The Maresin 1-LGR6 axis decreases respiratory syncytial virus-induced lung inflammation.

The resolution of infection is an active process with specific molecular and cellular mechanisms that temper inflammation and enhance pathogen clearance. Here, the specialized pro-resolving mediator (SPM) Maresin 1 (MaR1) inhibited respiratory syncytial virus (RSV)-induced inflammation. inlerleukin-13 production from type 2 innate lymphoid cells (ILC) and CD4 T helper type 2 cells was decreased by exogenous MaR1. In addition, MaR1 increased amphiregulin production and decreased RSV viral transcripts to promote resolution. MaR1 also promoted interferon-ß production in mouse lung tissues and also in pediatric lung slices. MaR1 significantly inhibited the RSV-triggered aberrant inflammatory phenotype in FoxP3-expressing Tregs. The receptor for MaR1, leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6), was constitutively expressed on Tregs. Following RSV infection, mice lacking Lgr6 had exacerbated type 2 immune responses with an increased viral burden and blunted responses to MaR1. Together, these findings have uncovered a multi-pronged protective signaling axis for MaR1-Lgr6, improving Tregs's suppressive function and upregulating host antiviral genes resulting in decreased viral burden and pathogen-mediated inflammation, ultimately promoting restoration of airway mucosal homeostasis.

Lgr6 marks nail stem cells and is required for digit tip regeneration.

The tips of the digits of some mammals, including human infants and mice, are capable of complete regeneration after injury. This process is reliant on the presence of the overlaying nail organ and is mediated by a proliferative blastema. Epithelial Wnt/ß-catenin signaling has been shown to be necessary for mouse digit tip regeneration. Here, we report on Lgr5 and Lgr6 (leucine-rich repeat-containing G protein-coupled receptor 5 and 6), two important agonists of the Wnt pathway that are known to be markers of several epithelial stem cell populations. We find that Lgr5 is expressed in a dermal population of cells adjacent to the specialized epithelia surrounding the keratinized nail plate. Moreover, Lgr5-expressing cells contribute to this dermis, but not the blastema, during digit tip regeneration. In contrast, we find that Lgr6 is expressed within cells of the nail matrix portion of the nail epithelium, as well as in a subset of cells in the bone and eccrine sweat glands. Genetic lineage analysis reveals that Lgr6-expressing cells give rise to the nail during homeostatic growth, demonstrating that Lgr6 is a marker of nail stem cells. Moreover, Lgr6-expressing cells contribute to the blastema, suggesting a potential direct role for Lgr6-expressing cells during digit tip regeneration. This role is confirmed by analysis of Lgr6-deficient mice, which have both a nail and bone regeneration defect.

Are fingernails a key to unlocking the puzzle of mammalian limb regeneration?

Some mammalian digit tips, including those of mice and human children, can regenerate following amputation, whereas mammalian limb regeneration does not occur. One major difference between the digit tip and the rest of the limb is the presence of the nail, which is necessary for this type of regeneration. This couples well with the finding that canonical Wnt signalling and Lgr6, an agonist of Wnt signalling that marks nail stem cells, are necessary for digit tip regeneration. This viewpoint essay discusses the role of the nail in digit tip regeneration and explores whether nail stem cells and their presumptive niche can be solely accountable for why regeneration is possible in the digit tip, but not the rest of the limb.

A novel intergenic ETnII-ß insertion mutation causes multiple malformations in polypodia mice.

Mouse early transposon insertions are responsible for ~10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd), a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-ß early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5' LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development.

Inducible genetic system for the axolotl.

Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.

Mouse digit tip regeneration is mediated by fate-restricted progenitor cells.

Regeneration of appendages is frequent among invertebrates as well as some vertebrates. However, in mammals this has been largely relegated to digit tip regeneration, as found in mice and humans. The regenerated structures are formed from a mound of undifferentiated cells called a blastema, found just below the site of amputation. The blastema ultimately gives rise to all of the tissues in the regenerate, excluding the epidermis, and has classically been thought of as a homogenous pool of pluripotent stem cells derived by dedifferentiation of stump tissue, although this has never been directly tested in the context of mammalian digit tip regeneration. Successful digit tip regeneration requires that the level of amputation be within the nail bed and depends on expression of Msx1. Because Msx1 is strongly expressed in the nail bed mesenchyme, it has been proposed that the Msx1-expressing cells represent a pluripotent cell population for the regenerating digit. In this report, we show that Msx1 is dynamically expressed during digit tip regeneration, and it does not mark a pluripotent stem cell population. Moreover, we show that both the ectoderm and mesoderm contain fate-restricted progenitor populations that work in concert to regenerate their own lineages within the digit tip, supporting the hypothesis that the blastema is a heterogeneous pool of progenitor cells.

Regeneration-specific promoter switching facilitates Mest expression in the mouse digit tip to modulate neutrophil response.

The mouse digit tip regenerates following amputation, a process mediated by a cellularly heterogeneous blastema. We previously found the gene Mest to be highly expressed in mesenchymal cells of the blastema and a strong candidate pro-regenerative gene. We now show Mest digit expression is regeneration-specific and not upregulated in post-amputation fibrosing proximal digits. Mest homozygous knockout mice exhibit delayed bone regeneration though no phenotype is found in paternal knockout mice, inconsistent with the defined maternal genomic imprinting of Mest. We demonstrate that promoter switching, not loss of imprinting, regulates biallelic Mest expression in the blastema and does not occur during embryogenesis, indicating a regeneration-specific mechanism. Requirement for Mest expression is tied to modulating neutrophil response, as revealed by scRNAseq and FACS comparing wildtype and knockout blastemas. Collectively, the imprinted gene Mest is required for proper digit tip regeneration and its blastema expression is facilitated by promoter switching for biallelic expression.

Toeing the line between regeneration and fibrosis.

Understanding the remarkable capacity of vertebrates to naturally regenerate injured body parts has great importance for potential translation into human therapeutic applications. As compared to other vertebrates, mammals have low regenerative capacity for composite tissues like the limb. However, some primates and rodents can regenerate the distal tips of their digits following amputation, indicating that at least very distal mammalian limb tissues are competent for innate regeneration. It follows that successful digit tip regenerative outcome is highly dependent on the location of the amputation; those proximal to the position of the nail organ do not regenerate and result in fibrosis. This distal regeneration versus proximal fibrosis duality of the mouse digit tip serves as a powerful model to investigate the driving factors in determining each process. In this review, we present the current understanding of distal digit tip regeneration in the context of cellular heterogeneity and the potential for different cell types to function as progenitor cells, in pro-regenerative signaling, or in moderating fibrosis. We then go on to discuss these themes in the context of what is known about proximal digit fibrosis, towards generating hypotheses for these distinct healing processes in the distal and proximal mouse digit.

Skin-type-dependent development of murine mechanosensory neurons.

Mechanosensory neurons innervating the skin underlie our sense of touch. Fast-conducting, rapidly adapting mechanoreceptors innervating glabrous (non-hairy) skin form Meissner corpuscles, while in hairy skin, they associate with hair follicles, forming longitudinal lanceolate endings. How mechanoreceptors develop axonal endings appropriate for their skin targets is unknown. We report that mechanoreceptor morphologies across different skin regions are indistinguishable during early development but diverge post-natally, in parallel with skin maturation. Neurons terminating along the glabrous and hairy skin border exhibit hybrid morphologies, forming both Meissner corpuscles and lanceolate endings. Additionally, molecular profiles of neonatal glabrous and hairy skin-innervating neurons largely overlap. In mouse mutants with ectopic glabrous skin, mechanosensory neurons form end-organs appropriate for the altered skin type. Finally, BMP5 and BMP7 are enriched in glabrous skin, and signaling through type I bone morphogenetic protein (BMP) receptors in neurons is critical for Meissner corpuscle morphology. Thus, mechanoreceptor morphogenesis is flexibly instructed by target tissues.

Dynamic expression of two thrombospondins during axolotl limb regeneration.

The molecular processes underlying regeneration remain largely unknown. Several potential factors have been elucidated by focusing on the regenerative function of genes originally identified in a developmental context. A complementary approach is to consider the roles of factors involved in wound healing. Here we focus on the Thrombospondins, a family of secreted extracellular matrix proteins that have been implicated in skin wound healing in mammals. We show that a subset of Thrombospondins are expressed at distinct times and in particular cell types during axolotl limb regeneration. Our studies have revealed the axolotl orthologs of thrombospondin-1 (tsp-1) and thrombospondin-4 (tsp-4) are highly upregulated during limb regeneration in patterns both distinct and similar to larval limb development. Our data suggest that thrombospondins may be key regulators of limb regeneration in axolotl, while their activation appears to be relegated solely to wound healing in vertebrates that have lost the ability to regenerate limbs.

Mouse LGR6 regulates osteogenesis in vitro and in vivo through differential ligand use.

Leucine-rich repeat containing G-protein-coupled receptor 6 (LGR6) is a marker of osteoprogenitor cells and is dynamically expressed during in vitro osteodifferentation of mouse and human mesenchymal stem cells (MSCs). While the Lgr6 genomic locus has been associated with osteoporosis in human cohorts, the precise molecular function of LGR6 in osteogenesis and maintenance of bone mass are not yet known. In this study, we performed in vitro Lgr6 knockdown and overexpression experiments in murine osteoblastic cells and find decreased Lgr6 levels results in reduced osteoblast proliferation, differentiation, and mineralization. Consistent with these data, overexpression of Lgr6 in these cells leads to significantly increased proliferation and osteodifferentiation. To determine whether these findings are recapitulated in vivo, we performed microCT and ex vivo osteodifferentiation analyses using our newly generated CRISPR-Cas9 mediated Lgr6 mouse knockout allele (Lgr6-KO). We find that ex vivo osteodifferentiation of Lgr6-KO primary MSCs is significantly reduced, and 8 week-old Lgr6-KO mice have less trabecular bone mass as compared to Lgr6 wildtype controls, indicating that Lgr6 is necessary for normal osteogenesis and bone mass. Towards mechanism, we analyzed in vitro signaling in the context of two LGR6 ligands, RSPO2 and MaR1. We find that RSPO2 stimulates LGR6-mediated WNT/ß-catenin signaling whereas MaR1 stimulates LGR6-mediated cAMP activity, suggesting two ligand-dependent functions for LGR6 receptor signaling during osteogenesis. Collectively, this study reveals that Lgr6 is necessary for wildtype levels of proliferation and differentiation of osteoblasts, and achieving normal bone mass.

Mammalian Digit Tip Regeneration: Moving from Phenomenon to Molecular Mechanism.

In this review, we present the current state of knowledge surrounding mammalian digit tip regeneration. We discuss the origin and formation of the blastema, a structure integral to digit tip regeneration, as well as recent insights driven by single-cell RNA sequencing into the molecular markers and cellular composition of the blastema. The digit tip is a composite of many different tissue types and we address what is known about the role of these separate tissues in regeneration of the whole digit tip. Specifically, we discuss the most extensively studied tissues in the digit tip: bone, nail epithelium, and peripheral nerves. We also address how known molecular pathways in limb development can inform research into digit tip regeneration. Overall, the mouse digit tip is an excellent model of complex mammalian regeneration that can provide insight into inducing regeneration in human tissues.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Cellular Heterogeneity and Lineage Restriction during Mouse Digit Tip Regeneration at Single-Cell Resolution.

Innate regeneration following digit tip amputation is one of the few examples of epimorphic regeneration in mammals. Digit tip regeneration is mediated by the blastema, the same structure invoked during limb regeneration in some lower vertebrates. By genetic lineage analyses, the digit tip blastema has been defined as a population of heterogeneous, lineage-restricted progenitor cells. These previous studies, however, do not comprehensively evaluate blastema heterogeneity or address lineage restriction of closely related cell types. In this report, we present single-cell RNA sequencing of over 38,000 cells from mouse digit tip blastemas and unamputated control digit tips and generate an atlas of the cell types participating in digit tip regeneration. We computationally define differentiation trajectories of vascular, monocytic, and fibroblastic lineages over regeneration, and while our data confirm broad lineage restriction of progenitors, our analysis reveals 67 genes enriched in blastema fibroblasts including a novel regeneration-specific gene, Mest.

Cellular Plasticity in Musculoskeletal Development, Regeneration, and Disease.

In this review, we highlight themes from a recent workshop focused on "Plasticity of Cell Fate in Musculoskeletal Tissues" held at the Orthopaedic Research Society's 2019 annual meeting. Experts in the field provided examples of mesenchymal cell plasticity during normal musculoskeletal development, regeneration, and disease. A thorough understanding of the biology underpinning mesenchymal cell plasticity may offer a roadmap for promoting regeneration while attenuating pathologic differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:708-718, 2020.

Evidence for Lgr6 as a Novel Marker of Osteoblastic Progenitors in Mice.

Bone marrow-derived mesenchymal stem cells are an important source of osteoblasts critical for both bone homeostasis and repair. The ability to isolate, or specifically target, mesenchymal stem cells committed to the osteogenic lineage is necessary for orthopedic translational therapy efforts; however the precise molecular signature of these cells remains elusive. Previously, we identified a population of osteoprogenitor cells expressing the Wnt signaling agonist Lgr6, which contributes to the development and regeneration of the mouse digit tip bone. In our present study we build upon this data and investigate the expression of Lgr6 more broadly in the skeleton. We find that Lgr6, and closely related Lgr4, are expressed in mouse primary calvarial cells, bone marrow cells, and bone marrow-derived mesenchymal stem cells. In addition, our data demonstrates that Lgr4 expression is modestly increased throughout the differentiation and mineralization of mesenchymal stem cells. In contrast, we find Lgr6 expression to be strikingly increased upon osteogenic induction and subsequently decreased upon differentiation and mineralization. These findings provide evidence for Lgr6 as a novel marker of osteoprogenitor cells in bone marrow, which could prove useful for isolation of this population toward future research and clinical applications.

BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood gene expression in mouse embryonic distal limbs and genital bud.

We previously demonstrated that a approximately 1 Mb domain of genes upstream of and including Hoxa13 is co-expressed in the developing mouse limbs and genitalia. A highly conserved non-coding sequence, mmA13CNS, was shown to be insufficient in transgenic mice to direct precise Hoxa13-like expression in the limb buds or genital bud, although some LacZ expression from the transgene was reproducibly found in these tissues. In this report, we used beta-globin minimal promoter LacZ recombinant BAC transgenes encompassing mmA13CNS to identify a single critical region involved in mouse Hoxa13-like embryonic genital bud expression. By analyzing the expression patterns of these overlapping BAC clones in transgenic mice, we show that at least two sequences remote to the HoxA cluster are required collectively to drive Hoxa13-like expression in developing distal limbs. Given that the paralogous posterior HoxD and neighboring genes have been shown to be under the influence of long-range distal limb and genital bud enhancers, we hypothesize that both long-range enhancers have one ancestral origin, which diverged in both sequence and function after the HoxA/D cluster duplication.

Expanded HOXA13 polyalanine tracts in a monotreme.

The N-terminal region of human HOXA13 has seven discrete polyalanine tracts. Our previous analysis of these tracts in multiple major vertebrate clades suggested that three are mammal-specific. We now report the N-terminal HOXA13 repetitive tract structures in the monotreme Tachyglossus aculeatus (echidna). Contrary to our expectations, echidna HOXA13 possesses a unique set of polyalanine tracts and an unprecedented polyglycine tract. The data support the conclusion that the emergence of expanded polyalanine tracts in proteins occurred very early in the stem lineage that gave rise to mammals, between 162 and 315 Ma.

Digit Tip Regeneration and Beyond: Schwann Cell Progenitors to the Rescue.

Digit tip regeneration following amputation is an innate response in some mammals, including mice. In this issue of Cell Stem Cell, Johnston et al. (2016) show that Schwann cell precursors are necessary for this process and can rescue regeneration in denervated digit tips through secretion of pro-regenerative factors including OSM and PDGF-AA.