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Salamanders serve as important tetrapod models for developmental, regeneration and evolutionary studies. An extensive molecular toolkit makes the Mexican axolotl ( Ambystoma mexicanum ) a key representative salamander for molecular investigations. Here we report the sequencing and assembly of the 32-gigabase-pair axolotl genome using an approach that combined long-read sequencing, optical mapping and development of a new genome assembler (MARVEL). We observed a size expansion of introns and intergenic regions, largely attributable to multiplication of long terminal repeat retroelements. We provide evidence that intron size in developmental genes is under constraint and that species-restricted genes may contribute to limb regeneration. The axolotl genome assembly does not contain the essential developmental gene Pax3 . However, mutation of the axolotl Pax3 paralogue Pax7 resulted in an axolotl phenotype that was similar to those seen in Pax3 −/− and Pax7 −/− mutant mice. The axolotl genome provides a rich biological resource for developmental and evolutionary studies. Sequencing and assembly of the 32-Gb genome of the Mexican axolotl reveals that it lacks the developmental gene Pax3 , which is essential in other vertebrates; the genome sequence could improve our understanding of the evolution of the axolotl’s remarkable regenerative capabilities. Axolotl genome sequence Elly Tanaka, Eugene Myers and colleagues report the genome sequence of the axolotl, a model organism for developmental, regeneration and evolutionary studies. To sequence and assemble this large and complex genome, the authors used a combination of long- and short-read sequencing, optical mapping and a new genome assembly pipeline, MARVEL, optimized for long-read sequencing of complex genomes. The genome assembly shows an expansion of long terminal repeat retroelements and the presence of a large HoxA cluster, but also a reduction in the number of Pax-family genes in the genome of this popular salamander.

Gastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show that an early pulse of retinoic acid (RA), together with later Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites and diverse cell types, including neural crest, neural progenitors, renal progenitors and myocytes. Through in silico staging based on single-cell RNA sequencing, we find that human RA-gastruloids progress further than other human or mouse embryo models, aligning to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis. Hamazaki, Yang et al. report that an early pulse of retinoic acid robustly induces human gastruloids with a neural tube, segmented somites and more advanced cell types than conventional gastruloids.

Optimal cell-based therapies for the treatment of muscle degenerative disorders should not only regenerate fibers but provide a quiescent satellite cell pool ensuring long-term maintenance and regeneration. Conditional expression of Pax3/Pax7 in differentiating pluripotent stem cells (PSCs) allows the generation of myogenic progenitors endowed with enhanced regenerative capacity. To identify the molecular determinants underlying their regenerative potential, we performed transcriptome analyses of these cells along with primary myogenic cells from several developmental stages. Here we show that in vitro-generated PSC-derived myogenic progenitors possess a molecular signature similar to embryonic/fetal myoblasts. However, compared with fetal myoblasts, following transplantation they show superior myofiber engraftment and ability to seed the satellite cell niche, respond to multiple reinjuries, and contribute to long-term regeneration. Upon engraftment, the transcriptome of reisolated Pax3/Pax7–induced PSC-derived myogenic progenitors changes toward a postnatal molecular signature, particularly in genes involved in extracellular matrix remodeling. These findings demonstrate that Pax3/Pax7–induced myogenic progenitors remodel their molecular signature and functionally mature upon in vivo exposure to the adult muscle environment.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers. There is lack of therapies targeting the PAX3-FOXO1 fusion oncogene in fusion-positive rhabdomyosarcoma (FP-RMS). Here, the authors identify and characterise an inhibitor with highest inhibition of histone lysine demethylase 3B that suppresses PAX3-FOXO1 activity in FP-RMS.

Adult stem cells are essential for tissue homeostasis. In skeletal muscle, muscle stem cells (MuSCs) reside in a quiescent state, but little is known about the mechanisms that control homeostatic turnover. Here we show that, in mice, the variation in MuSC activation rate among different muscles (for example, limb versus diaphragm muscles) is determined by the levels of the transcription factor Pax3. We further show that Pax3 levels are controlled by alternative polyadenylation of its transcript, which is regulated by the small nucleolar RNA U1. Isoforms of the Pax3 messenger RNA that differ in their 3′ untranslated regions are differentially susceptible to regulation by microRNA miR206, which results in varying levels of the Pax3 protein in vivo. These findings highlight a previously unrecognized mechanism of the homeostatic regulation of stem cell fate by multiple RNA species.

Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl , a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb -like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates. The Iberian ribbed newt Pleurodeles waltl has a wide spectrum of regeneration abilities. Here, Elewa et al . sequence its ~20 Gb genome and transcriptome to investigate the molecular features underlying its regenerative capacities.

Rhabdomyosarcoma (RMS), a malignancy of impaired myogenic differentiation, is the most common soft tissue pediatric cancer. PAX3-FOXO1 oncofusions drive the majority of the clinically more aggressive fusion-positive rhabdomyosarcoma (FP-RMS). Recent studies have established an epigenetic basis for PAX3-FOXO1-driven oncogenic processes. However, details of PAX3-FOXO1 epigenetic mechanisms, including interactions with, and dependence on, other chromatin and transcription factors, are incompletely understood. We previously identified a novel disease-promoting epigenetic axis in RMS, involving the histone demethylase KDM3A and the ETS1 transcription factor, and demonstrated that this epigenetic axis interfaces with PAX3-FOXO1 both phenotypically and transcriptomically, including co-regulation of biological processes and genes important to FP-RMS progression. In this study, we demonstrate that KDM3A and ETS1 colocalize with PAX3-FOXO1 to enhancers of important disease-promoting genes in FP-RMS, including FGF8, IL4R, and MEST, as well as PODXL, which we define herein as a new FP-RMS-promoting gene. We show that ETS1, which is induced by both PAX3-FOXO1 and KDM3A, exists in complex with PAX3-FOXO1, and augments PAX3-FOXO1 chromatin occupancy. We further show that the PAX3-FOXO1/ETS1 complex can be disrupted by the clinically relevant small molecule inhibitor YK-4-279. YK-4-279 displaces PAX3-FOXO1 from chromatin and interferes with PAX3-FOXO1-dependent gene regulation, resulting in potent inhibition of growth and invasive properties in FP-RMS, along with downregulation of FGF8, IL4R, MEST and PODXL expression. We additionally show that, in some FP-RMS, KDM3A also increases PAX3-FOXO1 levels. Together, our studies illuminate mechanisms of action of the KDM3A/ETS1 regulatory module, and reveal novel targetable mechanisms of PAX3-FOXO1 chromatin complex regulation, in FP-RMS.

Canonical (H3.1/H3.2) and noncanonical (H3.3) histone 3 K27M-mutant gliomas have unique spatiotemporal distributions, partner alterations and molecular profiles. The contribution of the cell of origin to these differences has been challenging to uncouple from the oncogenic reprogramming induced by the mutation. Here, we perform an integrated analysis of 116 tumors, including single-cell transcriptome and chromatin accessibility, 3D chromatin architecture and epigenomic profiles, and show that K27M-mutant gliomas faithfully maintain chromatin configuration at developmental genes consistent with anatomically distinct oligodendrocyte precursor cells (OPCs). H3.3K27M thalamic gliomas map to prosomere 2-derived lineages. In turn, H3.1K27M ACVR1-mutant pontine gliomas uniformly mirror early ventral NKX6-1 + /SHH-dependent brainstem OPCs, whereas H3.3K27M gliomas frequently resemble dorsal PAX3 + /BMP-dependent progenitors. Our data suggest a context-specific vulnerability in H3.1K27M-mutant SHH-dependent ventral OPCs, which rely on acquisition of ACVR1 mutations to drive aberrant BMP signaling required for oncogenesis. The unifying action of K27M mutations is to restrict H3K27me3 at PRC2 landing sites, whereas other epigenetic changes are mainly contingent on the cell of origin chromatin state and cycling rate. Comprehensive genomic profiling of human H3K27M mutant diffuse midline gliomas, combining single-cell RNA sequencing and ATAC sequencing with bulk ChIP sequencing and other data, proposes distinct oligodendrocyte populations as potential cells of origin.

We report a case of a 67-year-old male patient with a sinonasal tumor that showed areas of classic biphenotypic sinonasal sarcoma (BSNS) which in some sections sharply transitioned into high-grade rhabdomyosarcoma. Immunohistochemically, the conventional BSNS parts showed S100 protein, SMA, PAX7, and focal MyoD1 expression, whereas desmin and myogenin were negative. In contrast, the cells in high-grade areas expressed desmin, MyoD1, myogenin, and PAX7, while being negative for S100 protein and SMA. Using the Archer FusionPlex assay, the classical PAX3::MAML3 gene fusion was detected. FISH for PAX3 and MAML3 confirmed a break of these genes in both components. Despite aggressive therapy, the tumor progression resulted in the patient’s death. The herein presented case, together with 2 previously published cases of BSNS with high-grade transformation, helps to better understand this novel phenomenon. Although the risk for such transformation appears low, it has important clinical and diagnostic implications which are discussed.

Waardenburg syndrome (WS), also known as auditory-pigmentary syndrome, is the most common cause of syndromic hearing loss (HL), which accounts for approximately 2–5% of all patients with congenital hearing loss. WS is classified into four subtypes depending on the clinical phenotypes. Currently, pathogenic mutations of PAX3, MITF, SOX10, EDN3, EDNRB or SNAI2 are associated with different subtypes of WS. Although supportive techniques like hearing aids, cochlear implants, or other assistive listening devices can alleviate the HL symptom, there is no cure for WS to date. Recently major progress has been achieved in preclinical studies of genetic HL in animal models, including gene delivery and stem cell replacement therapies. This review focuses on the current understandings of pathogenic mechanisms and potential biological therapeutic approaches for HL in WS, providing strategies and directions for implementing WS biological therapies, as well as possible problems to be faced, in the future.