Adrenergic receptor signaling induced by Klf15, a regulator of regeneration enhancer, promotes kidney reconstruction.

Despite the recent discovery of tissue regeneration enhancers in highly regenerative animals, upstream and downstream genetic programs connected by these enhancers still remain unclear. Here, we performed a genome-wide analysis of enhancers and associated genes in regenerating nephric tubules of Xenopus laevis. Putative enhancers were identified using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) analyses. Their target genes were predicted based on their proximity to enhancers on genomic DNA and consistency of their transcriptome profiles to ATAC-seq/ChIP-seq profiles of the enhancers. Motif enrichment analysis identified the central role of Krüppel-like factors (Klf) in the enhancer. Klf15, a member of the Klf family, directly binds enhancers and stimulates expression of regenerative genes, including adrenoreceptor alpha 1A (adra1a), whereas inhibition of Klf15 activity results in failure of nephric tubule regeneration. Moreover, pharmacological inhibition of Adra1a-signaling suppresses nephric tubule regeneration, while its activation promotes nephric tubule regeneration and restores organ size. These results indicate that Klf15-dependent adrenergic receptor signaling through regeneration enhancers plays a central role in the genetic network for kidney regeneration.

Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3.

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 â€‹kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 â€‹kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

Development of a heat-stable alkaline phosphatase reporter system for cis-regulatory analysis and its application to 3D digital imaging of Xenopus embryonic tissues.

Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of ß-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.

X-ray micro-computed tomography of Xenopus tadpole reveals changes in brain ventricular morphology during telencephalon regeneration.

Xenopus tadpoles serve as an exceptional model organism for studying post-embryonic development in vertebrates. During post-embryonic development, large-scale changes in tissue morphology, including organ regeneration and metamorphosis, occur at the organ level. However, understanding these processes in a three-dimensional manner remains challenging. In this study, the use of X-ray micro-computed tomography (microCT) for the three-dimensional observation of the soft tissues of Xenopus tadpoles was explored. The findings revealed that major organs, such as the brain, heart, and kidneys, could be visualized with high contrast by phosphotungstic acid staining following fixation with Bouin's solution. Then, the changes in brain shape during telencephalon regeneration were analyzed as the first example of utilizing microCT to study organ regeneration in Xenopus tadpoles, and it was found that the size of the amputated telencephalon recovered to >80% of its original length within approximately 1 week. It was also observed that the ventricles tended to shrink after amputation and maintained this state for at least 3 days. This shrinkage was transient, as the ventricles expanded to exceed their original size within the following week. Temporary shrinkage and expansion of the ventricles, which were also observed in transgenic or fluorescent dye-injected tadpoles with telencephalon amputation, may be significant in tissue homeostasis in response to massive brain injury and subsequent repair and regeneration. This established method will improve experimental analyses in developmental biology and medical science using Xenopus tadpoles.

Phenotype-genotype relationships in Xenopus sox9 crispants provide insights into campomelic dysplasia and vertebrate jaw evolution.

Since CRISPR-based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non-syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame-shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in-frame deletion mutations expected to delete part of the jawed vertebrate-specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype-genotype relationships behind syndromic diseases and examining the tissue-specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution.

Optimization of CRISPR/Cas9-mediated gene disruption in Xenopus laevis using a phenotypic image analysis technique.

The CRISPR/Cas9 method has become popular for gene disruption experiments in Xenopus laevis. However, the experimental conditions that influence the efficiency of CRISPR/Cas9 remain unclear. To that end, we developed an image analysis technique for the semi-quantitative evaluation of the pigment phenotype resulting from the disruption of tyrosinase genes in X. laevis using a CRISPR/Cas9 approach, and then examined the effects of varying five experimental parameters (timing of the CRISPR reagent injection into developing embryos; amount of Cas9 mRNA in the injection reagent; total injection volume per embryo; number of injection sites per embryo; and the culture temperature of the injected embryos) on the gene disruption efficiency. The results of this systematic analysis suggest that the highest possible efficiency of target gene disruption can be achieved by injecting a total of 20 nL of the CRISPR reagent containing 1500 pg of Cas9 mRNA or 4 ng of Cas9 protein into two separate locations (10 nL each) of one-cell stage embryos cultured at 22°C. This study also highlights the importance of balancing the experimental parameters for increasing gene disruption efficiency and provides valuable insights into the optimal conditions for applying the CRISPR/Cas9 system to new experimental organisms.

Regeneration enhancers: A clue to reactivation of developmental genes.

During tissue and organ regeneration, cells initially detect damage and then alter nuclear transcription in favor of tissue/organ reconstruction. Until recently, studies of tissue regeneration have focused on the identification of relevant genes. These studies show that many developmental genes are reused during regeneration. Concurrently, comparative genomics studies have shown that the total number of genes does not vastly differ among vertebrate taxa. Moreover, functional analyses of developmental genes using various knockout/knockdown techniques demonstrated that the functions of these genes are conserved among vertebrates. Despite these data, the ability to regenerate damaged body parts varies widely between animals. Thus, it is important to determine how regenerative transcriptional programs are triggered and why animals with low regenerative potential fail to express developmental genes after injury. Recently, we discovered relevant enhancers and named them regeneration signal-response enhancers (RSREs) after identifying their activation mechanisms in a Xenopus laevis transgenic system. In this review, we summarize recent studies of injury/regeneration-associated enhancers and then discuss their mechanisms of activation.

Asymmetrically reduced expression of hand1 homeologs involving a single nucleotide substitution in a cis-regulatory element.

During vertebrate evolution, whole genome duplications resulted in a number of duplicated genes, some of which eventually changed their expression patterns and/or levels via alteration of cis-regulatory sequences. However, the initial process involved in such cis-regulatory changes remains unclear. Therefore, we investigated this process by analyzing the duplicated hand1 genes of Xenopus laevis (hand1.L and hand1.S), which were generated by allotetraploidization 17-18 million years ago, and compared these with their single ortholog in the ancestral-type diploid species X. tropicalis. A dN/dS analysis indicated that hand1.L and hand1.S are still under purifying selection, and thus, their products appear to retain ancestral functional properties. RNA-seq and in situ hybridization analyses revealed that hand1.L and hand1.S have similar expression patterns to each other and to X. tropicalis hand1, but the hand1.S expression level was much lower than the hand1.L expression level in the primordial heart. A comparative sequence analysis, luciferase reporter analysis, ChIP-PCR analysis, and transgenic reporter analysis showed that a single nucleotide substitution in the hand1.S promoter was responsible for the reduced expression in the heart. These findings demonstrated that a small change in the promoter sequence can trigger diversification of duplicated gene expression prior to diversification of their encoded protein functions in a young duplicated genome.

Co-accumulation of cis-regulatory and coding mutations during the pseudogenization of the Xenopus laevis homoeologs six6.L and six6.S.

Common models for the evolution of duplicated genes after genome duplication are subfunctionalization, neofunctionalization, and pseudogenization. Although the crucial roles of cis-regulatory mutations in subfunctionalization are well-documented, their involvement in pseudogenization and/or neofunctionalization remains unclear. We addressed this issue by investigating the evolution of duplicated homeobox genes, six6.L and six6.S, in the allotetraploid frog Xenopus laevis. Based on a comparative expression analysis, we observed similar eye-specific expression patterns for the two loci and their single ortholog in the ancestral-type diploid species Xenopus tropicalis. However, we detected lower levels of six6.S expression than six6.L expression. The six6.S enhancer sequence was more highly diverged from the orthologous enhancer of X. tropicalis than the six6.L enhancer, and showed weaker activity in a transgenic reporter assay. Based on a phylogenetic analysis of the protein sequences, we observed greater divergence between X. tropicalis Six6 and Six6.S than between X. tropicalis Six6 and Six6.L, and the observed mutations were reminiscent of a microphthalmia mutation in human SIX6. Misexpression experiments showed that six6.S has weaker eye-enlarging activity than six6.L, and targeted disruption of six6.L reduced the eye size more significantly than that of six6.S. These results suggest that enhancer attenuation stimulates the accumulation of hypomorphic coding mutations, or vice versa, in one duplicated gene copy and facilitates pseudogenization. We also underscore the value of the allotetraploid genome of X. laevis as a resource for studying latent pathogenic mutations.

Cyclic AMP-regulated opposing and parallel effects of serotonin and dopamine on phototaxis in the Marmorkrebs (marbled crayfish).

Phototactic behaviours are observed from prokaryotes to amphibians and are a basic form of orientation. We showed that the marbled crayfish displays phototaxis in which the behavioural response reversed from negative to positive depending on external light conditions. Animals reared in a 12-L/12-D light cycle showed negative phototaxis during daytime and positive phototaxis during night-time. Animals reared under constant light conditioning showed negative phototaxis during day- and night-time, while animals reared under constant dark conditioning showed positive phototaxis during day- and night-time. Injection of serotonin leads to a reversal of negative to positive phototaxis in both light/dark-reared and light/light-reared animals while injection of dopamine induced reversed negative phototaxis in dark/dark-reared animals. Four hours of dark adaptation were enough for light/dark-reared animals to reverse phototaxis from negative to positive. Injection of a serotonin 5HT1 receptor antagonist blocked the reverse phototaxis while serotonin 5HT2 receptor antagonists had no effects. Similarly, dark/dark-reared animals reversed to showing negative phototaxis after 4 h of light adaptation. Injection of a dopamine DA1 receptor antagonist blocked this reverse phototaxis, while dopamine DA2 receptor antagonists had no effects. Injection of a cAMP analogue into light/dark-reared animals blocked reverse phototaxis after dark adaptation, while adenylate cyclase inhibitor in dark/dark-reared animals blocked reverse phototaxis after light adaptation. These results strongly suggest that serotonin mediates positive phototaxis owing to decreased cAMP levels, while dopamine-mediated negative phototaxis occurs due to increased cAMP levels. Supporting this, the ratio of serotonin to dopamine in the brain was much higher in dark/dark-reared than light/dark-reared animals.

Identification of tumor-related genes via RNA sequencing of tumor tissues in Xenopus tropicalis.

Cancer treatment is still challenging because the disease is often caused by multiple mutations. Although genomic studies have identified many oncogenes and tumor suppressor genes, gene sets involved in tumorigenesis remain poorly understood. Xenopus, a genus of aquatic frogs, is a useful model to identify gene sets because it can be genetically and experimentally analyzed. Here, we analyzed gene expression in tumor tissues of three individuals in Xenopus tropicalis and identified 55 differentially expressed genes (DEGs). Gene ontology (GO) analysis showed that the upregulated genes in the tumor tissues were enriched in GO terms related to the extracellular matrix and collagen fibril organization. Hierarchical clustering showed that the gene expression patterns of tumor tissues in X. tropicalis were comparable to those of human connective, soft, and subcutaneous tissue-derived cancers. Additionally, pathway analysis revealed that these DEGs were associated with multiple pathways, including the extracellular matrix, collagen fibril organization, MET signaling, and keratan sulfate. We also found that the expression tendency of some DEGs that have not been well analyzed in the cancer field clearly determines the prognosis of human cancer patients. This study provides a remarkable reference for future experimental work on X. tropicalis to identify gene sets involved in human cancer.

Arid3a regulates nephric tubule regeneration via evolutionarily conserved regeneration signal-response enhancers.

Amphibians and fish have the ability to regenerate numerous tissues, whereas mammals have a limited regenerative capacity. Despite numerous developmental genes becoming reactivated during regeneration, an extensive analysis is yet to be performed on whether highly regenerative animals utilize unique cis-regulatory elements for the reactivation of genes during regeneration and how such cis-regulatory elements become activated. Here, we screened regeneration signal-response enhancers at the lhx1 locus using Xenopus and found that the noncoding elements conserved from fish to human function as enhancers in the regenerating nephric tubules. A DNA-binding motif of Arid3a, a component of H3K9me3 demethylases, was commonly found in RSREs. Arid3a binds to RSREs and reduces the H3K9me3 levels. It promotes cell cycle progression and causes the outgrowth of nephric tubules, whereas the conditional knockdown of arid3a using photo-morpholino inhibits regeneration. These results suggest that Arid3a contributes to the regeneration of nephric tubules by decreasing H3K9me3 on RSREs.

Identification of distal enhancers for Six2 expression in pronephros.

The embryonic nephric mesenchyme contains pluripotent progenitor cells. Six2, a homeodomain transcription factor, is expressed in a subset of the nephric mesenchyme, and it functions to maintain a progenitor state by suppressing nephrogenesis. Despite the functional significance of Six2 in nephric development, its regulatory mechanisms remain unclear. To identify the cis-regulatory elements for Six2, we focused on the evolutionarily conserved sequences known as conserved noncoding sequences (CNSs) associated with the Six2 locus. Transgenic experiments using Xenopus laevis embryos revealed that three of the eight CNSs located within a 317-kb segment of the Six2 genomic locus were nephric enhancers. Motif analysis of transcription factors combined with phylogenetic footprinting revealed the enrichment of putative T-cell factor (Tcf)-, Hox-, and SWI/SNF complex helicase-like transcription factor (Hltf)- and AT-rich interactive domain 3A (Arid3a)-binding motif sequences in these enhancers.