A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

Nail-associated mesenchymal cells contribute to and are essential for dorsal digit tip regeneration.

Here, we ask why the nail base is essential for mammalian digit tip regeneration, focusing on the inductive nail mesenchyme. We identify a transcriptional signature for these cells that includes Lmx1b and show that the Lmx1b-expressing nail mesenchyme is essential for blastema formation. We use a combination of Lmx1bCreERT2-based lineage-tracing and single-cell transcriptional analyses to show that the nail mesenchyme contributes cells for two pro-regenerative mechanisms. One group of cells maintains their identity and regenerates the new nail mesenchyme. A second group contributes specifically to the dorsal blastema, loses their nail mesenchyme phenotype, acquires a blastema transcriptional state that is highly similar to blastema cells of other origins, and ultimately contributes to regeneration of the dorsal but not ventral dermis and bone. Thus, the regenerative necessity for an intact nail base is explained, at least in part, by a requirement for the inductive nail mesenchyme.

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.

Restoration of hippocampal neural precursor function by ablation of senescent cells in the aging stem cell niche.

Senescent cells are responsible, in part, for tissue decline during aging. Here, we focused on CNS neural precursor cells (NPCs) to ask if this is because senescent cells in stem cell niches impair precursor-mediated tissue maintenance. We demonstrate an aging-dependent accumulation of senescent cells, largely senescent NPCs, within the hippocampal stem cell niche coincident with declining adult neurogenesis. Pharmacological ablation of senescent cells via acute systemic administration of the senolytic drug ABT-263 (Navitoclax) caused a rapid increase in NPC proliferation and neurogenesis. Genetic ablation of senescent cells similarly activated hippocampal NPCs. This acute burst of neurogenesis had long-term effects in middle-aged mice. One month post-ABT-263, adult-born hippocampal neuron numbers increased and hippocampus-dependent spatial memory was enhanced. These data support a model where senescent niche cells negatively influence neighboring non-senescent NPCs during aging, and ablation of these senescent cells partially restores neurogenesis and hippocampus-dependent cognition.

A finger on the pulse of regeneration: insights into the cellular mechanisms of adult digit tip regeneration.

In mammals, multi-tissue regeneration is largely restricted to the distal portion of the digit tip and involves the formation of a blastema, a transient, proliferating cell mass that reforms the diverse tissues of the digit. Historically little was known about the mammalian blastema but with recent advances in single cell transcriptomic approaches and genetic lineage tracing, a more precise understanding of this critical structure has begun to emerge. In this review we summarise the cellular mechanisms underlying adult mammalian digit tip regeneration. We posit that understanding how some mammals naturally regenerate complex tissues will lead to strategies for enhancing regenerative abilities in humans.

Introductions to the community: Early-career researchers in the time of COVID-19.

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.

Cellular and molecular mechanisms that regulate mammalian digit tip regeneration.

Digit tip regeneration is one of the few examples of true multi-tissue regeneration in an adult mammal. The key step in this process is the formation of the blastema, a transient proliferating cell mass that generates the different cell types of the digit to replicate the original structure. Failure to form the blastema results in a lack of regeneration and has been postulated to be the reason why mammalian limbs cannot regrow following amputation. Understanding how the blastema forms and functions will help us to determine what is required for mammalian regeneration to occur and will provide insights into potential therapies for mammalian tissue regeneration and repair. This review summarizes the cellular and molecular mechanisms that influence murine blastema formation and govern digit tip regeneration.

Single-Cell Profiling Shows Murine Forebrain Neural Stem Cells Reacquire a Developmental State when Activated for Adult Neurogenesis.

The transitions from developing to adult quiescent and activated neural stem cells (NSCs) are not well understood. Here, we use single-cell transcriptional profiling and lineage tracing to characterize these transitions in the murine forebrain. We show that the two forebrain NSC parental populations, embryonic cortex and ganglionic eminence radial precursors (RPs), are highly similar even though they make glutamatergic versus gabaergic neurons. Both RP populations progress linearly to transition from a highly active embryonic to a dormant adult stem cell state that still shares many similarities with embryonic RPs. When adult NSCs of either embryonic origin become reactivated to make gabaergic neurons, they acquire a developing ganglionic eminence RP-like identity. Thus, transitions from embryonic RPs to adult NSCs and back to neuronal progenitors do not involve fundamental changes in cell identity, but rather reflect conversions between activated and dormant NSC states that may be determined by the niche environment.

Peripheral Nerve Single-Cell Analysis Identifies Mesenchymal Ligands that Promote Axonal Growth.

Peripheral nerves provide a supportive growth environment for developing and regenerating axons and are essential for maintenance and repair of many non-neural tissues. This capacity has largely been ascribed to paracrine factors secreted by nerve-resident Schwann cells. Here, we used single-cell transcriptional profiling to identify ligands made by different injured rodent nerve cell types and have combined this with cell-surface mass spectrometry to computationally model potential paracrine interactions with peripheral neurons. These analyses show that peripheral nerves make many ligands predicted to act on peripheral and CNS neurons, including known and previously uncharacterized ligands. While Schwann cells are an important ligand source within injured nerves, more than half of the predicted ligands are made by nerve-resident mesenchymal cells, including the endoneurial cells most closely associated with peripheral axons. At least three of these mesenchymal ligands, ANGPT1, CCL11, and VEGFC, promote growth when locally applied on sympathetic axons. These data therefore identify an unexpected paracrine role for nerve mesenchymal cells and suggest that multiple cell types contribute to creating a highly pro-growth environment for peripheral axons.

Acquisition of a Unique Mesenchymal Precursor-like Blastema State Underlies Successful Adult Mammalian Digit Tip Regeneration.

Here, we investigate the origin and nature of blastema cells that regenerate the adult murine digit tip. We show that Pdgfra-expressing mesenchymal cells in uninjured digits establish the regenerative blastema and are essential for regeneration. Single-cell profiling shows that the mesenchymal blastema cells are distinct from both uninjured digit and embryonic limb or digit Pdgfra-positive cells. This unique blastema state is environmentally determined; dermal fibroblasts transplanted into the regenerative, but not non-regenerative, digit express blastema-state genes and contribute to bone regeneration. Moreover, lineage tracing with single-cell profiling indicates that endogenous osteoblasts or osteocytes acquire a blastema mesenchymal transcriptional state and contribute to both dermis and bone regeneration. Thus, mammalian digit tip regeneration occurs via a distinct adult mechanism where the regenerative environment promotes acquisition of a blastema state that enables cells from tissues such as bone to contribute to the regeneration of other mesenchymal tissues such as the dermis.

Schwann cell precursors contribute to skeletal formation during embryonic development in mice and zebrafish.

Immature multipotent embryonic peripheral glial cells, the Schwann cell precursors (SCPs), differentiate into melanocytes, parasympathetic neurons, chromaffin cells, and dental mesenchymal populations. Here, genetic lineage tracing revealed that, during murine embryonic development, some SCPs detach from nerve fibers to become mesenchymal cells, which differentiate further into chondrocytes and mature osteocytes. This occurred only during embryonic development, producing numerous craniofacial and trunk skeletal elements, without contributing to development of the appendicular skeleton. Formation of chondrocytes from SCPs also occurred in zebrafish, indicating evolutionary conservation. Our findings reveal multipotency of SCPs, providing a developmental link between the nervous system and skeleton.

Interleukin-6 Regulates Adult Neural Stem Cell Numbers during Normal and Abnormal Post-natal Development.

Circulating systemic factors can regulate adult neural stem cell (NSC) biology, but the identity of these circulating cues is still being defined. Here, we have focused on the cytokine interleukin-6 (IL-6), since increased circulating levels of IL-6 are associated with neural pathologies such as autism and bipolar disorder. We show that IL-6 promotes proliferation of post-natal murine forebrain NSCs and that, when the IL-6 receptor is inducibly knocked out in post-natal or adult neural precursors, this causes a long-term decrease in forebrain NSCs. Moreover, a transient circulating surge of IL-6 in perinatal or adult mice causes an acute increase in neural precursor proliferation followed by long-term depletion of adult NSC pools. Thus, IL-6 signaling is both necessary and sufficient for adult NSC self-renewal, and acute perturbations in circulating IL-6, as observed in many pathological situations, have long-lasting effects on the size of adult NSC pools.

The senescence-associated secretory phenotype induces cellular plasticity and tissue regeneration.

Senescence is a form of cell cycle arrest induced by stress such as DNA damage and oncogenes. However, while arrested, senescent cells secrete a variety of proteins collectively known as the senescence-associated secretory phenotype (SASP), which can reinforce the arrest and induce senescence in a paracrine manner. However, the SASP has also been shown to favor embryonic development, wound healing, and even tumor growth, suggesting more complex physiological roles than currently understood. Here we uncover timely new functions of the SASP in promoting a proregenerative response through the induction of cell plasticity and stemness. We show that primary mouse keratinocytes transiently exposed to the SASP exhibit increased expression of stem cell markers and regenerative capacity in vivo. However, prolonged exposure to the SASP causes a subsequent cell-intrinsic senescence arrest to counter the continued regenerative stimuli. Finally, by inducing senescence in single cells in vivo in the liver, we demonstrate that this activates tissue-specific expression of stem cell markers. Together, this work uncovers a primary and beneficial role for the SASP in promoting cell plasticity and tissue regeneration and introduces the concept that transient therapeutic delivery of senescent cells could be harnessed to drive tissue regeneration.

Detection of Senescence Markers During Mammalian Embryonic Development.

Senescence-associated ß-galactosidase (SAß-gal) is a convenient histological technique used to identify senescent cells. Its ease of use is helpful to initially screen and detect senescent cells in heterogeneous cell populations both in vitro and in vivo. However, SAß-gal staining is not an unequivocal marker of the senescent state, and diagnosis of such usually requires additional markers demonstrating an absence of proliferation and expression of cell-cycle inhibitors. Nonetheless, SAß-gal remains one of the most widely used biomarkers of senescent cells. Recently, by measuring SAß-gal activity, the expression of the cyclin-dependent kinase inhibitor p21 (waf1/cip1) and demonstrating a lack of proliferation, we identified senescent cells in the developing embryo. This chapter describes the methods for identifying cellular senescence in the embryo, detailing protocols for the detection of SAß-gal activity in both sections and at the whole mount level, and immunohistochemistry protocols for the detection of additional biomarkers of senescence.

Dedifferentiated Schwann Cell Precursors Secreting Paracrine Factors Are Required for Regeneration of the Mammalian Digit Tip.

Adult mammals have lost multi-tissue regenerative capacity, except for the distal digit, which is able to regenerate via mechanisms that remain largely unknown. Here, we show that, after adult mouse distal digit removal, nerve-associated Schwann cell precursors (SCPs) dedifferentiate and secrete growth factors that promote expansion of the blastema and digit regeneration. When SCPs were dysregulated or ablated, mesenchymal precursor proliferation in the blastema was decreased and nail and bone regeneration were impaired. Transplantation of exogenous SCPs rescued these regeneration defects. We found that SCPs secrete factors that promote self-renewal of mesenchymal precursors, and we used transcriptomic and proteomic analysis to define candidate factors. Two of these, oncostatin M (OSM) and platelet-derived growth factor AA (PDGF-AA), are made by SCPs in the regenerating digit and rescued the deficits in regeneration caused by loss of SCPs. As all peripheral tissues contain nerves, these results could have broad implications for mammalian tissue repair and regeneration.

Developing senescence to remodel the embryo.

Cellular senescence is an irreversible form of cell cycle arrest that has been linked to several pathological conditions. In particular, senescence can function as a tumor suppressor mechanism, but is also thought to contribute to organismal aging. Paradoxically however, through the secretion of various factors, collectively termed the senescence-associated secretory phenotype (SASP), senescent cells can also have tumor-promoting and tissue-remodeling functions. In addition, senescent cells can play beneficial roles in tissue repair and wound healing, and reconciling these contradictory features from an evolutionary standpoint has been challenging. Moreover, senescent cells had not previously been documented in non-pathological conditions. Recently however, 2 studies have identified cellular senescence as a programmed mechanism that contributes to tissue patterning and remodeling during normal embryonic development. These findings have significant implications for our understanding of cellular senescence and help to clarify the paradoxes and the evolutionary origin of this process.

Senescence is a developmental mechanism that contributes to embryonic growth and patterning.

Senescence is a form of cell-cycle arrest linked to tumor suppression and aging. However, it remains controversial and has not been documented in nonpathologic states. Here we describe senescence as a normal developmental mechanism found throughout the embryo, including the apical ectodermal ridge (AER) and the neural roof plate, two signaling centers in embryonic patterning. Embryonic senescent cells are nonproliferative and share features with oncogene-induced senescence (OIS), including expression of p21, p15, and mediators of the senescence-associated secretory phenotype (SASP). Interestingly, mice deficient in p21 have defects in embryonic senescence, AER maintenance, and patterning. Surprisingly, the underlying mesenchyme was identified as a source for senescence instruction in the AER, whereas the ultimate fate of these senescent cells is apoptosis and macrophage-mediated clearance. We propose that senescence is a normal programmed mechanism that plays instructive roles in development, and that OIS is an evolutionarily adapted reactivation of a developmental process.

Age-associated inflammation inhibits epidermal stem cell function.

Altered stem cell homeostasis is linked to organismal aging. However, the mechanisms involved remain poorly understood. Here we report novel alterations in hair follicle stem cells during skin aging, including increased numbers, decreased function, and an inability to tolerate stress. Performing high-throughput RNA sequencing on aging stem cells, cytokine arrays, and functional assays, we identify an age-associated imbalance in epidermal Jak-Stat signaling that inhibits stem cell function. Collectively, this study reveals a role for the aging epidermis in the disruption of cytokine and stem cell homeostasis, suggesting that stem cell decline during aging may be part of broader tumor-suppressive mechanisms.

High incidence of recurrent copy number variants in patients with isolated and syndromic Müllerian aplasia.

BACKGROUND: Congenital malformations involving the Müllerian ducts are observed in around 5% of infertile women. Complete aplasia of the uterus, cervix, and upper vagina, also termed Müllerian aplasia or Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, occurs with an incidence of around 1 in 4500 female births, and occurs in both isolated and syndromic forms. Previous reports have suggested that a proportion of cases, especially syndromic cases, are caused by variation in copy number at different genomic loci. METHODS: In order to obtain an overview of the contribution of copy number variation to both isolated and syndromic forms of Müllerian aplasia, copy number assays were performed in a series of 63 cases, of which 25 were syndromic and 38 isolated. RESULTS: A high incidence (9/63, 14%) of recurrent copy number variants in this cohort is reported here. These comprised four cases of microdeletion at 16p11.2, an autism susceptibility locus not previously associated with Müllerian aplasia, four cases of microdeletion at 17q12, and one case of a distal 22q11.2 microdeletion. Microdeletions at 16p11.2 and 17q12 were found in 4/38 (10.5%) cases with isolated Müllerian aplasia, and at 16p11.2, 17q12 and 22q11.2 (distal) in 5/25 cases (20%) with syndromic Müllerian aplasia. CONCLUSION: The finding of microdeletion at 16p11.2 in 2/38 (5%) of isolated and 2/25 (8%) of syndromic cases suggests a significant contribution of this copy number variant alone to the pathogenesis of Müllerian aplasia. Overall, the high incidence of recurrent copy number variants in all forms of Müllerian aplasia has implications for the understanding of the aetiopathogenesis of the condition, and for genetic counselling in families affected by it.

Genomic and genic deletions of the FOX gene cluster on 16q24.1 and inactivating mutations of FOXF1 cause alveolar capillary dysplasia and other malformations.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare, neonatally lethal developmental disorder of the lung with defining histologic abnormalities typically associated with multiple congenital anomalies (MCA). Using array CGH analysis, we have identified six overlapping microdeletions encompassing the FOX transcription factor gene cluster in chromosome 16q24.1q24.2 in patients with ACD/MPV and MCA. Subsequently, we have identified four different heterozygous mutations (frameshift, nonsense, and no-stop) in the candidate FOXF1 gene in unrelated patients with sporadic ACD/MPV and MCA. Custom-designed, high-resolution microarray analysis of additional ACD/MPV samples revealed one microdeletion harboring FOXF1 and two distinct microdeletions upstream of FOXF1, implicating a position effect. DNA sequence analysis revealed that in six of nine deletions, both breakpoints occurred in the portions of Alu elements showing eight to 43 base pairs of perfect microhomology, suggesting replication error Microhomology-Mediated Break-Induced Replication (MMBIR)/Fork Stalling and Template Switching (FoSTeS) as a mechanism of their formation. In contrast to the association of point mutations in FOXF1 with bowel malrotation, microdeletions of FOXF1 were associated with hypoplastic left heart syndrome and gastrointestinal atresias, probably due to haploinsufficiency for the neighboring FOXC2 and FOXL1 genes. These differences reveal the phenotypic consequences of gene alterations in cis.