The BAF chromatin remodeling complex licenses planarian stem cells access to ectodermal and mesodermal cell fates.

BACKGROUND: The flatworm planarian, Schmidtea mediterranea, has a large population of adult stem cells (ASCs) that replace any cell type during tissue turnover or regeneration. How planarian ASCs (called neoblasts) manage self-renewal with the ability to produce daughter cells of different cell lineages (multipotency) is not well understood. Chromatin remodeling complexes ultimately control access to DNA regions of chromosomes and together with specific transcription factors determine whether a gene is transcribed in a given cell type. Previous work in planarians determined that RNAi of core components of the BAF chromatin remodeling complex, brg1 and smarcc2, caused increased ASCs and failed regeneration, but how these cellular defects arise at the level of gene regulation in neoblasts is unknown. RESULTS: Here, we perform ATAC and RNA sequencing on purified neoblasts, deficient for the BAF complex subunits brg-1 and smarcc2. The data demonstrate that the BAF complex promotes chromatin accessibility and facilitates transcription at target loci, as in other systems. Interestingly, we find that the BAF complex enables access to genes known to be required for the generation of mesoderm- and ectoderm-derived lineages, including muscle, parenchymal cathepsin, neural, and epithelial lineages. BAF complex knockdowns result in disrupted differentiation into these cell lineages and functional consequences on planarian regeneration and tissue turnover. Notably, we did not detect a role for the BAF complex in neoblasts making endodermal lineages. CONCLUSIONS: Our study provides functional insights into how the BAF complex contributes to cell fate decisions in planarian ASCs in vivo.

Different positive end expiratory pressure and tidal volume controls on lung protection and inflammatory factors during surgical anesthesia.

BACKGROUND: Mechanical ventilation can lead to the severe impairment of the metabolic pathway of alveolar surfactants, inactivating alveolar surfactants and significantly reducing lung-chest compliance. The cardiopulmonary function of elderly patients usually reduced to a certain extent, and there are lung complications after surgical anesthesia, just like lung barotrauma caused by mechanical ventilation, atelectasis and postoperative hypoxemia. AIM: To investigate the effects of different positive end expiratory pressures (PEEPs) and tidal volumes (VTs) on respiratory function, the degree of the inflammatory response and hemodynamic indexes in patients undergoing surgery under general anesthesia. METHODS: A total of 120 patients undergoing surgery for gastric or colon cancer under general anesthesia in Xinghua People's Hospital from January 2017 to January 2021 were randomly divided into Group A and Group B, with 60 cases in each group. The ventilation mode in Group A was VT (6.0 mL/kg) + PEEP (5.0 cmH2O), while that in Group B was VT (6.0 mL/kg) + PEEP (8.0 cmH2O). Blood gas parameters, respiratory mechanical parameters, inflammatory response indicators, hemodynamic indicators and related complications were compared between the two groups. RESULTS: There were no significant differences in PaCO2, PaO2, oxygen or the examined indexes at T0 between group A and group B (P > 0.05). The measured PaO2 value of patients in group A at T3 was higher than that in group B, and the difference was significant (P < 0.05). There were no significant differences in peak airway pressure (Ppeak), mean airway pressure or dynamic pulmonary compliance (Cdyn) at T0 between group A and group B (P > 0.05). The measured Ppeak value of patients in group A at T1 was higher than that in group B, and the difference was significant (P < 0.05). The measured Cdyn value at T1 and T2 was greater than that in group B (P < 0.05). Before surgery, there were no significant differences in tumor necrosis factor-α (TNF-α), interleukin (IL)-6 or IL-10 between group A and group B (P > 0.05). After 4 h, the measured values of TNF-α and IL-6 in group A were lower than those in group B, and the differences were significant (P < 0.05). The IL-10 Level in group A was higher than that in group B (P < 0.05). At T0, there were no significant differences in cardiac output, cardiac index (CI), stroke volume index (SVI) or mean arterial pressure between group A and group B (P > 0.05). The measured values of CI and SVI at T2 in patients in group A were higher than those in group B, and the differences were significant (P < 0.05). CONCLUSION: For patients undergoing surgery for gastric or colon cancer under general anesthesia, the VT (6.0 mL/kg) + PEEP (5.0 cmH2O) regimen was more effective than the VT (6.0 mL/kg) + PEEP (8.0 cmH2O) regimen in protecting the lung function and ventilatory function of patients, and it had better effects on maintaining hemodynamic stability and reducing inflammatory reactions.

Smed-myb-1 Specifies Early Temporal Identity during Planarian Epidermal Differentiation.

The planarian epidermis provides an excellent model to explore adult stem cell (ASC) lineage development due to well-characterized and distinct spatiotemporal phases during lineage progression. Using flow cytometry-isolated cells enriched in epidermal progenitors, we performed transcriptional profiling and RNAi screening to uncover regulators of epidermal differentiation. We identified a MYB-type transcription factor (Smed-myb-1) required for the specification of the first temporal phase of post-mitotic maturation. Knockdown of myb-1 abolished the early progenitor phase of differentiation without ceasing production of subsequent epidermal progenitor states or homeostatic turnover and regeneration of the epidermis. Further examination revealed accelerated maturation of ASC descendants, with premature entry into subsequent progeny phases and, ultimately, the epidermis. These results demonstrate that a spatiotemporal shift in lineage progression occurs in the absence of the early progenitor state after myb-1 RNAi, and identify myb-1 as a critical regulator of the early temporal window in stepwise specification during planarian epidermal differentiation.

(Neo)blast from the past: new insights into planarian stem cell lineages.

Collectively, planarian stem cells (neoblasts) are totipotent and are required for tissue homeostasis and regeneration. Recent work has begun to test the long-standing question of whether all neoblasts have the same potential, or whether they actually represent molecularly distinct subpopulations with distinct tissue restriction. Here, we summarize the current state of the field in neoblast lineage organization. It is clear that at least some neoblasts are totipotent, whereas other neoblasts represent functionally distinct molecular subclasses with restricted potential. In addition to neoblast subclasses, tissue-specific progenitors have also been identified, though their ability to proliferate is largely unknown. Together, neoblast lineage development, subclasses, and cell hierarchies are becoming elucidated, showing the complex regulation required for proper tissue homeostasis and regeneration in planarians.

A mex3 homolog is required for differentiation during planarian stem cell lineage development.

Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment.

The Retinoblastoma pathway regulates stem cell proliferation in freshwater planarians.

Freshwater planarians are flatworms of the Lophotrochozoan superphylum and are well known for their regenerative abilities, which rely on a large population of pluripotent adult stem cells. However, the mechanisms by which planarians maintain a precise population of adult stem cells while balancing proliferation and cell death, remain to be elucidated. Here we have identified, characterized, and functionally tested the core Retinoblastoma (Rb) pathway components in planarian adult stem cell biology. The Rb pathway is an ancient and conserved mechanism of proliferation control from plants to animals and is composed of three core components: an Rb protein, and a transcription factor heterodimer of E2F and DP proteins. Although the planarian genome contains all components of the Rb pathway, we found that they have undergone gene loss from the ancestral state, similar to other species in their phylum. The single Rb homolog (Smed-Rb) was highly expressed in planarian stem cells and was required for stem cell maintenance, similar to the Rb-homologs p107 and p130 in vertebrates. We show that planarians and their phylum have undergone the most severe reduction in E2F genes observed thus far, and the single remaining E2F was predicted to be a repressive-type E2F (Smed-E2F4-1). Knockdown of either Smed-E2F4-1 or its dimerization partner Dp (Smed-Dp) by RNAi resulted in temporary hyper-proliferation. Finally, we showed that known Rb-interacting genes in other systems, histone deacetylase 1 and cyclinD (Smed-HDAC1; Smed-cycD), were similar to Rb in expression and phenotypes when knocked down by RNAi, suggesting that these established interactions with Rb may also be conserved in planarians. Together, these results showed that planarians use the conserved components of the Rb tumor suppressor pathway to control proliferation and cell survival.

A comparative transcriptomic analysis reveals conserved features of stem cell pluripotency in planarians and mammals.

Many long-lived species of animals require the function of adult stem cells throughout their lives. However, the transcriptomes of stem cells in invertebrates and vertebrates have not been compared, and consequently, ancestral regulatory circuits that control stem cell populations remain poorly defined. In this study, we have used data from high-throughput RNA sequencing to compare the transcriptomes of pluripotent adult stem cells from planarians with the transcriptomes of human and mouse pluripotent embryonic stem cells. From a stringently defined set of 4,432 orthologs shared between planarians, mice and humans, we identified 123 conserved genes that are ≥5-fold differentially expressed in stem cells from all three species. Guided by this gene set, we used RNAi screening in adult planarians to discover novel stem cell regulators, which we found to affect the stem cell-associated functions of tissue homeostasis, regeneration, and stem cell maintenance. Examples of genes that disrupted these processes included the orthologs of TBL3, PSD12, TTC27, and RACK1. From these analyses, we concluded that by comparing stem cell transcriptomes from diverse species, it is possible to uncover conserved factors that function in stem cell biology. These results provide insights into which genes comprised the ancestral circuitry underlying the control of stem cell self-renewal and pluripotency.

Knockdown of SPARC leads to decreased cell-cell adhesion and lens cataracts during post-gastrula development in Xenopus laevis.

SPARC is a multifunctional matricellular glycoprotein with complex, transient tissue distribution during embryonic development. In Xenopus laevis embryos, zygotic activation of SPARC is first detected during late gastrulation, undergoing rapid changes in its spatiotemporal distribution throughout organogenesis. Injections of anti-sense Xenopus SPARC morpholinos (XSMOs) into 2- and 4-cell embryos led to a dose-dependent dissociation of embryos during neurula and tailbud stages of development. Animal cap explants derived from XSMO-injected embryos also dissociated, resulting in the formation of amorphous ciliated microspheres. At low doses of XSMOs, lens cataracts were formed, phenocopying that observed in Sparc-null mice. At XSMOs concentrations that did not result in a loss of axial tissue integrity, adhesion between myotomes at intersomitic borders was compromised with a reduction in SPARC concentration. The combined data suggest a critical requirement for SPARC during post-gastrula development in Xenopus embryos and that SPARC, directly or indirectly, promotes cell-cell adhesion in vivo.

RET-mediated gene expression pattern is affected by isoform but not oncogenic mutation.

The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) is caused by mutations of the RET receptor tyrosine kinase and is characterized by medullary thyroid carcinoma. MEN 2 subtypes have distinct mutational spectrums and vary in severity. The most severe disease subtype, MEN 2B, is associated with a specific RET mutation (M918T) that has been predicted to alter downstream signaling and target gene expression patterns. We used gene expression microarray analysis to identify target genes modulated by RET. We compared two oncogenic RET mutants, associated with MEN 2A (2ARET) or MEN 2B (2BRET) disease subtypes, that are predicted to have distinct downstream target genes. We showed that overall, 2ARET and 2BRET modulated genes with similar functional ontologies. Further, when we validated our microarray data by quantitative real time PCR, we did not detect major differences in gene expression associated with these mutants when differences in receptor activity levels were considered. We did, however, detect differences in gene expression induced by two RET COOH-terminal isoforms, RET9 and RET51, irrespective of the RET form present (wildtype, 2ARET, or 2BRET). Our data suggest that similar transcriptional programs contribute to all forms of MEN 2 but that differences in target gene expression may contribute to developmental pattern differences observed between RET isoforms.