Regeneration Initiation Research Articles

Multiple roles for the hippo effector yap in gut regeneration and cancer initiation

ABSTRACTThe Hippo signaling effector Yes-associated protein (Yap) is known for its potent control of tissue growth. Our recent work now shows that Yap promotes regeneration in the intestine by reprogramming intestinal stem cells and blocking their terminal differentiation. Similarly, in tumor-initiating cells Yap regenerative signaling synergizes with Wnt activation to drive adenoma formation.

ITRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR) remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

IN VITRO Regeneration of Bina Mungbean Varieties

The experiment was conducted to develop an efficient protocol for in vitro regeneration of mungbean (Vignaradiata) on the aspect of regeneration potentiality of two mungbean varieties (BINA mung 5 and BINA mung 7) as influenced by different combinations of growth regulators supplemented with MS medium. Cotyledon explant of both varieties was used for the present study. Data were collected for various characters of callus initiation, shoot regeneration and root proliferation. Initiation of callus (%) and required days for its initiation and weight of callus were influenced significantly due to the effect of varieties where BINA mung 5 produced more callus induction (40.36%) at minimum requiring time (18.27 days) including heavier sizes of callus (1.54 g) than BINA mung 7 when BINA mung 5 further recorded the longest root (2.92 cm) compare to BINA mung 7. Effect of treatments of the present study were significantly influenced the whole characters regarding callus culture, shoot regeneration and root proliferation. The highest percentage of callus (88.44%) within minimum time (12.53 days) including larger sizes callus (3.521 g) were produced in 1.0 mg L–1 BAP + 2.5 mg L–1 NAA among the treatments while the highest percentage of regenerated shoot (83.44%) at minimum requiring time (17.59 days) and more shoots (7.69 callus–1) were obtained in 1.0 mg L–1 BAP + 2.0 mg L– 1 NAA. Root induction (82.50%), number of roots plantlet–1 (8.469) with minimum requiring time for initiation (14.13 days) and root length (5.250 cm) were the highest in 0.2 mg L–1 IAA + 1.0 mg L–1 kinetin + 0.2 mg L–1 BAP. Incase of interaction, percentage of callus initiation (89.38 %) was the highest in BINA mung 5 treated by 1.0 mg L–1 BAP + 2.5 mg L–1 NAA at requiring minimum time (12.38 days) while same treatment produced the larger callus (3.581 g) among the interactions. The highest percentage (84.38%) and number (7.813 callus–1) of shoot with minimum requiring time (17.50 days) were found from BINA mung 5 treated by 1.0 mg L–1 BAP + 2.0 mg L–1 NAA. Similarly, the longest shoot (5.58 cm) was produced from the BINA mung 5 treated by 1.0 mg L–1 BAP + 2.0 mg L–1 NAA. However, root induction (%), roots plantlet–1, days required for root initiation and root length were statistically similar among the whole interaction treatments due to non significant variation. This result mentioned that the variety BINA mung 5 was better than BINA mung 7 for callus induction, shoot regeneration and root initiation while 1.0 mg L–1 BAP + 2.5 mg L–1 NAA, 1.0 mg L–1 BAP + 2.0 mg L–1 NAA and 0.2 mg L–1 IAA + 1.0 mg L–1 kinetin + 0.2 mg L–1 BAP supplemented with MS medium were the best combinations for better callusing, higher ability of shoot regeneration and root proliferation.DOI: http://dx.doi.org/10.3329/jesnr.v7i2.22203 J. Environ. Sci. & Natural Resources, 7(2): 47-52 2014

An Integrated Approach for the Identification of HNF4α-Centered Transcriptional Regulatory Networks During Early Liver Regeneration

Background/Aims: Hepatocyte nuclear factor-4α (HNF4α), the liver enriched transcription factor (TF), is one of the major regulators of hepatocyte differentiation and proliferation. However, how HNF4α participate in liver regeneration after partial hepatectomy (PH) remains largely unknown. In order to identify the HNF4α-centered regulatory network, we applied an integrated analytic strategy, in which, TF array, mRNA microarray, bioinformatic analysis and ChIP-on-chip assays were integrated. Methods/Results: The TF signatures from MOUSE OATFA (TF-array) platform revealed that the activity of HNF4α was significantly reduced and 17 other TFs showed increased activity at 4 h post PH. Then the ChIP-on-chip analysis on HNF4α were combined with mRNA expression profiling to select the possible HNF4α target genes during early liver regeneration, which were then sub-grouped according to their signaling pathways. More specifically, the HNF4α target genes with the gene ontology (GO) terms of cytokine-cytokine receptor, Jak-STAT, MAPK, toll-like receptor and insulin signaling pathways were further analyzed with an advanced bioinformatics tool named oPOSSUM to identify TF binding sites occupancy and predict the co-regulatory relationship between TFs and targets. Furthermore, we identified that repressed HNF4α during the early phase of liver regeneration may contribute cooperatively to the induction of immediate early genes, such as, c-fos, c-jun and stat3. Conclusion: our data indicate that HNF4α may play an inhibitory role on the induction of specific promitogenic genes and liver regeneration initiation. The integrated approach of mRNA/OATFA/ChIP-DSL/oPOSSUM analysis may help us better characterize the target genes and co-regulatory network of HNF4α during the early stage of liver regeneration.

Bimodal regulation of ICR1 levels generates self-organizing auxin distribution.

Auxin polar transport, local maxima, and gradients have become an important model system for studying self-organization. Auxin distribution is regulated by auxin-dependent positive feedback loops that are not well-understood at the molecular level. Previously, we showed the involvement of the RHO of Plants (ROP) effector INTERACTOR of CONSTITUTIVELY active ROP 1 (ICR1) in regulation of auxin transport and that ICR1 levels are posttranscriptionally repressed at the site of maximum auxin accumulation at the root tip. Here, we show that bimodal regulation of ICR1 levels by auxin is essential for regulating formation of auxin local maxima and gradients. ICR1 levels increase concomitant with increase in auxin response in lateral root primordia, cotyledon tips, and provascular tissues. However, in the embryo hypophysis and root meristem, when auxin exceeds critical levels, ICR1 is rapidly destabilized by an SCF(TIR1/AFB) [SKP, Cullin, F-box (transport inhibitor response 1/auxin signaling F-box protein)]-dependent auxin signaling mechanism. Furthermore, ectopic expression of ICR1 in the embryo hypophysis resulted in reduction of auxin accumulation and concomitant root growth arrest. ICR1 disappeared during root regeneration and lateral root initiation concomitantly with the formation of a local auxin maximum in response to external auxin treatments and transiently after gravitropic stimulation. Destabilization of ICR1 was impaired after inhibition of auxin transport and signaling, proteasome function, and protein synthesis. A mathematical model based on these findings shows that an in vivo-like auxin distribution, rootward auxin flux, and shootward reflux can be simulated without assuming preexisting tissue polarity. Our experimental results and mathematical modeling indicate that regulation of auxin distribution is tightly associated with auxin-dependent ICR1 levels.

Cell-State Transitions Regulated by SLUG Are Critical for Tissue Regeneration and Tumor Initiation

SummaryPerturbations in stem cell activity and differentiation can lead to developmental defects and cancer. We use an approach involving a quantitative model of cell-state transitions in vitro to gain insights into how SLUG/SNAI2, a key developmental transcription factor, modulates mammary epithelial stem cell activity and differentiation in vivo. In the absence of SLUG, stem cells fail to transition into basal progenitor cells, while existing basal progenitor cells undergo luminal differentiation; together, these changes result in abnormal mammary architecture and defects in tissue function. Furthermore, we show that in the absence of SLUG, mammary stem cell activity necessary for tissue regeneration and cancer initiation is lost. Mechanistically, SLUG regulates differentiation and cellular plasticity by recruiting the chromatin modifier lysine-specific demethylase 1 (LSD1) to promoters of lineage-specific genes to repress transcription. Together, these results demonstrate that SLUG plays a dual role in repressing luminal epithelial differentiation while unlocking stem cell transitions necessary for tumorigenesis.

Cell-State Transitions Regulated by SLUG Are Critical for Tissue Regeneration and Tumor Initiation

Cell-State Transitions Regulated by SLUG Are Critical for Tissue Regeneration and Tumor Initiation

Tissue absence initiates regeneration through Follistatin-mediated inhibition of Activin signaling

Regeneration is widespread, but mechanisms that activate regeneration remain mysterious. Planarians are capable of whole-body regeneration and mount distinct molecular responses to wounds that result in tissue absence and those that do not. A major question is how these distinct responses are activated. We describe a follistatin homolog (Smed-follistatin) required for planarian regeneration. Smed-follistatin inhibition blocks responses to tissue absence but does not prevent normal tissue turnover. Two activin homologs (Smed-activin-1 and Smed-activin-2) are required for the Smed-follistatin phenotype. Finally, Smed-follistatin is wound-induced and expressed at higher levels following injuries that cause tissue absence. These data suggest that Smed-follistatin inhibits Smed-Activin proteins to trigger regeneration specifically following injuries involving tissue absence and identify a mechanism critical for regeneration initiation, a process important across the animal kingdom. DOI:http://dx.doi.org/10.7554/eLife.00247.001.

A molecular wound response program associated with regeneration initiation in planarians

Planarians are capable of regenerating any missing body part and present an attractive system for molecular investigation of regeneration initiation. The gene activation program that occurs at planarian wounds to coordinate regenerative responses remains unknown. We identified a large set of wound-induced genes during regeneration initiation in planarians. Two waves of wound-induced gene expression occurred in differentiated tissues. The first wave includes conserved immediate early genes. Many second-wave genes encode conserved patterning factors required for proper regeneration. Genes of both classes were generally induced by wounding, indicating that a common initial gene expression program is triggered regardless of missing tissue identity. Planarian regeneration uses a population of regenerative cells (neoblasts), including pluripotent stem cells. A class of wound-induced genes was activated directly within neoblasts, including the Runx transcription factor-encoding runt-1 gene. runt-1 was required for specifying different cell types during regeneration, promoting heterogeneity in neoblasts near wounds. Wound-induced gene expression in neoblasts, including that of runt-1, required SRF (serum response factor) and sos-1. Taken together, these data connect wound sensation to the activation of specific cell type regeneration programs in neoblasts. Most planarian wound-induced genes are conserved across metazoans, and identified genes and mechanisms should be important broadly for understanding wound signaling and regeneration initiation.

Planarian regeneration involves distinct stem cell responses to wounds and tissue absence

Regeneration requires signaling from a wound site for detection of the wound and a mechanism that determines the nature of the injury to specify the appropriate regenerative response. Wound signals and tissue responses to wounds that elicit regeneration remain poorly understood. Planarians are able to regenerate from essentially any type of injury and present a novel system for the study of wound responses in regeneration initiation. Newly developed molecular and cellular tools now enable study of regeneration initiation using the planarian Schmidtea mediterranea. Planarian regeneration requires adult stem cells called neoblasts and amputation triggers two peaks in neoblast mitoses early in regeneration. We demonstrate that the first mitotic peak is a body-wide response to any injury and that a second, local, neoblast response is induced only when injury results in missing tissue. This second response was characterized by recruitment of neoblasts to wounds, even in areas that lack neoblasts in the intact animal. Subsequently, these neoblasts were induced to divide and differentiate near the wound, leading to formation of new tissue. We conclude that there exist two functionally distinct signaling phases of the stem cell wound response that distinguish between simple injury and situations that require the regeneration of missing tissue.

Experimental study on liver regeneration after simultaneous partial hepatectomy and pancreatectomy

In this study, we performed hepatectomy and pancreatectomy to assess the physiological contribution of the pancreas, especially in terms of endocrine function to hepatic regeneration. Group 1 Wistar rats underwent 70% hepatectomy and group 2 rats underwent 70% hepatectomy plus 50% pancreatectomy. The time course assessment of liver regeneration rates obtained by Fishback’s formula demonstrated a difference in rates between the two groups as early as day 3 or day 7 after surgery. Since levels of both PCNA-positive cells and serum transforming growth factor-α (TGF-α) were significantly higher in the hepatectomy only group, we could prove the difference of liver regeneration between the two groups. We have concluded that pancreatectomy retards the liver regeneration initiation processes occurring from 24 h to 3 days after evisceration. Glucagon–insulin molar ratios most significantly differed between the two groups 3 days after evisceration in the present study. This result was due to increased glucagon level of group 2 at day 3 after evisceration. Our findings suggest that 50% partial pancreatectomy inhibits the rate of hepatic regeneration, thereby altering the supply of pancreatic hormones, especially glucagon.

Regeneration of thallus fragments of Rhodochorton purpureum (Rhodophyceae, Nemalionales)

Abstract Fragments of Rhodochorton purpureum filaments regenerated frequently in culture. Most fragments produced an adhesive rhizoid from one cut end of the filament before a shoot or another rhizoid developed from the opposite cut end. Intact apical cells on fragments did not appear to inhibit regeneration or branch initiation. Similar patterns of regeneration were observed in media of 7, 17, and 31 ‰ salinity, but filaments grew most rapidly in 17‰. The patterns of regeneration observed suggest that the dispersion of R. purpureum in intertidal habitats may result from thallus fragmentation.