Ventral Epithelium Research Articles

Comparisons of developmental processes of air-breathing organs among terrestrial isopods (Crustacea, Oniscidea): implications for their evolutionary origins

BackgroundThe acquisition of air-breathing organs is one of the key innovations for terrestrialization in animals. Terrestrial isopods, a crustacean lineage, can be interesting models to study the evolution of respiratory organs, as they exhibit varieties of air-breathing structures according to their habitats. However, the evolutionary processes and origins of these structures are unclear, due to the lack of information about their developmental processes. To understand the developmental mechanisms, we compared the developmental processes forming different respiratory structures in three isopod species, i.e., ‘uncovered lungs’ in Nagurus okinawaensis (Trachelipodidae), ‘dorsal respiratory fields’ in Alloniscus balssi (Alloniscidae), and pleopods without respiratory structures in Armadilloniscus cf. ellipticus (Detonidae).ResultsIn N. okinawaensis with uncovered lungs, epithelium and cuticle around the proximal hemolymph sinus developed into respiratory structures at post-manca juvenile stages. On the other hand, in Al. balssi with dorsal respiratory fields, the region for the future respiratory structure was already present at manca 1 stage, immediately after hatching, where the lateral protrusion of ventral epithelium occurred, forming the respiratory structure. Furthermore, on pleopods in Ar. cf. ellipticus, only thickened dorsal cuticle and the proximal hemolymph sinus developed during postembryonic development without special morphogenesis.ConclusionsThis study shows that the respiratory structures in terrestrial isopods develop primarily by postembryonic epithelial modifications, but the epithelial positions developing into respiratory structures differ between uncovered lungs and dorsal respiratory fields. This suggests that these two types of respiratory structures do not result from simple differences in the degree of development. Future analysis of molecular developmental mechanisms will help determine whether these are the result of heterotopic changes or have different evolutionary origins. Overall, this study provides fundamental information for evolutionary developmental studies of isopod respiratory organs.

A novel protein Moat prevents ectopic epithelial folding by limiting Bazooka/Par3-dependent adherens junctions.

Contractile myosin and cell adhesion work together to induce tissue shape changes, but how they are patterned to achieve diverse morphogenetic outcomes remains unclear. Epithelial folding occurs via apical constriction, mediated by apical contractile myosin engaged with adherens junctions, as in Drosophila ventral furrow formation. While it has been shown that a multicellular gradient of myosin contractility determines folding shape, the impact of multicellular patterning of adherens junction levels on tissue folding is unknown. We identified a novel Drosophila genemoat essential for differential apical constriction and folding behaviors across the ventral epithelium which contains both folding ventral furrow and nonfolding ectodermal anterior midgut (ectoAMG). We show that Moat functions to downregulate polarity-dependent adherens junctions through inhibiting cortical clustering of Bazooka/Par3 proteins. Such downregulation of polarity-dependent junctions is critical for establishing a myosin-dependent pattern of adherens junctions, which in turn mediates differential apical constriction in the ventral epithelium. Inmoatmutants, abnormally high levels of polarity-dependent junctions promote ectopic apical constriction in cells with low-level contractile myosin, resulting in expansion of infolding from ventral furrow to ectoAMG, and flattening of ventral furrow constriction gradient. Our results demonstrate that tissue-scale distribution of adhesion levels patterns apical constriction and establishes morphogenetic boundaries.

Mechanism underlying the anti-diabetic potential of bee venom as compared to bone marrow mesenchymal stem cells in the diabetic rat tongue.

Diabetes mellitus (DM) is a critical chronic metabolic disease. Several treatment modalities are currently under investigation. Both bee venom (BV) and bone marrow mesenchymal stem cells (BMSCs) can possibly offer an approach for treating type I diabetes. The aim of the present study was to investigate the mechanism underlying the anti-diabetic effect of BV as compared to BMSCs on the tongue mucosa of diabetic rats. A total of 52 male albino rats were used in the current study. The rats were randomly assigned into 4 groups: group 1 (control); group 2 (streptozocin (STZ)); group 3 (BV-treated); and group 4 (BMSC-treated). Diabetes mellitus was induced via an intraperitoneal (IP) injection of STZ in the rats from groups 2, 3 and 4. Following the diagnosis of DM, the rats in group 3 were injected with a daily dose of 0.5 mg/kg of BV, while the rats in group 4 were treated with a single injection of BMSCs. All rats were euthanized after 4 weeks, and their tongues were dissected and divided into halves. The right halves of the tongues were utilized for the histological examination, followed by morphometric analysis. In contrast, the left halves were used to detect the local gene expression of transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF). Group 2 revealed marked disruption in the morphology of the fungiform and filiform papillae, and atrophic epithelial changes in both dorsal and ventral surface epithelium as compared to other groups. Group 4 showed a significantly larger number of taste buds, and a higher gene expression of TGF-β1 and VEGF as compared to groups 2 and 3. Additionally, BV and BMSCs effectively increased the thickness of dorsal and ventral surface epithelium as compared to group 2. Treatment with BMSCs was associated with significant improvement in the morphology and number of lingual epithelial cells and taste buds in the tongues of diabetic rats as compared to BV-treated rats, which was due to the local upregulation of TGF-β1 and VEGF gene expression.

Cell adhesion marker expression dynamics during fusion of the optic fissure

Tissue fusion is a critical process that is repeated in multiple contexts during embryonic development and shares common attributes to processes such as wound healing and metastasis. Ocular coloboma is a developmental eye disorder that presents as a physical gap in the ventral eye, and is a major cause of childhood blindness. Coloboma results from fusion failure between opposing ventral retinal epithelia, but there are major knowledge gaps in our understanding of this process at the molecular and cell behavioural levels. Here we catalogue the expression of cell adhesion proteins: N-cadherin, E-cadherin, R-cadherin, ZO-1, and the EMT transcriptional activator and cadherin regulator SNAI2, in the developing chicken embryonic eye. We find that fusion pioneer cells at the edges of the fusing optic fissure have unique and dynamic expression profiles for N-cad, E-cad and ZO-1, and that these are temporally preceded by expression of SNAI2. This highlights the unique properties of these cells and indicates that regulation of cell adhesion factors may be a critical process in optic fissure closure.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Long-term effect of subacute ruminal acidosis on the morphology and function of rumen epithelial barrier in lactating goats

Grain-induced subacute ruminal acidosis (SARA) impairs rumen epithelial barrier function, but it is yet to be determined if SARA can cause persistent damage to the morphology and function of the rumen epithelial barrier. The objective of the present study was to investigate if SARA has persistent effects on the morphological structure and permeability of ruminal epithelium and the expression of the genes involved in epithelial barrier function using a lactating goat model. Twelve mid-lactating Saanen goats with rumen cannulas were randomly assigned to 1 of 2 groups: control group (Ctrl, n=4) fed a basal diet with a non-fiber carbohydrate (NFC) to neutral detergent fiber (NDF) ratio of 1.40, and SARA group (SARA, n=8) fed the same basal diet but with increasing NFC to NDF ratio from 1.4 to 1.79, 2.31, and 3.23 overtime to induce SARA. At the end of the SARA challenge (post-SARA), 4 goats were randomly selected from the SARA group and fed only hay mixture adlibitum for another 4 weeks to allow for restitution (post-SARA). Ruminal pH was continuously recorded to monitor the severity of SARA. Samples of the ventral ruminal epithelium were collected after slaughter to examine the structural and functional changes of the ruminal epithelium using transmission electron microscopy (TEM), Ussing chambers, qRT-PCR, and Western bolt analyses. Compared with the Ctrl group, ruminal papilla length, width, surface area and thickness of stratum corneum increased (P<0.05), while stratum spinosum and basale thickness, and total depth of the epithelium decreased (P<0.05) in the SARA group. These changes diminished or tended to return to the levels of the Ctrl group in the post-SARA group (P>0.05). The SARA challenge also decreased cellular junction and widened the intercellular space between epithelial cells. Rumen transepithelial short-circuit current (Isc), tissue conductance (Gt), and mucosa-to-serosa flux of paracellular horseradish peroxidase (HRP) all increased (P<0.05) both in the SARA and post-SARA groups, which indicates that SARA can induce a sustained increase in epithelial permeability and barrier dysfunction. Moreover, the mRNA and protein expressions of CLDN1, OCLN and ZO-1 were down-regulated (P<0.01) in both the SARA and post-SARA groups. The results of this study showed that SARA could result in sustained epithelial barrier dysfunction, at both structural and functional levels, which is associated with decreased expression of rumen epithelial tight junction proteins, and the restitution of rumen epithelial barrier function is slower than that of its morphology.

Placozoan fiber cells: mediators of innate immunity and participants in wound healing

Placozoa is a phylum of non-bilaterian marine animals. These small, flat organisms adhere to the substrate via their densely ciliated ventral epithelium, which mediates mucociliary locomotion and nutrient uptake. They have only six morphological cell types, including one, fiber cells, for which functional data is lacking. Fiber cells are non-epithelial cells with multiple processes. We used electron and light microscopic approaches to unravel the roles of fiber cells in Trichoplax adhaerens, a representative member of the phylum. Three-dimensional reconstructions of serial sections of Trichoplax showed that each fiber cell is in contact with several other cells. Examination of fiber cells in thin sections and observations of live dissociated fiber cells demonstrated that they phagocytose cell debris and bacteria. In situ hybridization confirmed that fiber cells express genes involved in phagocytic activity. Fiber cells also are involved in wound healing as evidenced from microsurgery experiments. Based on these observations we conclude that fiber cells are multi-purpose macrophage-like cells. Macrophage-like cells have been described in Porifera, Ctenophora, and Cnidaria and are widespread among Bilateria, but our study is the first to show that Placozoa possesses this cell type. The phylogenetic distribution of macrophage-like cells suggests that they appeared early in metazoan evolution.

New data on the ultrastructure of the proboscis in females of Bonellia viridis (Annelida: Bonellinae)

Although Bonellia viridis is the best studied echiurid species, many features of its anatomy and ultrastructure are still unclear. Detailed data on the organisation of the proboscis may help us to understand the echiurid biology and some aspects of their feeding behaviour. In this study, a detailed description of the proboscis epithelium of females of B. viridis is provided. According to histological specificities, four zones of proboscis stem can be distinguished: dorsal, ventral, and two lateral. These zones differ from each other in ciliation and abundance of bonellin-producing and gland cells. Each zone provides certain functions: protective for dorsal, transport of food particles for ventral, and agglutination of food particles for lateral. Ciliated cells have been described in the epithelium of the dorsal side of the proboscis stem for the first time. These cells probably supply the slow ciliary movements of the proboscis. For the first time, the organisation of epithelial cells is described: these cells contain many pinocytotic vesicles, multivesicular bodies, and phagosomes, whose presence may provide evidence of the ability of epithelial cells to consume nutrients. The absence of continuous basal lamina under the ventral epithelium is ascertained and may be explained by the activity of bonellin-producing cells, which constantly penetrate between epithelial cells. In bonellin-producing cells, voluminous perinuclear space and the contribution of rough endoplasmic reticulum into the formation of bonellin-containing granules are detected for the first time. For the first time, the neurite bundles extending from lateral nerve cords to gland cells and bonellin-producing cells are discovered.

Therapeutic effect of iodised serum milk protein, lycopene and their combination on benign prostatic hyperplasia induced in rats.

Benign prostatic hyperplasia (BPH) is a common chronic disease in ageing men. Synthetic inhibitors of 5α-reductase commonly used in BPH treatment have limited effectiveness and may cause side effects. Evaluation of iodised serum milk protein and lycopene therapeutic effect in rat BPH model was the aim of the present study. BPH was induced in male Wistar rats by surgical castration and subsequent testosterone administrations (25mg/kg, 7 injections). Rats with induced BPH received lycopene (5mg/kg), iodised serum milk protein (200µg/kg) or their combination for 1month daily. The efficacy of the treatment was evaluated by the prostate weight, prostatic index and ventral lobe epithelium thickness. In lycopene and iodised serum milk protein-treated rats, prostate weight and prostatic index were significantly reduced compared to control group; and lycopene and iodised serum milk protein used in combination yielded an additive effect. Thus, further investigation of combined supplementation with micronutrients and plant-derived substances in BPH models may help to find new opportunities or its safe and effective treatment.

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos.

Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.

Quantitative Proteomic Analysis of the Slime and Ventral Mantle Glands of the Striped Pyjama Squid (Sepioloidea lineolata).

Cephalopods are known to produce an extensive range of secretions including ink, mucus, and venom. Sepiadariidae, a family of small, benthic bobtail squids, are notable for the high volume of viscous slime they emit when stressed. One species, Sepioloidea lineolata (striped pyjama squid), is covered with glands along the perimeter of the ventral mantle, and these structures are hypothesized to be the source of its slime. Using label-free quantitative proteomics, we analyzed five tissue types (dorsal and ventral mantle muscle, dorsal and ventral epithelium, and ventral mantle glands) and the slime from four individuals. In doing so, we were able to determine the relationship between the slime and the tissues as well as highlight proteins that were specifically identified within the slime and ventral mantle glands. A total of 28 proteins were identified to be highly enriched in slime, and these were composed of peptidases and protease inhibitors. Seven of these proteins contained predicted signal peptides, indicating classical secretion, with four proteins having no identifiable domains or similarity to any known proteins. The ventral mantle glands also appear to be the tissue with the closest overall proteomic composition to the slime; therefore, it is likely that the slime originates, at least in part, from these glands.

Bacterial communities in the solid, liquid, dorsal, and ventral epithelium fractions of yak (Bos grunniens) rumen.

Yak (Bos grunniens) is an important and dominant livestock species in the challenging environment of the Qinghai–Tibetan Plateau. Rumen microbiota of the solid, liquid, and epithelium fractions play key roles in nutrient metabolism and contribute to host adaptation in ruminants. However, there is a little knowledge of the microbiota in these rumen fractions of yak. Therefore, we collected samples of solid, liquid, dorsal, and ventral epithelium fractions from five female yaks, then amplified bacterial 16S rRNA gene V4 regions and sequenced them using an Illumina MiSeq platform. Principal coordinates analysis detected significant differences in bacterial communities between the liquid, solid, and epithelium fractions, and between dorsal and ventral epithelium fractions. Rikenellaceae RC9, the families Lachnospiraceae and Ruminococcaceae, and Fibrobacter spp. were the abundant and enriched bacteria in solid fraction, while the genera Prevotella and Prevotellaceae UCG 003 were higher in the liquid fraction. Campylobacter spp., Comamonas spp., Desulfovibrio spp., and Solobacterium spp. were significantly higher in dorsal epithelium, while Howardella spp., Prevotellaceae UCG 001, Ruminococcaceae UCG 005, and Treponema 2 were enriched in the ventral epithelium. Comparison of predictive functional profiles among the solid, liquid, and dorsal, and ventral epithelium fractions also revealed significant differences. Microbiota in the ventral fraction of yak rumen also significantly differ from reported microbiota of cattle. In conclusion, our results improve our knowledge of the taxonomic composition and roles of yak rumen microbiota.

C. elegans LRP-2 functions in vulval precursor cell polarity.

The C. elegans vulva is formed from divisions of three vulval precursor cells (VPCs) – P5.p, P6.p, and P7.p – arranged along the anteroposterior axis in the ventral epithelium (Sulston and Horvitz, 1977). Previous analyses show the orientation of P5.p and P7.p descendants is determined by the interaction of multiple Wnt signals. Specifically, in the absence of all Wnts, the VPCs display a randomized orientation, which is likely the default (Green et al., 2008; Minor et al. 2013). Two separate Wnts from the anchor cell, LIN-44 and MOM-2 acting through receptors LIN-17/Frizzled and LIN-18/Ryk, respectively, regulate P7.p orientation (Ferguson et al., 1987; Sternberg and Horvitz, 1988; Sawa et al., 1996; Inoue et al., 2004; Gleason et al., 2006). In the absence of these signals the orientation of the progeny of P7.p mimic those of P5.p and face toward the posterior of the worm, a phenotype referred to as posterior-reversed vulval lineage (P-Rvl). This posterior orientation is dependent on the instructive signal EGL-20, a Wnt expressed in the tail acting through CAM-1/ROR and VANG-1/Van Gogh, and is referred to as “ground polarity” (Green et al., 2008). Here we examine the role of a low-density lipoprotein receptor, lrp-2, and its role in controlling the orientation of P7.p daughter cells. To investigate this interaction double mutants were constructed with both alleles of lrp-2 and lin-17(n671) (Table 1). Much like cam-1(gm122) and vang-1(ok1142), both alleles of lrp-2 suppress the lin-17(n671) phenotype from 74 to approximately 50% P-Rvl leading us to hypothesize that lrp-2 functions in the same pathway as cam-1 and vang-1. Furthering this hypothesis we have shown that, like cam-1 and vang-1, lrp-2 controls the localization of SYS-1/b-catenin (Minor and Sternberg, 2019). To ensure that this phenotype was a result of loss of lrp-2 function as opposed to background effects we injected a fosmid (WRM0617cA02) containing the full-length sequence of lrp-2 and found that it does rescue the double mutant phenotype of lin-17(n671); lrp-2(gk272) from 55 to 73%. In order to better test this hypothesis a triple mutant was constructed between lin-17(n671), lrp-2(gk272), and cam-1(gm122) (Table 1). This triple mutant displays the same P-Rvl penetrance as both the lin-17(n671); lrp-2(gk272) and lin-17(n671); cam-1(gm122) double mutants confirming that lrp-2 functions in the same pathway as cam-1.

Real-Time In Vivo Imaging of the Developing Pupal Wing Tissues in the Pale Grass Blue Butterfly Zizeeria maha: Establishing the Lycaenid System for Multiscale Bioimaging.

To systematically analyze biological changes with spatiotemporal dynamics, it is important to establish a system that is amenable for real-time in vivo imaging at various size levels. Herein, we focused on the developing pupal wing tissues in the pale grass blue butterfly, Zizeeria maha, as a system of choice for a systematic multiscale approach in vivo in real time. We showed that the entire pupal wing could be monitored throughout development using a high-resolution bright-field time-lapse imaging system under the forewing-lift configuration; we recorded detailed dynamics of the dorsal and ventral epithelia that behaved independently for peripheral adjustment. We also monitored changes in the dorsal hindwing at the compartmental level and directly observed evaginating scale buds. We also employed a confocal laser microscopy system with multiple fluorescent dyes for three-dimensional observations at the tissue and cellular levels. We discovered extensive cellular clusters that may be functionally important as a unit of cellular communication and differentiation. We also identified epithelial discal and marginal dents that may function during development. Together, this lycaenid forewing system established a foundation to study the differentiation process of epithelial cells and can be used to study biophysically challenging mechanisms such as the determination of color patterns and scale nanoarchitecture at the multiscale levels.

Social feeding behavior of Trichoplax adhaerens.

Animals have evolved different foraging strategies in which some animals forage independently and others forage in groups. The evolution of social feeding does not necessarily require cooperation; social feeding can be a beneficial individual-level strategy if it provides mutualistic benefits, for example though increasing the efficiency of resource extraction or processing. We found that Trichoplax adhaerens, the simplest multicellular animal ever described, engages in social feeding behavior. T. adhaerens lacks muscle tissue, nervous and digestive systems - yet is capable of aggregating and forming groups of closely connected individuals who collectively feed. The tight physical interactions between the animals are transitory and appear to serve the goal of staying connected to neighbors during the external digestion of algae when enzymes are released on the biofilm and nutrients are absorbed through the ventral epithelium. We found that T. adhaerens are more likely to engage in social feeding when the concentrations of algae are high - both in a semi-natural conditions and in vitro. It is surprising that T. adhaerens - an organism without a nervous system - is able to engage in this social feeding behavior. Whether this behavior is cooperative is still an open question. Nevertheless, the social feeding behavior of T. adhaerens, an early multicellular animal, suggests that sociality may have played an important role in the early evolution of animals. It also suggests that T. adhaerens could be used as a simple model organism for exploring questions regarding ecology and sociobiology.

Coupling between dynamic 3D tissue architecture and BMP morphogen signaling during Drosophila wing morphogenesis

At the level of organ formation, tissue morphogenesis drives developmental processes in animals, often involving the rearrangement of two-dimensional (2D) structures into more complex three-dimensional (3D) tissues. These processes can be directed by growth factor signaling pathways. However, little is known about how such morphological changes affect the spatiotemporal distribution of growth factor signaling. Here, using the Drosophila pupal wing, we address how decapentaplegic (Dpp)/bone morphogenetic protein (BMP) signaling and 3D wing morphogenesis are coordinated. Dpp, expressed in the longitudinal veins (LVs) of the pupal wing, initially diffuses laterally within both dorsal and ventral wing epithelia during the inflation stage to regulate cell proliferation. Dpp localization is then refined to the LVs within each epithelial plane, but with active interplanar signaling for vein patterning/differentiation, as the two epithelia appose. Our data further suggest that the 3D architecture of the wing epithelia and the spatial distribution of BMP signaling are tightly coupled, revealing that 3D morphogenesis is an emergent property of the interactions between extracellular signaling and tissue shape changes.

The ventral epithelium of Trichoplax adhaerens deploys in distinct patterns cells that secrete digestive enzymes, mucus or diverse neuropeptides.

ABSTRACTThe disk-shaped millimeter-sized marine animal, Trichoplax adhaerens, is notable because of its small number of cell types and primitive mode of feeding. It glides on substrates propelled by beating cilia on its lower surface and periodically pauses to feed on underlying microorganisms, which it digests externally. Here, a combination of advanced electron and light microscopic techniques are used to take a closer look at its secretory cell types and their roles in locomotion and feeding. We identify digestive enzymes in lipophils, a cell type implicated in external digestion and distributed uniformly throughout the ventral epithelium except for a narrow zone near its edge. We find three morphologically distinct types of gland cell. The most prevalent contains and secretes mucus, which is shown to be involved in adhesion and gliding. Half of the mucocytes are arrayed in a tight row around the edge of the ventral epithelium while the rest are scattered further inside, in the region containing lipophils. The secretory granules in mucocytes at the edge label with an antibody against a neuropeptide that was reported to arrest ciliary beating during feeding. A second type of gland cell is arrayed in a narrow row just inside the row of mucocytes while a third is located more centrally. Our maps of the positions of the structurally distinct secretory cell types provide a foundation for further characterization of the multiple peptidergic cell types in Trichoplax and the microscopic techniques we introduce provide tools for carrying out these studies.

Differential cell proliferation and cell death during the urethral groove formation in guinea pig model.

Urethral groove (UG) formation is an important step in penile formation. Because commonly used animal models do not have UG, the mechanisms of UG formation have never been discovered. We aim to discover the cellular mechanism of the UG formation using guinea pig model. Histology was used to study the ontogeny of UG. BrdU immunofluorescence was used to label proliferating cells, cell death was determined using LysoTracker Red and TUNEL staining, and stereology was used for quantification. To reveal Shh mRNA expression patterns, in situ hybridization was performed in guinea pig genital tubercles (GTs) and ShhGFPcre-LacZ-reporter mice were used for comparison. Cell proliferation in the outer layers and programmed cell death in the inner layers of urethral epithelium played key roles during urethral canal movement from dorsal to ventral aspect and final opening to form UG. Shh mRNA expression domain shifted out to the ventral surface of GT from proximal throughout to distal in guinea pigs, but was excluded from the ventral surface epithelium in midshaft and distal of mouse GT. Differential cell proliferation and cell death in developing urethral epithelium lead to UG formation and Shh expression in ventral surface epithelium of GT may play an important role.

Developmental dynamics of butterfly wings: real-time in vivo whole-wing imaging of twelve butterfly species

Colour pattern development of butterfly wings has been studied from several different approaches. However, developmental changes in the pupal wing tissues have rarely been documented visually. In this study, we recorded real-time developmental changes of the pupal whole wings of 9 nymphalid, 2 lycaenid, and 1 pierid species in vivo, from immediately after pupation to eclosion, using the forewing-lift method. The developmental period was roughly divided into four sequential stages. At the very early stage, the wing tissue was transparent, but at the second stage, it became semi-transparent and showed dynamic peripheral adjustment and slow low-frequency contractions. At this stage, the wing peripheral portion diminished in size, but simultaneously, the ventral epithelium expanded in size. Likely because of scale growth, the wing tissue became deeply whitish at the second and third stages, followed by pigment deposition and structural colour expression at the fourth stage. Some red or yellow (light-colour) areas that emerged early were “overpainted” by expanding black areas, suggesting the coexistence of two morphogenic signals in some scale cells. The discal spot emerged first in some nymphalid species, as though it organised the entire development of colour patterns. These results indicated the dynamic wing developmental processes common in butterflies.

Local Epithelial Fracture and Healing Mechanics Dictate Morphogenesis and Asexual Reproduction in Trichoplax adhaerens

Epithelial tissues play a crucial role in determining shape and form in all adult animals by providing robust support to organs and organ systems. However, in some situations, epithelia are also known to be dynamic structures that display a fluid-like behavior with local cell rearrangements (intercalation events) regulated by genetic programs during embryo morphogenesis. Here, we have discovered a novel case of non-canonical morphogenesis in an early diverging animal - the Trichoplax adhaerens. It is one of the simplest known multicellular animals with a flat, pancake-shaped body plan. Although the animal lacks neurons and muscles, it exhibits complex behavioral traits reminiscent of higher animals. Using live microscopy, we reveal that adult animals are capable of real-time (∼seconds) extreme ‘amoeboid’ shape changes exhibiting both solid-like (elastic) and liquid-like (plastic) tissue behavior. To quantitatively study this phenomenon, we developed a novel technique for long duration (∼hours) and large-scale (∼mm) morphogenetic tracking of thousands of cells using micro-bead tags. Single cell tracking analysis of these organism-scale flow fields has revealed a surprising fluid-like motion - with characteristic features such as vortices and shear regions, accompanied by local non-affine (disordered) cell rearrangements. We show that these disordered cell movements arise from local micro-fractures in the ventral epithelium. Remarkably, it turns out that these micro-fractures are generated physiologically, and are regulated by global mechanical forces generated by the animal's natural movements. These micro-fractures can enlarge to form macro-fractures that may heal over time and lead to permanent organismal shape change, thus demonstrating a direct link between micro-scale fractures and macro organism-scale morphogenesis. Finally, we show that epithelial fracture and healing mechanics govern two key biologically important phenomena observed in this animal - asexual reproduction by fission, and multi-scale tissue mixing.