The TRESK (K2P18.1, KCNK18) background potassium channel is expressed in primary sensory neurons and has been reported to contribute to the regulation of pain sensations. In the present study, we examined the interaction of TRESK with NDFIP1 (Nedd4 family-interacting protein 1) in the Xenopus oocyte expression system by two-electrode voltage clamp and biochemical methods. We showed that the coexpression of NDFIP1 abolished the TRESK current under the condition where the other K+ channels were not affected. Mutations in the three PPxY motifs of NDFIP1, which are responsible for the interaction with the Nedd4 ubiquitin ligase, prevented a reduction in the TRESK current. Furthermore, the overexpression of a dominant-negative Nedd4 construct in the oocytes coexpressing TRESK with NDFIP1 partially reversed the down-modulating effect of the adaptor protein on the K+ current. The biochemical data were also consistent with the functional results. An interaction between epitope-tagged versions of TRESK and NDFIP1 was verified by co-immunoprecipitation experiments. The coexpression of NDFIP1 with TRESK induced the ubiquitination of the channel protein. Altogether, the results suggest that TRESK is directly controlled by and highly sensitive to the activation of the NDFIP1-Nedd4 system. The NDFIP1-mediated reduction in the TRESK component may induce depolarization, increase excitability, and attenuate the calcium dependence of the membrane potential by reducing the calcineurin-activated fraction in the ensemble background K+ current.

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development, including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and include regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth.

Evolutionary novelties entail the origin of morphologies that enable new functions. These features can arise through changes to gene function and regulation. One key novelty is the fused rod at the end of the vertebral column in anurans, the urostyle. This feature is composed of a coccyx and a hypochord, both of which ossify during metamorphosis. To elucidate the genetic basis of these features, we used laser capture microdissection of these tissues and did RNA-seq and ATAC-seq at three developmental stages in tadpoles of Xenopus tropicalis. RNA-seq reveals that the coccyx and hypochord have two different molecular signatures. Neuronal (TUBB3) and muscle markers (MYH3) are upregulated in coccygeal tissues, whereas T-box genes (TBXT, TBXT.2), corticosteroid stress hormones (CRCH.1) and matrix metallopeptidases (MMP1, MMP8 and MMP13) are upregulated in the hypochord. ATAC-seq reveals potential regulatory regions that are observed in proximity to candidate genes that regulate ossification identified from RNA-seq. Even though an ossifying hypochord is only present in anurans, this ossification between the vertebral column and the notochord resembles a congenital vertebral anomaly seen prenatally in humans caused by an ectopic expression of the TBXT/TBXT.2 gene. This work opens the way to functional studies that can elucidate anuran bauplan evolution.

Protocadherin 19 (Pcdh19) is a homophilic cell adhesion molecule and is involved in a variety of neuronal functions. Here, we tested whether Pcdh19 has a regulatory role in axon guidance using the developing Xenopus retinotectal system. We performed targeted microinjections of a translation blocking antisense morpholino oligonucleotide to knock down the expression of Pcdh19 selectively in the central nervous system. Knocking down Pcdh19 expression resulted in navigational errors of retinal ganglion cell (RGC) axons specifically at the optic chiasm. Instead of projecting to the contralateral optic tectum, RGC axons in the Pcdh19-depleted embryo misprojected ipsilaterally. Although incorrectly delivered into the ipsilateral brain hemisphere, these axons correctly reached the optic tectum. These data suggest that Pcdh19 has a critical role in preventing mixing of RGC axons originating from the opposite eyes at the optic chiasm, highlighting the importance of cell adhesion in bundling of RGC axons.

In response to DNA double-strand breaks or oxidative stress, ATM-dependent DNA damage response (DDR) is activated to maintain genome integrity. However, it remains elusive whether and how DNA single-strand breaks (SSBs) activate ATM. Here, we provide direct evidence in Xenopus egg extracts that ATM-mediated DDR is activated by a defined SSB structure. Our mechanistic studies reveal that APE1 promotes the SSB-induced ATM DDR through APE1 exonuclease activity and ATM recruitment to SSB sites. APE1 protein can form oligomers to activate the ATM DDR in Xenopus egg extracts in the absence of DNA and can directly stimulate ATM kinase activity in vitro. Our findings reveal distinct mechanisms of the ATM-dependent DDR activation by SSBs in eukaryotic systems and identify APE1 as a direct activator of ATM kinase.

DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.

The zinc finger and BTB domain-containing 11 gene (zbtb11) is expressed in the Xenopus anterior neuroectoderm, but the molecular nature of the Zbtb11 protein during embryonic development remains to be elucidated. Here, we show the role of Zbtb11 in anterior patterning of the neuroectoderm and the cooperative action with the transcription factor Otx2. Both overexpression and knockdown of zbtb11 caused similar phenotypes: expanded expression of the posterior gene gbx2 in the neural plate, and later microcephaly with reduced eyes, suggesting that a proper level of zbtb11 expression is necessary for normal patterning of the neuroectoderm, including eye formation. Co-immunoprecipitation assays showed that Zbtb11 formed a complex with itself and with a phosphomimetic and repressive form of Otx2, suggesting that Zbtb11 forms a dimer or oligomer and interacts with Otx2 in a phosphorylation-dependent manner. Reporter analysis further showed that Zbtb11 enhanced the activity of the phosphomimetic Otx2 to repress a silencer element of the posterior gene meis3. These data suggest that Zbtb11 coordinates with phosphorylated Otx2 to specify the anterior neuroectoderm by repressing posterior genes.

The SRY HMG box transcription factor Sox21 plays multiple critical roles in neurogenesis, with its function dependent on concentration and developmental stage. In the allotetraploid Xenopus laevis, there are two homeologs of sox21, namely sox21.S and sox21.L. Previous studies focused on Sox21.S, but its amino acid sequence is divergent, lacking conserved poly-A stretches and bearing more similarity with ancestral homologs. In contrast, Sox21.L shares higher sequence similarity with mouse and chick Sox21. To determine if Sox21.S and Sox21.L have distinct functions, we conducted gain and loss-of-function studies in Xenopus embryos. Our studies revealed that Sox21.S and Sox21.L are functionally redundant, but Sox21.L is more effective at driving changes than Sox21.S. These results also support our earlier findings in ectodermal explants, demonstrating that Sox21 function is dose-dependent. While Sox21 is necessary for primary neuron formation, high levels prevent their formation. Strikingly, these proteins autoregulate, with high levels of Sox21.L reducing sox21.S and sox21.L mRNA levels, and decreased Sox21.S promoting increased expression of sox21.L. Our findings shed light on the intricate concentration-dependent roles of Sox21 homeologs in Xenopus neurogenesis.

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two β-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.

Peptidoglycan recognition proteins (PGRPs) are a family of multifunctional proteins playing vital roles in PGN metabolism and antibacterial defense, and their functions have been well-characterized in mammals, bony fishes, and insects. However, the information about the functions of amphibian long-type PGRP is rather limited. Here, we identified and cloned a long-type PGRP gene (named Xl-PGRP-L) from African clawed frog, Xenopus laevis. Xl-PGRP-L gene was detected in all orangs/tissues examined, and was rapidly induced in intestine, liver, and lung following the stimulation of PGN. Sequence analysis showed that Xl-PGRP-L possesses four Zn2+-binding residues (His358, Tyr395, His470, and Cys478) required for amidase activity of catalytic PGRPs, and assays for amidase activity revealed that recombinant Xl-PGRP-L cloud degrade PGN in a Zn2+-dependent manner, indicating that Xl-PGRP-L is belonging to catalytic PGRPs. In addition, Xl-PGRP-L have antibacterial activity against Gram-negative bacteria Edwardsiella tarda and Gram-positive bacteria Streptococcus agalactiae. The present investigation represents the first characterization regarding the biological activities of amphibian long-type PGRPs, thus contributes to a better understanding of the functions of tetrapod PGRPs and the molecular mechanisms of amphibian antibacterial defense.