Bone regeneration via skeletal cell lineage plasticity: All hands mobilized for emergencies: Quiescent mature skeletal cells can be activated in response to injury and robustly participate in bone regeneration through cellular plasticity.

An emerging concept is that quiescent mature skeletal cells provide an important cellular source for bone regeneration. It has long been considered that a small number of resident skeletal stem cells are solely responsible for the remarkable regenerative capacity of adult bones. However, recent in vivo lineage-tracing studies suggest that all stages of skeletal lineage cells, including dormant pre-adipocyte-like stromal cells in the marrow, osteoblast precursor cells on the bone surface and other stem and progenitor cells, are concomitantly recruited to the injury site and collectively participate in regeneration of the damaged skeletal structure. Lineage plasticity appears to play an important role in this process, by which mature skeletal cells can transform their identities into skeletal stem cell-like cells in response to injury. These highly malleable, long-living mature skeletal cells, readily available throughout postnatal life, might represent an ideal cellular resource that can be exploited for regenerative medicine.

Ultra-low frequency labeling of single cell lineages in Drosophila.

We describe a new methodology for genetically labeling single cell lineages in Drosophila called DMARCM. The system offers ultra-low frequency labeling, linear induction, consistent labeling among individuals and virtually no background signal. We compare this technique to an existing approach, which has been widely adopted. We demonstrate how application of DMARCM in the gastrointestinal epithelium permits the effects of labeling frequency on tumorigenic stem cell growth to be distinguished in an established tumor model.

Meristem development and activity in gametophytes of the model fern, Ceratopteris richardii.

Ceratopteris richardii is a model fern species widely used to analyze various developmental processes and their regulation in gametophytes. The form of mature C. richardii gametophytes depends on the activity of the marginal meristem, but knowledge on meristem formation and structure is limited. Therefore, we analyzed cellular events accompanying the development of gametophytes using cell lineage and proliferation analyses to explain the establishment and functioning of the marginal meristem. We show that: i) gametophytes are devoid of the apical initial cell or the apical cell-based meristem in the early developmental stages; ii) the cells that are predestined to form the marginal meristem divide according to a stable pattern; iii) only one transient initial cell is present in the marginal meristem, and the selection of a new functioning initial cell is related to a stable sequence of its divisions. Our results contribute to a better understanding of the developmental events underlying gametophyte growth and marginal meristem functioning in Ceratopteris. The principles, which were established in this study and enabled the identification of functioning initial cells, can be applied to analyze genetic and/or physiological mechanism(s) governing meristem maintenance in vascular plants, both in developmental and evolutionary contexts.

Noise-driven growth rate gain in clonal cellular populations.

Cellular populations in both nature and the laboratory are composed of phenotypically heterogeneous individuals that compete with each other resulting in complex population dynamics. Predicting population growth characteristics based on knowledge of heterogeneous single-cell dynamics remains challenging. By observing groups of cells for hundreds of generations at single-cell resolution, we reveal that growth noise causes clonal populations of Escherichia coli to double faster than the mean doubling time of their constituent single cells across a broad set of balanced-growth conditions. We show that the population-level growth rate gain as well as age structures of populations and of cell lineages in competition are predictable. Furthermore, we theoretically reveal that the growth rate gain can be linked with the relative entropy of lineage generation time distributions. Unexpectedly, we find an empirical linear relation between the means and the variances of generation times across conditions, which provides a general constraint on maximal growth rates. Together, these results demonstrate a fundamental benefit of noise for population growth, and identify a growth law that sets a "speed limit" for proliferation.

Patterning and morphogenesis of the intricate but stereotyped oikoplastic epidermis of the appendicularian, Oikopleura dioica.

Mechanisms for morphogenetic processes that generate complex patterns in a reproducible manner remain elusive. Live imaging provides a powerful tool to record cell behaviors. The appendicularian, Oikopleura dioica, is a planktonic tunicate that has a rapid developmental speed, small number of cells (less than 3500 cells in a juvenile), and a transparent body. The trunk epidermis, called the oikoplastic epithelium (OE), has elaborate cellular arrangements showing a complex pattern to secrete so-called "house" made of extracellular components. The OE is characterized by invariant number, size, and shape of the monolayer epithelial cells. Pattern formation is achieved during 5h of larval development without growth of the body, making this a suitable system for live imaging of a two-dimensional (2D) sheet. First, we subdivided the OE and defined several domains by cellular resolution, and systematically gave names to the constituent cells, since there is no variation among individuals. Time-lapse imaging of the epidermal cells revealed region-specific pattern formation processes. Each identified domain served as a compartment into which distribution of descendant cells of founder cells is restricted. Regulation of orientation, timing, and the number of rounds of cell divisions, but not cell death and migration, was a critical mechanism for determination of final cell arrangement and size. In addition, displacement of epithelial sheet plates was observed in the Eisen domain. Stem-cell-like cell divisions, whereby large mother stem cells generate a chain of small daughter cells, were involved in formation of the Nasse region and ventral sensory organ. These are the first examples of this kind of stem-cell-like cell division in deuterostomes. Furthermore, labeling of the left or right blastomere of the two-cell-stage embryo, which roughly gives rise to the left or right side of the body, respectively, revealed that the boundary of the descendant cells does not match with the midline of the trunk epidermis. Left and right descendants largely invade into the opposite side in an invariant way, suggesting the possibility that specification of the OE cell identities may occur later in development, most probably around hatching, and depending on cell position in the OE epithelial sheet. These detailed descriptions of OE patterning processes provide basic and essential information to analyze further cell behaviors in the generation of elaborate and intricate but stereotyped 2D cellular patterns in this advantageous model system for developmental and cell biological studies in chordates.

Drosophila intermediate neural progenitors produce lineage-dependent related series of diverse neurons.

Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain.

Mathematical study of pattern formation accompanied by heterocyst differentiation in multicellular cyanobacterium.

The filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest models of a multicellular system showing cellular differentiation. In nitrogen-deprived culture, undifferentiated vegetative cells differentiate into heterocysts at ~10-cell intervals along the cellular filament. As undifferentiated cells divide, the number of cells between heterocysts (segment length) increases, and a new heterocyst appears in the intermediate region. To understand how the heterocyst pattern is formed and maintained, we constructed a one-dimensional cellular automaton (CA) model of the heterocyst pattern formation. The dynamics of vegetative cells is modeled by a stochastic transition process including cell division, differentiation and increase of cell age (maturation). Cell division and differentiation depend on the time elapsed after the last cell division, the "cell age". The model dynamics was mathematically analyzed by a two-step Markov approximation. In the first step, we determined steady state of cell age distribution among vegetative cell population. In the second step, we determined steady state distribution of segment length among segment population. The analytical solution was consistent with the results of numerical simulations. We then compared the analytical solution with the experimental data, and quantitatively estimated the immeasurable intercellular kinetics. We found that differentiation is initially independent of cellular maturation, but becomes dependent on maturation as the pattern formation evolves. Our mathematical model and analysis enabled us to quantify the internal cellular dynamics at various stages of the heterocyst pattern formation.

Differentiation ability of Gli1+ cells during orthodontic tooth movement.

Orthodontic tooth movement (OTM) induces bone formation on the alveolar bone of the tension side; however, the mechanism of osteoblast differentiation is not fully understood. Gli1 is an essential transcription factor for hedgehog signaling and functions in undifferentiated cells during embryogenesis. In this study, we examined the differentiation of Gli1+ cells in the periodontal ligament (PDL) during OTM using a lineage-tracing analysis. After the final administration of tamoxifen for 2 days to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice, Gli1/Tomato+ cells were rarely observed near endomucin+ blood vessels in the PDL. Osteoblasts lining the alveolar bone did not exhibit Gli1/Tomato fluorescence. To move the first molar of iGli1/Tomato mice medially, nickel-titanium closed-coil springs were attached between the upper anterior alveolar bone and the first molar. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased along with numerous PCNA+ cells in the PDL of the tension side. As some Gli1/Tomato+ cells exhibited positive expression of osterix, an osteoblast differentiation marker, Gli1+ cells probably differentiated into osteoblast progenitor cells. On day 10, the newly formed bone labeled by calcein administration during OTM was detected on the surface of the original alveolar bone of the tension side. Gli1/Tomato+ cells expressing osterix localized to the surface of the newly formed bone. In contrast, in the PDL of the compression side, Gli1/Tomato+ cells proliferated before day 10 and expressed type I collagen, suggesting that the Gli1+ cells also differentiated into fibroblasts. Collectively, these results demonstrate that Gli1+ cells in the PDL can differentiate into osteoblasts at the tension side and may function in bone remodeling as well as fibril formation in the PDL during OTM.

Bone formation ability of Gli1+ cells in the periodontal ligament after tooth extraction.

During the process of socket healing after tooth extraction, osteoblasts appear in the tooth socket and form alveolar bone; however, the source of these osteoblasts is still uncertain. Recently, it has been demonstrated that cells expressing Gli1, a downstream factor of sonic hedgehog signaling, exhibit stem cell properties in the periodontal ligament (PDL). Therefore, in the present study, the differentiation ability of Gli1+-PDL cells after tooth extraction was analyzed using Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice. After the final administration of tamoxifen to iGli1/Tomato mice, Gli1/Tomato+ cells were rarely detected in the PDL. One day after the tooth extraction, although inflammatory cells appeared in the tooth socket, Periostin+ PDL-like tissues having a few Gli1/Tomato+ cells remained near the alveolar bone. Three days after the extraction, the number of Gli1/Tomato+ cells increased as evidenced by numerous PCNA+ cells in the socket. Some of these Gli1/Tomato+ cells expressed BMP4 and Phosphorylated (P)-Smad1/5/8. After seven days, the Osteopontin+ bone matrix was formed in the tooth socket apart from the alveolar bone. Many Gli1/Tomato+ osteoblasts that were positive for Runx2+ were arranged on the surface of the newly formed bone matrix. In the absence of Gli1+-PDL cells in Gli1-CreERT2/Rosa26-loxP-stop-loxP-tdDTA (iGli1/DTA) mice, the amount of newly formed bone matrix was significantly reduced in the tooth socket. Therefore, these results collectively suggest that Gli1+-PDL cells differentiate into osteoblasts to form the bone matrix in the tooth socket; thus, this differentiation might be regulated, at least in part, by bone morphogenetic protein (BMP) signaling.

Retroviral lineage analysis reveals dual contribution from ectodermal placodes and neural crest cells to avian olfactory sensory and GnRH neurons.

The origin of the neurons and glia in the olfactory system of vertebrates has been controversial, with different cell types attributed to being of ectodermal placode versus neural crest lineage, depending upon the species. Here, we use replication incompetent avian (RIA) retroviruses to perform prospective cell lineage analysis of either presumptive olfactory placode or neural crest cells during early development of the chick embryo. Surprisingly, the results reveal a dual contribution from both the olfactory placode and neural crest cells to sensory neurons in the nose and Gonadotropin Releasing Hormone (GnRH) neurons migrating to the olfactory bulb. We also confirm that olfactory ensheathing glia are solely derived from the neural crest. Finally, our results show that neural crest cells and olfactory placode cells contribute to p63 positive cells, likely to be basal stem cells of the olfactory epithelium. Taken together, these finding provide evidence for previously unknown contributions of neural crest cells to some cell types in the chick olfactory system and help resolve previous discrepancies in the literature.

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina.

Direct reprogramming of retinal Müller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Müller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Müller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.

Overview of MARCM-Related Technologies in Drosophila Neurobiological Research.

Mosaic analysis with a repressible cell marker (MARCM)-related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.

Growth plate skeletal stem cells and their transition from cartilage to bone.

The growth plate is an essential component of endochondral bone development. Not surprisingly, the growth plate and its surrounding structure, the perichondrium, contain a wealth of skeletal stem cells (SSCs) and progenitor cells that robustly contribute to bone development. Recent in vivo lineage-tracing studies using mouse genetic models provide substantial insight into the diversity and versatility of these skeletal stem and progenitor cell populations, particularly shedding light on the importance of the transition from cartilage to bone. Chondrocytes and perichondrial cells are inseparable twins that develop from condensing undifferentiated mesenchymal cells during the fetal stage; although morphologically and functionally distinct, these cells ultimately serve for the same goal, that is, to make bone bigger and stronger. Even in the postnatal stage, a small subset of growth plate chondrocytes can transform into osteoblasts and marrow stromal cells; this is in part fueled by a unique type of SSCs maintained in the resting zone of the growth plate, which continue to self-renew for the long term. Here, we discuss diverse skeletal stem and progenitor cell populations in the growth plate and the perichondrium and their transition from cartilage to bone.

A Cell Segmentation/Tracking Tool Based on Machine Learning.

The ability to gain quantifiable, single-cell data from time-lapse microscopy images is dependent upon cell segmentation and tracking. Here, we present a detailed protocol for obtaining quality time-lapse movies and introduce a method to identify (segment) and track cells based on machine learning techniques (Fiji's Trainable Weka Segmentation) and custom, open-source Python scripts. To provide a hands-on experience, we provide datasets obtained using the aforementioned protocol.