Secreted inhibitors drive the loss of regeneration competence in Xenopus limbs.

Absence of a specialized wound epidermis is hypothesized to block limb regeneration in higher vertebrates. However, the factors preventing its formation in regeneration-incompetent animals are poorly understood. To characterize the endogenous molecular and cellular regulators of specialized wound epidermis formation in Xenopus laevis tadpoles, and the loss of their regeneration competency during development, we used single-cell transcriptomics and ex vivo regenerating limb cultures. Transcriptomic analysis revealed that the specialized wound epidermis is not a novel cell state, but a re-deployment of the apical-ectodermal-ridge (AER) programme underlying limb development. Enrichment of secreted inhibitory factors, including Noggin, a morphogen expressed in developing cartilage/bone progenitor cells, are identified as key inhibitors of AER cell formation in regeneration-incompetent tadpoles. These factors can be overridden by Fgf10, which operates upstream of Noggin and blocks chondrogenesis. These results indicate that manipulation of the extracellular environment and/or chondrogenesis may provide a strategy to restore regeneration potential in higher vertebrates.

The myeloid lineage is required for the emergence of a regeneration-permissive environment following Xenopus tail amputation.

Regeneration-competent vertebrates are considered to suppress inflammation faster than non-regenerating ones. Hence, understanding the cellular mechanisms affected by immune cells and inflammation can help develop strategies to promote tissue repair and regeneration. Here, we took advantage of naturally occurring tail regeneration-competent and -incompetent developmental stages of Xenopus tadpoles. We first establish the essential role of the myeloid lineage for tail regeneration in the regeneration-competent tadpoles. We then reveal that upon tail amputation there is a myeloid lineage-dependent change in amputation-induced apoptosis levels, which in turn promotes tissue remodelling, and ultimately leads to the relocalization of the regeneration-organizing cells responsible for progenitor proliferation. These cellular mechanisms failed to be executed in regeneration-incompetent tadpoles. We demonstrate that regeneration incompetency is characterized by inflammatory myeloid cells whereas regeneration competency is associated with reparative myeloid cells. Moreover, treatment of regeneration-incompetent tadpoles with immune-suppressing drugs restores myeloid lineage-controlled cellular mechanisms. Collectively, our work reveals the effects of differential activation of the myeloid lineage on the creation of a regeneration-permissive environment and could be further exploited to devise strategies for regenerative medicine purposes.

Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival.

BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. METHOD: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. RESULTS: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. CONCLUSION: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma. Video Abstract.

To regenerate or not to regenerate: Vertebrate model organisms of regeneration-competency and -incompetency.

Why only certain species can regenerate their appendages (e.g. tails and limbs) remains one of the biggest mysteries of nature. Unlike anuran tadpoles and salamanders, humans and other mammals cannot regenerate their limbs, but can only regrow lost digit tips under specific circumstances. Numerous hypotheses have been postulated to explain regeneration-incompetency in mammals. By studying model organisms that show varying regenerative abilities, we now have more opportunities to uncover what contributes to regeneration-incompetency and functionally test which perturbations restore appendage regrowth. Particularly, Xenopus laevis tail and limb, and mouse digit tip model systems exhibit naturally occurring variations in regenerative capacities. Here, we discuss major hypotheses that are suggested to contribute to regeneration-incompetency, and how species with varying regenerative abilities reflect on these hypotheses.

Multi-species atlas resolves an axolotl limb development and regeneration paradox.

Humans and other tetrapods are considered to require apical-ectodermal-ridge (AER) cells for limb development, and AER-like cells are suggested to be re-formed to initiate limb regeneration. Paradoxically, the presence of AER in the axolotl, a primary model organism for regeneration, remains controversial. Here, by leveraging a single-cell transcriptomics-based multi-species atlas, composed of axolotl, human, mouse, chicken, and frog cells, we first establish that axolotls contain cells with AER characteristics. Further analyses and spatial transcriptomics reveal that axolotl limbs do not fully re-form AER cells during regeneration. Moreover, the axolotl mesoderm displays part of the AER machinery, revealing a program for limb (re)growth. These results clarify the debate about the axolotl AER and the extent to which the limb developmental program is recapitulated during regeneration.

Tissues and Cell Types of Appendage Regeneration: A Detailed Look at the Wound Epidermis and Its Specialized Forms.

Therapeutic implementation of human limb regeneration is a daring aim. Studying species that can regrow their lost appendages provides clues on how such a feat can be achieved in mammals. One of the unique features of regeneration-competent species lies in their ability to seal the amputation plane with a scar-free wound epithelium. Subsequently, this wound epithelium advances and becomes a specialized wound epidermis (WE) which is hypothesized to be the essential component of regenerative success. Recently, the WE and specialized WE terminologies have been used interchangeably. However, these tissues were historically separated, and contemporary limb regeneration studies have provided critical new information which allows us to distinguish them. Here, I will summarize tissue-level observations and recently identified cell types of WE and their specialized forms in different regeneration models.

Appendage regeneration is context dependent at the cellular level.

Species that can regrow their lost appendages have been studied with the ultimate aim of developing methods to enable human limb regeneration. These examinations highlight that appendage regeneration progresses through shared tissue stages and gene activities, leading to the assumption that appendage regeneration paradigms (e.g. tails and limbs) are the same or similar. However, recent research suggests these paradigms operate differently at the cellular level, despite sharing tissue descriptions and gene expressions. Here, collecting the findings from disparate studies, I argue appendage regeneration is context dependent at the cellular level; nonetheless, it requires (i) signalling centres, (ii) stem/progenitor cell types and (iii) a regeneration-permissive environment, and these three common cellular principles could be more suitable for cross-species/paradigm/age comparisons.

AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation.

BACKGROUND: The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown. RESULTS: We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes. CONCLUSIONS: Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.