphragm. Our method provides a convenient approach and present a clear innervation pattern that increases the reliability of the assessment of NMJ phenotypes in the diaphragm. ConclusionsThis simple method for whole-mount immunostaining of the adult diaphragm will allow researchers to perform a detailed analysis of the neuromuscular system in mouse models.

Modulating glutathione thiol status alters pancreatic β-cell morphogenesis in the developing zebrafish (Danio rerio) embryo

Emerging evidence suggests that redox-active chemicals perturb pancreatic islet development. To better understand potential mechanisms for this, we used zebrafish (Danio rerio) embryos to investigate roles of glutathione (GSH; predominant cellular redox buffer) and the transcription factor Nrf2a (Nfe2l2a; zebrafish Nrf2 co-ortholog) in islet morphogenesis. We delineated critical windows of susceptibility to redox disruption of β-cell morphogenesis, interrogating embryos at 24, 48 and 72 h post fertilization (hpf) and visualized Nrf2a expression in the pancreas using whole-mount immunohistochemistry at 96 hpf. Chemical GSH modulation at 48 hpf induced significant islet morphology changes at 96 hpf. Pro-oxidant exposures to tert-butylhydroperoxide (77.6 μM; 10-min at 48 hpf) or tert-butylhydroquinone (1 μM; 48-56 hpf) decreased β-cell cluster area at 96 hpf. Conversely, exposures to antioxidant N-acetylcysteine (bolsters GSH pools; 100 μM; 48-72 hpf) or sulforaphane (activates Nrf2a; 20 μM; 48-72 hpf) significantly increased islet areas. Nrf2a was also stabilized in β-cells: 10-min exposures to 77.6 μM tert-butylhydroperoxide significantly increased Nrf2a protein compared to control islet cells that largely lack stabilized Nrf2a; 10-min exposures to higher (776 μM) tert-butylhydroperoxide concentration stabilized Nrf2a throughout the pancreas. Using biotinylated-GSH to visualize in situ protein glutathionylation, islet cells displayed high protein glutathionylation, indicating oxidized GSH pools. The 10-min high (776 μM) tert-butylhydroperoxide exposure (induced Nrf2a globally) decreased global protein glutathionylation at 96 hpf. Mutant fish expressing inactive Nrf2a were protected against tert-butylhydroperoxide-induced abnormal islet morphology. Our data indicate that disrupted redox homeostasis and Nrf2a stabilization during pancreatic β-cell development impact morphogenesis, with implications for disease states at later life stages. Our work identifies a potential molecular target (Nrf2) that mediates abnormal β-cell morphology in response to redox disruptions. Moreover, our findings imply that developmental exposure to exogenous stressors at distinct windows of susceptibility could diminish the reserve redox capacity of β-cells, rendering them vulnerable to later-life stresses and disease.

Involvement of Netrin/Unc-5 Interaction in Ciliary Beating and in Pattern Formation of the Ciliary Band-Associated Strand (CBAS) in the Sea Urchin, Hemicentrotus pulcherrimus.

The GABAergic neural circuit is involved in the motile activities of both larval and juvenile sea urchins. Therefore, its function is inherited beyond metamorphosis, despite large scale remodeling of larval organs during that period. However, the initial neural circuit formation mechanism is not well understood, including how glutamate decarboxylase-expressing blastocoelar cells (GADCs) construct the neural circuit along the circumoral ciliary band (a ciliary band-associated strand, CBAS) on the larval body surface. In this study, using whole-mount immunohistochemistry and 3D reconstructed imaging, the ontogenic process of CBAS patterning was studied by focusing on Netrin and the interaction with its receptor, Unc-5. During the early 2-arm pluteus stage, a small number of GADCs egress onto the apical surface of the larval ectoderm. Then, they line up on the circumoral side of the ciliary band, and by being inserted by a further number of GADCs, form longer multicellular strands along the Netrin stripe. Application of a synthetic peptide, CRFNMELYKLSGRKSGGVC of Hp-Netrin, that binds to the immunoglobulin domain of Unc-5 during the prism stage, causes stunted CBAS formation due to inhibition of GADC egression. This also results in reduced ciliary beating. Thus, the Netrin/Unc-5 interaction is involved in the construction and function of the CBAS.