The Heterotaxy Gene CCDC11 Is Important for Cytokinesis via RhoA Regulation.

Mutations in CCDC11 (cfap53) have been identified in multiple patients with heterotaxy (Htx), a disorder of left-right (LR) patterning of the internal organs. In Xenopus, depletion of Ccdc11 causes defects in LR patterning, recapitulating the patient phenotype. Upon Ccdc11 depletion, monociliated cells of the Left-Right Organizer (LRO) exhibit multiple cilia per cell. Unexpectedly, we found that Ccdc11 is necessary for successful cytokinesis, explaining the multiciliation phenotype observed in Ccdc11-depleted cells. The small GTPase RhoA is critical for cytokinesis, and our Ccdc11 depletion phenotypes are reminiscent of RhoA loss of function. Here, we demonstrate that during cytokinesis CCDC11 is localized to the cytokinetic contractile ring overlapping with RhoA, and CCDC11 regulates total RhoA protein levels. Our results connect CCDC11 to cytokinesis and LR patterning via RhoA regulation, providing a potential mechanism for heterotaxy disease pathogenesis.

Effect of Guar Gum Content on the Mechanical Properties of Laterite Soil for Subgrade Soil Application.

Using biopolymers for soil stabilization is favorable compared to more conventional methods because they are more environmentally friendly, cost-effective, and long-lasting. This study analyzes the physical properties of guar gum and laterite soil mixes. A comprehensive engineering study of guar gum-treated soil was conducted with the help of a brief experimental program. This study examined the effects of soil-guar gum interactions on the strengthening behavior of guar gum-treated soil mixtures using a series of laboratory tests. The treated laterite soil's dry density increased marginally, while its optimum moisture content decreased as the guar gum increased. Treatment with guar gum significantly enhanced the strength of laterite soil mixtures. For laterite soil with 2% guar gum, the unsoaked CBR increased by 148% and the soaked CBR increased by 192.36%. The cohesiveness and internal friction angle increased by 93.33% and 31.52%, respectively. These results show that using guar gum dramatically improves the strength of laterite soil, offering a more environmentally friendly and sustainable alternative to traditional soil additives. Using guar gum in T8 subgrade soil requires a 1395 mm pavement depth and costs INR 3.83 crores, 1.52 times more than laterite soil. For T9 subgrade soil, the depth was 1495 mm, costing INR 4.42 crores, 1.72 times more than laterite soil. This study introduces a novel approach to soil stabilization by employing guar gum, a biopolymer, to enhance the physical and mechanical properties of laterite soil. Furthermore, this study provides a detailed cost-benefit analysis for pavement applications, revealing the financial feasibility of using guar gum despite it requiring a marginally higher initial investment.

19th International Xenopus Conference Meeting Report: Latest developments and future perspectives.

The African clawed frog, Xenopus laevis, and the Western clawed frog, Xenopus tropicalis, have been foundational model organisms for establishing key principles of embryonic development. Today, the utility of Xenopus has been greatly expanded for studying a wide range of biological processes both in health and disease. Here, we describe the latest advancements from the Xenopus community, which span the molecular, cellular, tissue, and organismal scales.

Mechanisms of cilia regeneration in Xenopus multiciliated epithelium in vivo.

Cilia regeneration is a physiological event, and while studied extensively in unicellular organisms, it remains poorly understood in vertebrates. In this study, using Xenopus multiciliated cells (MCCs) as a model, we demonstrate that, unlike unicellular organisms, deciliation removes the transition zone (TZ) along with the ciliary axoneme. While MCCs immediately begin the regeneration of the ciliary axoneme, surprisingly, the assembly of TZ was delayed. Instead, ciliary tip proteins, Sentan and Clamp, were the first to localize to regenerating cilia. Using cycloheximide (CHX) to block new protein synthesis, we show that the TZ protein B9d1 is not a component of the cilia precursor pool and requires new transcription/translation providing insights into the delayed repair of TZ. Moreover, CHX treatment led MCCs to assemble fewer (~ ten compared to ~150 in controls) but about wild-type length (78% of WT) cilia by gradually concentrating ciliogenesis proteins like IFT43 at a select few basal bodies, highlighting the exciting possibility of protein transport between basal bodies to facilitate faster regeneration in cells with multiple cilia. In summary, we demonstrate that MCCs begin regeneration with the assembly of ciliary tip and axoneme followed by TZ, questioning the importance of TZ in motile ciliogenesis.

Mechanical stretch scales centriole number to apical area via Piezo1 in multiciliated cells.

How cells count and regulate organelle number is a fundamental question in cell biology. For example, most cells restrict centrioles to two in number and assemble one cilium; however, multiciliated cells (MCCs) synthesize hundreds of centrioles to assemble multiple cilia. Aberration in centriole/cilia number impairs MCC function and can lead to pathological outcomes. Yet how MCCs control centriole number remains unknown. Using Xenopus, we demonstrate that centriole number scales with apical area over a remarkable 40-fold change in size. We find that tensile forces that shape the apical area also trigger centriole amplification based on both cell stretching experiments and disruption of embryonic elongation. Unexpectedly, Piezo1, a mechanosensitive ion channel, localizes near each centriole suggesting a potential role in centriole amplification. Indeed, depletion of Piezo1 affects centriole amplification and disrupts its correlation with the apical area in a tension-dependent manner. Thus, mechanical forces calibrate cilia/centriole number to the MCC apical area via Piezo1. Our results provide new perspectives to study organelle number control essential for optimal cell function.

Xenopus to the rescue: A model to validate and characterize candidate ciliopathy genes.

Cilia are present on most vertebrate cells and play a central role in development, growth, and homeostasis. Thus, cilia dysfunction can manifest into an array of diseases, collectively termed ciliopathies, affecting millions of lives worldwide. Yet, our understanding of the gene regulatory networks that control cilia assembly and functions remain incomplete. With the advances in next-generation sequencing technologies, we can now rapidly predict pathogenic variants from hundreds of ciliopathy patients. While the pace of candidate gene discovery is exciting, most of these genes have never been previously implicated in cilia assembly or function. This makes assigning the disease causality difficult. This review discusses how Xenopus, a genetically tractable and high-throughput vertebrate model, has played a central role in identifying, validating, and characterizing candidate ciliopathy genes. The review is focused on multiciliated cells (MCCs) and diseases associated with MCC dysfunction. MCCs harbor multiple motile cilia on their apical surface to generate extracellular fluid flow inside the airway, the brain ventricles, and the oviduct. In Xenopus, these cells are external and present on the embryonic epidermal epithelia, facilitating candidate genes analysis in MCC development in vivo. The ability to introduce patient variants to study their effects on disease progression makes Xenopus a powerful model to improve our understanding of the underlying disease mechanisms and explain the patient phenotype.

Corticosterone Is Essential for Survival Through Frog Metamorphosis.

Thyroid hormone (TH) is required for frog metamorphosis, and corticosterone (CORT) increases TH signaling to accelerate metamorphic progression. However, a requirement for CORT in metamorphosis has been difficult to assess prior to the recent development of gene-editing technologies. We addressed this long-standing question using transcription activator-like effector nuclease (TALEN) gene disruption to knock out proopiomelanocortin (pomc) and disrupt CORT production in Xenopus tropicalis. As expected, mutant tadpoles had a reduced peak of plasma CORT at metamorphosis with correspondingly reduced expression of the CORT-response gene Usher syndrome type-1G (ush1g). Mutants had reduced rates of growth and development and exhibited lower expression levels of 2 TH response genes, Krüppel-like factor 9 (klf9) and TH receptor ß (thrb). In response to exogenous TH, mutants had reduced TH response gene induction and slower morphological change. Importantly, death invariably occurred during tail resorption, unless rescued by exogenous CORT and, remarkably, by exogenous TH. The ability of exogenous TH by itself to overcome death in pomc mutants indicates that the CORT-dependent increase in TH signaling may ensure functional organ transformation required for survival through metamorphosis and/or may shorten the nonfeeding metamorphic transition to avoid lethal inanition.

WDR5 regulates left-right patterning via chromatin-dependent and -independent functions.

Congenital heart disease (CHD) is a major cause of infant mortality and morbidity, yet the genetic causes and mechanisms remain opaque. In a patient with CHD and heterotaxy, a disorder of left-right (LR) patterning, a de novo mutation was identified in the chromatin modifier gene WDR5 WDR5 acts as a scaffolding protein in the H3K4 methyltransferase complex, but a role in LR patterning is unknown. Here, we show that Wdr5 depletion leads to LR patterning defects in Xenopus via its role in ciliogenesis. Unexpectedly, we find a dual role for WDR5 in LR patterning. First, WDR5 is expressed in the nuclei of monociliated cells of the LR organizer (LRO) and regulates foxj1 expression. LR defects in wdr5 morphants can be partially rescued with the addition of foxj1 Second, WDR5 localizes to the bases of cilia. Using a mutant form of WDR5, we demonstrate that WDR5 also has an H3K4-independent role in LR patterning. Guided by the patient phenotype, we identify multiple roles for WDR5 in LR patterning, providing plausible mechanisms for its role in ciliopathies like heterotaxy and CHD.

RPSA, a candidate gene for isolated congenital asplenia, is required for pre-rRNA processing and spleen formation in Xenopus.

A growing number of tissue-specific inherited disorders are associated with impaired ribosome production, despite the universal requirement for ribosome function. Recently, mutations in RPSA, a protein component of the small ribosomal subunit, were discovered to underlie approximately half of all isolated congenital asplenia cases. However, the mechanisms by which mutations in this ribosome biogenesis factor lead specifically to spleen agenesis remain unknown, in part due to the lack of a suitable animal model for study. Here we reveal that RPSA is required for normal spleen development in the frog, Xenopus tropicalis Depletion of Rpsa in early embryonic development disrupts pre-rRNA processing and ribosome biogenesis, and impairs expression of the key spleen patterning genes nkx2-5, bapx1 and pod1 in the spleen anlage. Importantly, we also show that whereas injection of human RPSA mRNA can rescue both pre-rRNA processing and spleen patterning, injection of human mRNA bearing a common disease-associated mutation cannot. Together, we present the first animal model of RPSA-mediated asplenia and reveal a crucial requirement for RPSA in pre-rRNA processing and molecular patterning during early Xenopus development.

WDR5 Stabilizes Actin Architecture to Promote Multiciliated Cell Formation.

The actin cytoskeleton is critical to shape cells and pattern intracellular organelles, which collectively drives tissue morphogenesis. In multiciliated cells (MCCs), apical actin drives expansion of the cell surface necessary to host hundreds of cilia. The apical actin also forms a lattice to uniformly distribute basal bodies. This apical actin network is dynamically remodeled, but the molecules that regulate its architecture remain poorly understood. We identify the chromatin modifier, WDR5, as a regulator of apical F-actin in MCCs. Unexpectedly in MCCs, WDR5 has a function independent of chromatin modification. We discover a scaffolding role for WDR5 between the basal body and F-actin. Specifically, WDR5 binds to basal bodies and migrates apically, where F-actin organizes around WDR5. Using a monomer trap for G-actin, we show that WDR5 stabilizes F-actin to maintain lattice architecture. In summary, we identify a non-chromatin role for WDR5 in stabilizing F-actin in MCCs.

RAPGEF5 Regulates Nuclear Translocation of ß-Catenin.

Canonical Wnt signaling coordinates many critical aspects of embryonic development, while dysregulated Wnt signaling contributes to common diseases, including congenital malformations and cancer. The nuclear localization of ß-catenin is the defining step in pathway activation. However, despite intensive investigation, the mechanisms regulating ß-catenin nuclear transport remain undefined. In a patient with congenital heart disease and heterotaxy, a disorder of left-right patterning, we previously identified the guanine nucleotide exchange factor, RAPGEF5. Here, we demonstrate that RAPGEF5 regulates left-right patterning via Wnt signaling. In particular, RAPGEF5 regulates the nuclear translocation of ß-catenin independently of both ß-catenin cytoplasmic stabilization and the importin ß1/Ran-mediated transport system. We propose a model whereby RAPGEF5 activates the nuclear GTPases, Rap1a/b, to facilitate the nuclear transport of ß-catenin, defining a parallel nuclear transport pathway to Ran. Our results suggest new targets for modulating Wnt signaling in disease states.

Genetic accommodation via modified endocrine signalling explains phenotypic divergence among spadefoot toad species.

Phenotypic differences among species may evolve through genetic accommodation, but mechanisms accounting for this process are poorly understood. Here we compare hormonal variation underlying differences in the timing of metamorphosis among three spadefoot toads with different larval periods and responsiveness to pond drying. We find that, in response to pond drying, Pelobates cultripes and Spea multiplicata accelerate metamorphosis, increase standard metabolic rate (SMR), and elevate whole-body content of thyroid hormone (the primary morphogen controlling metamorphosis) and corticosterone (a stress hormone acting synergistically with thyroid hormone to accelerate metamorphosis). In contrast, Scaphiopus couchii has the shortest larval period, highest whole-body thyroid hormone and corticosterone content, and highest SMR, and these trait values are least affected by pond drying among the three species. Our findings support that the atypically rapid and canalized development of S. couchii evolved by genetic accommodation of endocrine pathways controlling metamorphosis, showing how phenotypic plasticity within species may evolve into trait variation among species.

Corticosteroid signaling in frog metamorphosis.

Stress in fetal and larval life can impact later health and fitness in humans and wildlife. Long-term effects of early life stress are mediated by altered stress physiology induced during the process of relaying environmental effects on development. Amphibian metamorphosis has been an important model system to study the role of hormones in development in an environmental context. Thyroid hormone (TH) is necessary and sufficient to initiate the dramatic morphological and physiological changes of metamorphosis, but TH alone is insufficient to complete metamorphosis. Other hormones, importantly corticosteroid hormones (CSs), influence the timing and nature of post-embryonic development. Stressors or treatments with CSs delay or accelerate metamorphic change, depending on the developmental stage of treatment. Also, TH and CSs have synergistic, antagonistic, and independent effects on gene regulation. Importantly, the identity of the endogenous corticosteroid hormone or receptor underlying any gene induction or remodeling event has not been determined. Levels of both CSs, corticosterone and aldosterone, peak at metamorphic climax, and the corticosteroid receptors, glucocorticoid and mineralocorticoid receptors, have wide expression distribution among tadpole tissues. Conclusive experiments to identify the endogenous players have been elusive due to difficulties in experimental control of corticosteroid production and signaling. Current data are consistent with the hypothesis that the two CSs and their receptors serve largely overlapping functions in regulating metamorphosis and synergy with TH. Knowledge of the endogenous players is critical to understanding the basic mechanisms and significance of corticosteroid action in regulating post-embryonic development in environmental contexts.

Mechanisms and consequences of developmental acceleration in tadpoles responding to pond drying.

Many amphibian species exploit temporary or even ephemeral aquatic habitats for reproduction by maximising larval growth under benign conditions but accelerating development to rapidly undergo metamorphosis when at risk of desiccation from pond drying. Here we determine mechanisms enabling developmental acceleration in response to decreased water levels in western spadefoot toad tadpoles (Pelobates cultripes), a species with long larval periods and large size at metamorphosis but with a high degree of developmental plasticity. We found that P. cultripes tadpoles can shorten their larval period by an average of 30% in response to reduced water levels. We show that such developmental acceleration was achieved via increased endogenous levels of corticosterone and thyroid hormone, which act synergistically to achieve metamorphosis, and also by increased expression of the thyroid hormone receptor TRΒ, which increases tissue sensitivity and responsivity to thyroid hormone. However, developmental acceleration had morphological and physiological consequences. In addition to resulting in smaller juveniles with proportionately shorter limbs, tadpoles exposed to decreased water levels incurred oxidative stress, indicated by increased activity of the antioxidant enzymes catalase, superoxide dismutase, and glutathione peroxidase. Such increases were apparently sufficient to neutralise the oxidative damage caused by presumed increased metabolic activity. Thus, developmental acceleration allows spadefoot toad tadpoles to evade drying ponds, but it comes at the expense of reduced size at metamorphosis and increased oxidative stress.

Developmental programs and endocrine disruption in frog metamorphosis: the perspective from microarray analysis.

A major goal for understanding the role of thyroid hormone (TH) in development has been to identify genes regulated by TH in different tissues during frog metamorphosis. The exquisite dependence of metamorphosis on TH also provides a model to study TH endocrine disruption. To identify such TH-regulated genes and select biomarkers for TH endocrine disruption, global gene expression analyses in tadpoles using microarrays have been done in 21 studies, involving five frog species, seven organs, and four endocrine disrupting chemicals. As expected, each organ has a unique set of genes associated with its tissue-specific metamorphic outcome, and functions ascribed to many of these genes correspond to histological changes induced by TH. Also, the large number of transcription factors identified in microarrays is consistent with the molecular mechanisms of TH action. On the other hand, microarray analysis has also revealed interesting findings not predicted from previous morphological or molecular studies. Furthermore, endocrine disruption studies identified candidate biomarkers for TH disruption, and the mechanisms of action of several endocrine disrupting chemicals have been examined. The microarray studies described here have produced a wealth of data on gene expression that requires further functional studies to elucidate the roles of these genes in development and endocrine disruption.

Beyond synergy: corticosterone and thyroid hormone have numerous interaction effects on gene regulation in Xenopus tropicalis tadpoles.

Hormones play critical roles in vertebrate development, and frog metamorphosis has been an excellent model system to study the developmental roles of thyroid hormone (TH) and glucocorticoids. Whereas TH regulates the initiation and rate of metamorphosis, the actions of corticosterone (CORT; the main glucocorticoid in frogs) are more complex. In the absence of TH during premetamorphosis, CORT inhibits development, but in the presence of TH during metamorphosis, CORT synergizes with TH to accelerate development. Synergy at the level of gene expression is known for three genes in frogs, but the nature and extent of TH and CORT cross talk is otherwise unknown. Therefore, to examine TH and CORT interactions, we performed microarray analysis on tails from Xenopus tropicalis tadpoles treated with CORT, TH, CORT+TH, or vehicle for 18 h. The expression of 5432 genes was significantly altered in response to either or both hormones. Using Venn diagrams and cluster analysis, we identified 16 main patterns of gene regulation due to up- or down-regulation by TH and/or CORT. Many genes were affected by only one of the hormones, and a large proportion of regulated genes (22%) required both hormones. We also identified patterns of additive or synergistic, inhibitory, subtractive, and annihilatory regulation. A total of 928 genes (17%) were regulated by novel interactions between the two hormones. These data expand our understanding of the hormonal cross talk underlying the gene regulation cascade directing tail resorption and suggest the possibility that CORT affects not only the timing but also the nature of TH-dependent tissue transformation.

Corticotropin-releasing factor regulates the development in the direct developing frog, Eleutherodactylus coqui.

Direct developing frogs lack a free-living larval phase, such that miniature adults hatch directly from the eggs. Even under such extreme reorganization of the ancestral biphasic developmental pattern, direct developers still undergo thyroid hormone (TH)-dependent post-embryonic development. Hypothalamic regulation of TH synthesis and release plays a central role in controlling the timing of metamorphosis in biphasic developers. In particular, the neuropeptide corticotropin-releasing factor (CRF) regulates TH in tadpoles, but in adults, both thyrotropin-releasing hormone (TRH) and CRF regulate TH. Because direct developers lack a tadpole stage, it was not clear whether hypothalamic regulation of TH would be tadpole-like or adult-like prior to hatching. To test this, we injected pre-hatching Eleutherodactylus coqui daily with CRF, TRH or astressin (a CRF receptor blocker). CRF but not TRH significantly accelerated the developmental rate compared to controls. Astressin-treated animals showed a near complete developmental arrest, which confirmed that development requires CRF. To support the idea that CRF acts to regulate development in E. coqui via thyroid physiology, we showed the TH-direct response gene TRß is up-regulated 24 and 48 h after CRF injection. In addition, treatment with 50 nM T3 (triiodothyronine, the active form of TH) increased the developmental rate similar to CRF injections. Our results extend the evidence for a cryptic metamorphosis in direct developers by showing that neuroendocrine signaling is conserved between biphasic and direct developers. Furthermore, the conserved neuroendocrine regulation implies that changes at the peripheral level of hormone action underlie the evolution of the radically divergent development in direct developers.