MRLC controls apoptotic cell death and functions to regulate epidermal development during planarian regeneration and homeostasis.

Adult stem cells (ASCs) are pluripotent cells with the capacity to self-renew and constantly replace lost cells due to physiological turnover or injury. Understanding the molecular mechanisms of the precise coordination of stem cell proliferation and proper cell fate decision is important to regeneration and organismal homeostasis. The planarian epidermis provides a highly tractable model to study ASC complex dynamic due to the distinct spatiotemporal differentiation stages during lineage development. Here, we identified the myosin regulatory light chain (MRLC) homologue in the Dugesia japonica transcriptome. We found high expression levels of MRLC in wound region during regeneration and also expressed in late epidermal progenitors as an essential regulator of the lineage from neoblasts to mature epidermal cells. We investigated the function of MRLC using in situ hybridization, real-time polymerase chain reaction and double fluorescent and uncovered the potential mechanism. Knockdown of MRLC leads to a remarkable increase in cell death, causes severe abnormalities during regeneration and homeostasis and eventually leads to animal death. The global decrease in epidermal cell in MRLC RNAi animals induces accelerated epidermal proliferation and differentiation. Additionally, we find that MRLC is co-expressed with cdc42 and acts cooperatively to control the epidermal lineage development by affecting cell death. Our results uncover an important role of MRLC, as an inhibitor of apoptosis, involves in epidermal development.

Meis1 Controls the Differentiation of Eye Progenitor Cells and the Formation of Posterior Poles during Planarian Regeneration.

As a member of TALE family, Meis1 has been proven to regulate cell proliferation and differentiation during cell fate commitment; however, the mechanism is still not fully understood. The planarian, which has an abundance of stem cells (neoblasts) responsible for regenerating any organ after injury, is an ideal model for studying the mechanisms of tissue identity determination. Here, we characterized a planarian homolog of Meis1 from the planarian Dugesia japonica. Importantly, we found that knockdown of DjMeis1 inhibits the differentiation of neoblasts into eye progenitor cells and results in an eyeless phenotype with normal central nervous system. Furthermore, we observed that DjMeis1 is required for the activation of Wnt signaling pathway by promoting the Djwnt1 expression during posterior regeneration. The silencing of DjMeis1 suppresses the expression of Djwnt1 and results in the inability to reconstruct posterior poles. In general, our findings indicated that DjMeis1 acts as a trigger for the activation of eye and tail regeneration by regulating the differentiation of eye progenitor cells and the formation of posterior poles, respectively.

Djck1α Is Required for Proper Regeneration and Maintenance of the Medial Tissues in Planarians.

CK1α (Casein kinase 1α) is a member of the casein kinase 1(CK1) family that is involved in diverse cellular processes, but its functions remain unclear in stem cell development. Freshwater planarians are capable of whole-body regeneration, making it a classic model for the study of regeneration, tissue homeostasis, and polarity in vivo. To investigate the roles of CK1α in regeneration and homeostasis progress, we characterize a homolog of CK1α from planarian Dugesia japonica. We find that Djck1α, which shows an enriched expression pattern in the nascent tissues, is widely expressed especially in the medial regions of planarians. Knockdown of CK1α by RNAi presents a thicker body due to dorsal hyperplasia, along with defects in the medial tissues including nerve proliferation, missing epidermis, intestine disturbance, and hyper-proliferation during the progression of regeneration and homeostasis. Moreover, we find that the ck1α RNAi animals exhibit expansion of the midline marker slit. The eye deficiency induced by slit RNAi can be rescued by ck1α and slit double RNAi. These results suggest that ck1α is required for the medial tissue regeneration and maintenance in planarian Dugesia japonica by regulating the expression of slit, which helps to further investigate the regulation of planarian mediolateral axis.

Djsnon, a downstream gene of Djfoxk1, is required for the regeneration of the planarian central nervous system.

Regulators of adult neurogenesis are crucial targets for neuronal repair. Freshwater planarians are ideal model systems for studying neuronal regeneration as they can regenerate their entire central nervous system (CNS) using pluripotent adult stem cells. Here, we identified Djfoxk1 in planarian Dugesia japonica to be required for planarian CNS regeneration. Knockdown of Djfoxk1 inhibits the regeneration of the cephalic ganglia, resulting in the failure of eye regeneration. By RNAi screening of Djfoxk1 downstream genes, we identified Djsnon as another regulator of planarian neuronal regeneration. Inhibition of Djsnon with RNA interference (RNAi) results in similar phenotypes caused by Djfoxk1 RNAi without affecting cell proliferation and wound healing. Our findings show that Djsnon as a downstream gene of Djfoxk1 regulates the regeneration of the planarian CNS.

Actin restricts cell proliferation and promotes differentiation during planarian regeneration.

Actin is an integral component of the cytoskeleton, which plays an important role in various fundamental cellular processes, such as affecting the polarity of embryonic cells during embryonic development in various model organisms. Meanwhile, previous studies have demonstrated that the polymerization of the actin cytoskeleton can affect cell migration, proliferation, and differentiation. Actin polymerization state regulated osteogenic differentiation and affected cell proliferation. However, the function of actin in regenerative biology has not been thoroughly elucidated. The planarian flatworm, which contains a large number of adult somatic stem cells (neoblasts), is an ideal model organism to study regenerative biology. Here, we identified a homolog of actin in planarian Dugesia japonica and found that RNAi targeting actin during planarian regeneration results in the formation of protrusions on the dorsal side, where the division of phospho-H3 mitotic cells is increased. In addition, a decrease in differentiation is observed in regenerating tissues after Djactin RNAi. These results indicate that Djactin functions in proliferation and differentiation control in planarian regeneration.

Whole-genome sequence of the planarian Dugesia japonica combining Illumina and PacBio data.

Advances in stem cell biology have posed the challenges in revealing the mechanistic themes underlying whole tissues and organs formation during regeneration. The planarian Dugesia japonica is an ideal model organism for the study of regeneration and stem cell biology. However, the genome resources for this species are still limited. Here, we combined single-molecule real-time DNA sequencing platform Pacific Biosciences (PacBio) sequencing, Illumina paired-end sequencing and 10× Genomics linked reads data to obtain the whole-genome sequence of the planarian D. japonica. The final assembled D. japonica genome is 1.13G with contig N50 of 248.44 kb, and scaffold N50 of 652.52 kb. Repeat elements account for 64.92% of the genome, and 12,031 protein coding genes were annotated, of which 10,114 genes had at least one functional annotation, representing 84.07% of the total genes. We present a highly contiguous genome assembly of D. japonica. The D. japonica genome assembly, together with gene annotation and transcriptome data provide a valuable resource to investigate molecular mechanism of planarian and stem cell research.

Djnedd4L Is Required for Head Regeneration by Regulating Stem Cell Maintenance in Planarians.

SUMOylation and ubiquitylation are homologous processes catalyzed by homologous enzymes, and they are involved in nearly all aspects of eukaryotic biology. Planarians, which have the remarkable ability to regenerate their central nervous system (CNS), provide an excellent opportunity to investigate the molecular processes of CNS regeneration in vivo. In this study, we analyzed gene expression profiles during head regeneration with an RNA-seq-based screening approach and found that Djnedd4L and Djubc9 were required for head regeneration in planarians. RNA interference targeting of Djubc9 caused the phospho-H3 mitotic cells to decrease in quantity, or even become absent as a part of the Djubc9 RNAi phenotype, which also showed the collapse of the stem cell lineage along with the reduced expression of epidermal differentiation markers. Furthermore, we found that Djnedd4L RNAi induced increased cell division and promoted the premature differentiation during regeneration. Taken together, our findings show that Djubc9 and Djnedd4L are required for stem cell maintenance in the planarian Dugesia japonica, which helps to elucidate the role of SUMOylation and ubiquitylation in regulating the regeneration process.

CHIP promotes the activation of NF-κB signaling through enhancing the K63-linked ubiquitination of TAK1.

Transcriptional factor nuclear factor κB (NF-κB) can be activated by various intracellular or extracellular stimuli and its dysregulation leads to pathological conditions, such as neurodegenerative disorders, infection, and cancer. The carboxyl terminus of HSC70-interacting protein (CHIP), a pathogenic gene of spinocerebellar autosomal recessive 16 (SCAR16), plays an important roles in protein degradation, trafficking, and multiple signaling transductions. It has been reported that CHIP participates in the regulation of NF-κB signaling, and the mutant of CHIP (p.T246M) leads to the occurrence of SCAR16. However, the detailed mechanism of CHIP and CHIP (p.T246M) in the regulation of NF-κB signaling in neurological disorders remains unclear. Here, we found that CHIP promoted the activation of NF-κB signaling, while the knockdown had the opposite effect. Furthermore, CHIP interacted with TAK1 and targeted it for K63-linked ubiquitination. Finally, CHIP enhanced the interaction between TAK1 and NEMO. However, CHIP (p.T246M) couldn't upregulate NF-κB signaling, potentiate the ubiquitination of TAK1, and enhance the interactions. Taken together, our study demonstrated for the first time that CHIP positively regulates NF-κB signaling by targeting TAK1 and enhancing its K63-linked ubiquitination.

TBX2/3 is required for regeneration of dorsal-ventral and medial-lateral polarity in planarians.

The molecular mechanisms responsible for axis establishment during non-embryonic processes remain elusive. The planarian flatworm is an ideal model organism to study body axis polarization and patterning in vivo. Here, we identified a homolog of the TBX2/3 in the planarian Dugesia japonica. RNA interference (RNAi) knockdown of TBX2/3 results in the ectopic formation of protrusions in the midline of the dorsal surface which shows an abnormal expression of midline and ventral cell markers. Additionally, the TBX2/3 RNAi animals also show the duplication of expression of the boundary marker at the lateral edge. Furthermore, TBX2/3 is expressed in muscle cells and co-expressed with bmp4. Inhibition of bone morphogenetic protein (BMP) signaling reduces the expression of TBX2/3 at the midline. These results suggest that TBX2/3 RNAi results in phenotypic characters caused by inhibition of the BMP signal, indicating that TBX2/3 is required for DV and ML patterning, and might be a downstream gene of BMP signaling.

CHIP promotes Wnt signaling and regulates Arc stability by recruiting and polyubiquitinating LEF1 or Arc.

The carboxyl terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase, participates in many cellular processes such as protein degradation, trafficking, autophagy, apoptosis, and multiple signaling transductions. The mutant of CHIP (p.T246M) causes the spinocerebellar autosomal recessive 16 (SCAR16), a neurodegenerative disease characterized by spinocerebellar atrophy. Previous studies have shown that Wnt signaling and activity-regulated cytoskeleton-associated protein (Arc) play important roles in neurodegenerative diseases. However, the mechanisms by which CHIP regulates Wnt signaling and the stability of Arc that may affect SCAR16 are still unclear. We show that overexpression of CHIP promoted the activation of Wnt signaling, and enhanced the interaction between LEF1 and ß-catenin through heightening the K63-linked polyubiquitin chains attached to LEF1, while the knockdown of CHIP had the opposite effect. Moreover, we verified that Wnt signaling was inhibited in the rat models of SCAR16 induced by the CHIP (p.T246M) mutant. CHIP also accelerated the degradation of Arc and regulated the interaction between Arc and GSK3ß by heightening the K48- or K63-linked polyubiquitin chains, which further potentiated the interaction between GSK3ß and ß-catenin. Our data identify that CHIP is an undescribed regulator of Wnt signaling and Arc stability which may be related to the occurrence of SCAR16.

Tubgcp3 is a mitotic regulator of planarian epidermal differentiation.

Tubgcp3/GCP3 (The centrosomal protein γ-tubulin complex protein 3) is a component of the γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs), which play critical roles in mitotic spindle formation during mitosis. However, its function in stem cell development has not been thoroughly elucidated. The planarian flatworm, which contains a large number of adult somatic stem cells (neoblasts), is a unique model to study stem cell lineage development in vivo. Here, we identified a homolog of Tubgcp3 in planarian Dugesia japonica, and found that Tubgcp3 is required for the maintenance of epidermal lineage. RNAi targeting Tubgcp3 resulted in tissue homeostasis and regeneration defect. Knockdown of Tubgcp3 reduced cell divisions and led to a loss of the mature epidermal cells. Our findings indicate that Tubgcp3 is a mitotic regulator and plays a crucial role in planarian epidermal differentiation.

Crystal structure of the yeast heterodimeric ADAT2/3 deaminase.

BACKGROUND: The adenosine-to-inosine (A-to-I) editing in anticodons of tRNAs is critical for wobble base-pairing during translation. This modification is produced via deamination on A34 and catalyzed by the adenosine deaminase acting on tRNA (ADAT) enzyme. Eukaryotic ADATs are heterodimers composed of the catalytic subunit ADAT2 and the structural subunit ADAT3, but their molecular assemblies and catalytic mechanisms are largely unclear. RESULTS: Here, we report a 2.8-Å crystal structure of Saccharomyces cerevisiae ADAT2/3 (ScADAT2/3), revealing its heterodimeric assembly and substrate recognition mechanism. While each subunit clearly contains a domain resembling their prokaryotic homolog TadA, suggesting an evolutionary gene duplication event, they also display accessory domains for additional structural or functional purposes. The N-lobe of ScADAT3 exhibits a positively charged region with a potential role in the recognition and binding of tRNA, supported by our biochemical analysis. Interestingly, ScADAT3 employs its C-terminus to block tRNA's entry into its pseudo-active site and thus inactivates itself for deamination despite the preservation of a zinc-binding site, a mechanism possibly shared only among yeasts. CONCLUSIONS: Combining the structural with biochemical, bioinformatic, and in vivo functional studies, we propose a stepwise model for the pathway of deamination by ADAT2/3. Our work provides insight into the molecular mechanism of the A-to-I editing by the eukaryotic ADAT heterodimer, especially the role of ADAT3 in catalysis.

TRIM25 Promotes TNF-α-Induced NF-κB Activation through Potentiating the K63-Linked Ubiquitination of TRAF2.

As an important effector in response to various intracellular or extracellular stimuli, the NF-κB family extensively participates in a wide spectrum of biological events, and its dysregulation may result in many pathological conditions, such as microbial infection, tumor progression, and neurodegenerative disorders. Previous investigations showed that multiple types of ubiquitination play critical roles in the modulation of the NF-κB signaling pathway, yet the molecular mechanisms are still poorly understood. In the current study, we identified TRIM25, an E3 ubiquitin ligase, as a novel positive regulator in mediating NF-κB activation in human embryonic kidney 293T (HEK293T), HeLa cells, THP-1 cells, and PBMCs. The expression of TRIM25 promoted TNF-α-induced NF-κB signaling, whereas the knockdown had the opposite effect. Furthermore, TRIM25 interacted with TRAF2 and enhanced the K63-linked polyubiquitin chains attached to TRAF2. Moreover, TRIM25 bridged the interaction of TRAF2 and TAK1 or IKKß. To our knowledge, our study has identified a previously unrecognized role for TRIM25 in the regulation of NF-κB activation by enhancing the K63-linked ubiquitination of TRAF2.

Application of next-generation sequencing in resistance genes of neoadjuvant chemotherapy for esophageal cancer.

BACKGROUND: Neoadjuvant chemotherapy (NAC) has been proven to effectively improve the prognosis and long-term survival of patients with esophageal cancer (EC). But approximately 40% of patients are relatively insensitive to NAC. The mechanism underlying gene-induced resistance remains elusive. METHODS: We conducted a cohort of 13 NAC patients with different chemotherapy responses to identify gene mutations related to drug resistance by next-generation sequencing (NGS) of samples from patients with EC. We performed protein conformation on these mutant genes to figure out the possible mechanisms related to resistance. RESULTS: Our results indicated that missense mutations were commonly emerged in patients with partial response (PR) and stable disease (SD). Moreover, NOTCH1 gene, which is closely related to efficacy of chemotherapy, was further screened by comparing the changes in gene mutations before and after chemotherapy. Through protein conformational analysis, we found that missense mutations may cause changes in the ability of NOTCH1 receptor protein to bind ligands, which may cause abnormalities in the NOTCH1 pathway and make patients resistant to chemotherapy. CONCLUSIONS: We analyzed the effect of platinum-based NAC on gene mutations in patients with EC and find that mutations in somatic genes, especially the NOTCH1 gene, may be associated with NAC resistance in EC.

Gain and loss events in the evolution of the apolipoprotein family in vertebrata.

BACKGROUND: Various apolipoproteins widely distributed among vertebrata play key roles in lipid metabolism and have a direct correlation with human diseases as diagnostic markers. However, the evolutionary progress of apolipoproteins in species remains unclear. Nine human apolipoproteins and well-annotated genome data of 30 species were used to identify 210 apolipoprotein family members distributed among species from fish to humans. Our study focused on the evolution of nine exchangeable apolipoproteins (ApoA-I/II/IV/V, ApoC-I~IV and ApoE) from Chondrichthyes, Holostei, Teleostei, Amphibia, Sauria (including Aves), Prototheria, Marsupialia and Eutheria. RESULTS: In this study, we reported the overall distribution and the frequent gain and loss evolutionary events of apolipoprotein family members in vertebrata. Phylogenetic trees of orthologous apolipoproteins indicated evident divergence between species evolution and apolipoprotein phylogeny. Successive gain and loss events were found by evaluating the presence and absence of apolipoproteins in the context of species evolution. For example, only ApoA-I and ApoA-IV occurred in cartilaginous fish as ancient apolipoproteins. ApoA-II, ApoE, and ApoC-I/ApoC-II were found in Holostei, Coelacanthiformes, and Teleostei, respectively, but the latter three apolipoproteins were absent from Aves. ApoC-I was also absent from Cetartiodactyla. The apolipoprotein ApoC-III emerged in terrestrial animals, and ApoC-IV first arose in Eutheria. The results indicate that the order of the emergence of apolipoproteins is most likely ApoA-I/ApoA-IV, ApoE, ApoA-II, ApoC-I/ApoC-II, ApoA-V, ApoC-III, and ApoC-IV. CONCLUSIONS: This study reveals not only the phylogeny of apolipoprotein family members in species from Chondrichthyes to Eutheria but also the occurrence and origin of new apolipoproteins. The broad perspective of gain and loss events and the evolutionary scenario of apolipoproteins across vertebrata provide a significant reference for the research of apolipoprotein function and related diseases.

DjERas plays an important role in planarian regeneration and homeostasis.

The mechanisms of cell turnover including cell proliferation and cell differentiation were complex. Planarians possess amazing regeneration ability and undergo cell turnover throughout life. We identified a homologous gene of ERas by RNAi in Dugesia japonica. Knocking-down DjERas resulted in regeneration and homeostasis defects. Furthermore, we found that the expression of neoblasts and late progeny marker gene decreased in DjERas RNAi planarians. Our studies indicated that down-regulation of DjERas inhibited the proliferation and differentiation of stem cells through the conserved signaling pathway, resulted in the inability of the planarian to regenerate and maintain homeostasis. Our results suggest that DjERas plays a crucial role in the process of cell turnover.

TINP1 homolog is required for planarian regeneration.

The planarian flatworm is an ideal system for the study of regeneration in vivo. In this study, we focus on TINP1, which is one of the most conserved proteins in eukaryotic organisms. We found that TINP1 was expressed in parenchymal region through whole body as well as central nervous system (CNS) during the course of regeneration. RNA interference targeting DjTINP1 caused lysis defects in regenerating tissues and a decreased in cell division and expression levels of DjpiwiA and Djpcna. Furthermore, the expression levels of DjTINP1 were decreased when we inhibited the TGF-ß signal by knockdown of smad4, which is the sole co-smad and has been proved to control the blastema patterning and central nervous system (CNS) regeneration in planarians. These findings suggest that DjTINP1 participate in the maintenance of neoblasts and be required for proper cell proliferation in planarians as a downstream gene of the TGF-ß signal pathway.

Exportin-1 is required for the maintenance of the planarian epidermal lineage.

Nucleocytoplasmic transport is essential for normal cellular function that mediates cargo transport from the cytoplasm to the nucleus. However, the mechanisms of nucleocytoplasmic transport that integrate stem cell development remain largely unknown. Since it has a large population of stem cells, the planarian flatworm is an ideal system for the study of adult stem cell lineage development in vivo. Here, we focus on exportin-1, which is the most conserved nuclear export receptor. Homologs of exportin-1 have no currently known role in stem cell biology. RNA interference targeting exportin-1 caused a failure in anterior and posterior regeneration, and resulted in curly and lysis phenotypes in both intact and regenerating flatworms. During the course of exportin-1 RNAi phenotype, cell division was significantly decreased, and the expression of the epidermal cell markers (vimentin and laminB) were lost from the intact body. Additionally, the expression levels of the neoblast marker piwiA decreased. By contrast, the expression levels of the epidermal progenitor markers NB21.11e and AGAT1 increased. These results suggest that exportin-1 is required for the maintenance of the epidermal lineage in planarians. Inhibition of exportin-1 could promote the premature differentiation of neoblasts to the epidermal lineages, disrupting the proper epidermal maturation.

A planarian RPS3 homolog is critical to the modulation of planarian regeneration.

Planarians are an important model for stem cell and regeneration biology, because they can regenerate any missing body structure in a short time. As an important component of ribosomes, ribosomal proteins can synthesize proteins and play a central role in cell cycle checkpoint, cell survival/senescence/apoptosis, and organismal growth and development. In this study, we identified and amplified the homologous gene of RPS3 in Dugesia japonica. Double-stranded RNA mediated RNAi revealed that when the Dj-RPS3 function was lost by planarians; they did not form blastemas and died 100%. Further investigation, confirmed that Dj-RPS3 was involved in regulating the proliferative and early differentiation of neoblasts.

Down-regulate of Djrfc2 causes tissues hypertrophy during planarian regeneration.

Planarians are an ideal model organism for regeneration research due to their amazing ability to regenerate. DNA replication is crucial for genome stability. Replication factor C (RFC), which is a replication factor C-like complex and plays an important role during DNA replication in eukaryotes, has been reported as a wound response factor during planarian regeneration. However, how RFC controls regeneration in planarians by regulating DNA replication remains to be explained. Here, we used a two-dimensional electrophoresis (2-DE) proteomic approach to identify differentially expressed proteins in intact and regenerated planarians. Approximately 132 protein spots showed differences between intact and regenerative tissues. We selected 21 significantly expressed protein spots and processed them using TOF MS analysis. Finally, we cloned three of these candidate genes (Djhsp70, Djrfc2, Djfaim), focusing on the function of Djrfc2 during regeneration. We found that the distribution of Djrfc2 tends toward the wound site. RNA interference (RNAi) of Djrfc2 increases the number of dividing cells and the expression level of planarian neoblast marker genes, which may result in hyper-proliferation. Our studies use an available approach to directly study the regeneration dynamic at the protein level and provide further evidence to support a function of Djrfc2 in planarian regeneration.