Biology subjects

Zelzer, E.

Publications and source records attributed to Zelzer, E..

15 recordsLinked to original sources

bioRxiv2024

Wnt/β-Catenin coordinates muscle spindle development by regulating capsule differentiation, intrafusal fiber nuclei aggregation and proprioceptive nerve endings

Proprioception is essential for the regulation of posture, movement, and musculoskeletal integrity. The muscle spindle, a mechanosensory organ composed of multiple specialized tissues, detects stretch and provides proprioceptive feedback. Despite its importance, the molecular mechanisms that orchestrate the development of the spindle components remain poorly understood. Here, we reveal the involvement of the Wnt/{beta}-catenin pathway in muscle spindle development. We show that Wnt ligands and their Frizzled receptors are expressed in the developing spindle and that {beta}-catenin is active throughout development in capsule cells and in bag2 intrafusal fibers. Embryonic deletion of {beta}-catenin from capsule and intrafusal fibers induced widespread transcriptomic changes, which led to significant malformations, including impaired capsule cell differentiation, disrupted nuclear organization in intrafusal fibers, and disorganized proprioceptive nerve endings. Postnatal deletion of {beta}-catenin from intrafusal fibers resulted in abnormal nerve endings and impaired proprioceptive function, indicating that the development of proprioceptive afferents is regulated by {beta}-catenin through a non-cell-autonomous mechanism acting in bag2 fibers. Collectively, our findings position the Wnt/{beta}-catenin pathway as a central regulator that acts through both cell-autonomous and non-cell-autonomous mechanisms to coordinate the development of the various spindle tissues into a functional organ.

developmental biology↗

bioRxiv2024

Piezo2 in sensory neurons influences systemic and adipose tissue metabolism

Systemic metabolism ensures energy homeostasis through inter-organ crosstalk regulating thermogenic adipose tissue. Unlike the well-described inductive role of the sympathetic system, the inhibitory signal ensuring energy preservation remains poorly understood. Here, we show that, via the mechanosensor Piezo2, sensory neurons regulate morphological and physiological properties of brown and beige fat and prevent systemic hypermetabolism. Targeting Runx3/PV sensory neurons in independent genetic mouse models resulted in a systemic metabolic phenotype characterized by reduced body fat and increased insulin sensitivity and glucose tolerance. Deletion of Piezo2 in PV sensory neurons reproduced the phenotype, protected against high-fat diet-induced obesity and caused adipose tissue browning and beiging, likely driven by elevated norepinephrine levels. Finding that brown and beige fat are innervated by Runx3/PV sensory neurons expressing Piezo2, suggests a model where mechanical signals sensed by Piezo2 in sensory neurons protect energy storages and prevent a systemic metabolic phenotype. HighlightsLack of Runx3/PV sensory neurons reduces body fat and fasting glucose levels and increases glucose tolerance in mice Mechanosensitive ion channel PIEZO2 in PV sensory neurons plays an important role in systemic metabolism under physiological and pathological conditions PIEZO2 in PV sensory neurons regulates thermogenic programs and glucose uptake in brown and beige adipose tissues Brown and beige adipose tissues are innervated by Runx3/PV sensory neurons

physiology↗

bioRxiv2023

Bone elongation in the embryo occurs without column formation in the growth plate

Chondrocyte columns, which are a hallmark of growth plate architecture, play a central role in bone elongation. Columns are formed by clonal expansion following rotation of the division plane, resulting in a stack of cells oriented parallel to the growth direction. However, despite decades of research, column structure has thus far been studied only in two dimensions. To fill this knowledge gap, we analyzed hundreds of Confetti multicolor clones in growth plates of mouse embryos using a pipeline comprising 3D imaging and algorithms for morphometric analysis. Surprisingly, analysis of the elevation angles between neighboring pairs of cells revealed that most cells did not display the typical stacking pattern associated with column formation, implying incomplete rotation of the division plane. Morphological analysis revealed that although embryonic clones were elongated, they formed clusters oriented perpendicular to the growth direction. Analysis of growth plates of postnatal mice revealed both complex columns, composed of both ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Our finding that embryonic growth plates function without forming columns suggests that longitudinal bone growth is regulated by different cellular mechanisms during pre- and postnatal development. Moreover, the observed complex columnar and cluster arrangements may serve other, yet unknown morphogenetic functions. More broadly, our findings provide a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development.

developmental biology↗

bioRxiv2022

Molecular characterization of the intact muscle spindle using a multi-omics approach

The proprioceptive system is essential for the control of coordinated movement, posture and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease.

developmental biology↗

bioRxiv2022

Single-cell atlas of mouse limb development reveals a complex spatiotemporal dynamics of skeleton formation

Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern. Single-cell RNA sequencing of the developing mouse forelimb revealed three progenitor states: naive, proximal and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments. Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest a new model for limb skeleton development.

developmental biology↗

bioRxiv2022

The role of TGFβ signaling in Gli1+ tendon and enthesis cells

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGF{beta} signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGF{beta} signaling in Gli1+ cell function, the receptor for TGF{beta}, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGF{beta} signaling in these cells led to defects in enthesis formation by P56, including deficient bone morphometry underlying the enthesis and decreased mechanical properties. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGF{beta} controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGF{beta} directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

developmental biology↗

bioRxiv2022

A unique mineralizing pool of Gli1+ stem cells builds the tendon enthesis and demonstrates therapeutic potential

The enthesis, a fibrocartilaginous transition between tendon and bone, is necessary for the transfer of force from muscle to bone to produce joint motion. The enthesis is prone to injury due to mechanical demands, and it cannot regenerate. A better understanding of how the enthesis develops will lead to more effective therapies to prevent pathology and promote regeneration. Here, we used single-cell RNA sequencing to define the development transcriptome of the entheses over postnatal stages. Six resident cell types, including enthesis progenitors and mineralizing chondrocytes, were identified along with their transcription factor regulons and temporal regulation. Following our prior discovery of the necessity of Gli1-lineage cells for enthesis development and healing, we then examined their transcriptomes at single cell resolution and demonstrated their clonogenicity and multipotency. Transplantation of these Gli1- lineage cells to enthesis injuries improved healing, demonstrating their therapeutic potential for enthesis regeneration. HighlightsO_LIThe transcriptome and differentiation trajectory of enthesis stem cells during postnatal development are defined at single cell resolution. C_LIO_LITranscription factor regulons drive enthesis stem cell differentiation. C_LIO_LIGli1-lineage enthesis stem cells demonstrate in vivo and in vitro clonogenicity and multipotency. C_LIO_LITransplantation of Gli1-lineage enthesis stem cells to enthesis injuries improves healing. C_LI

developmental biology↗

bioRxiv2021

Neonatal enthesis healing involves non-inflammatory formation of acellular scar through ECM secretion by resident cells

Wound healing is a well-orchestrated process that typically recruits the immune and vascular systems to restore the structure and function of the injured tissue. Injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, thereby severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Here, we established an injury model to the Achilles entheses of neonatal mice to study the possibility that at an early age, the enthesis can heal more effectively. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, neonatal enthesis healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including ER stress, autophagy and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and TUNEL assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. Collectively, these findings reveal a novel healing mechanism in neonatal entheses, whereby local ECM secretion by resident cells forms an acellular ECM deposit in the absence of inflammation markers, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.

molecular biology↗

bioRxiv2020

3D MAPs discovers the morphological sequence chondrocytes undergo in the growth plate and the regulatory role of GDF5 in this process

The activity of the epiphyseal growth plates, which drive longitudinal growth of long bones, is dependent on the ability of chondrocytes to change their shape and size extensively as they differentiate. However, organ size, extracellular matrix density and cell number have hindered the study of chondrocyte morphology. Here, we describe a new pipeline called 3D Morphometric Analysis for Phenotypic significance (3D MAPs), which overcomes these obstacles. By using 3D MAPs, we have created an image database of hundreds of thousands of cells from orthologous long bones. Analysis of this database revealed the growth strategies that chondrocytes use during differentiation. We found that chondrocytes employed both allometric and isometric growth, and that allometric growth is achieved by changes either in volume or surface area along a specific cell axis in a zone-specific manner. Additionally, we discovered a new organization of chondrocytes within the growth plate, where cells are orientated such that their longest axis always aligns with the dorsal-ventral axis of the bone. To demonstrate the ability of 3D MAPs to explore mechanisms of growth plate regulation, we studied the abnormally short tibiae of Gdf5-null mice. 3D MAPs identified aberrant cellular growth behaviors which resulted in a 3-fold reduction in volumetric cell growth, as well as affected cell morphology and orientation, highlighting GDF5 as a new regulator of growth plate activity. Overall, our findings provide new insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover molecular and cellular mechanisms regulating this process. More broadly, this work provides a new framework for studying growth plate biology.

developmental biology↗

bioRxiv2020

BCKDK regulates the TCA cycle through PDC to ensure embryonic development in the absence of PDK family

Pyruvate dehydrogenase kinases (PDK1-4) inhibit the TCA cycle by phosphorylating pyruvate dehydrogenase complex (PDC). Here, we show that the PDK family is dispensable for the survival of murine embryonic development and that BCKDK serves as a compensatory mechanism by inactivating PDC. First, we knocked out all four Pdk genes one by one. Surprisingly, Pdk total KO embryos developed and were born in expected ratios, but died by postnatal day 4 due to hypoglycemia or ketoacidosis. Finding that PDC was phosphorylated in these embryos suggested that another kinase compensates for the PDK family. Bioinformatic analysis implicated brunch chain ketoacid dehydrogenase kinase (Bckdk), a key regulator of branched chain amino acids (BCAA) catabolism. Indeed, knockout of Bckdk and the Pdk family led to loss of PDC phosphorylation, increment in PDC activity, elevation of Pyruvate flux into the TCA and early embryonic lethality. These findings reveal a new regulatory crosstalk hardwiring BCAA and glucose catabolic pathways, which feed the TCA cycle.

molecular biology↗

bioRxiv2020

Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors

The connection between different tissues is vital for the development and function of any organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Kruppel-like factors transcription factors (KLF). To uncover the molecular identity of attachment cells, we first applied high-throughput RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ hybridization and single-molecule in situ hybridization, reveal that attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism allowing these cells to express these genes, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells. In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connect tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them.

developmental biology↗

bioRxiv2018

Multiscale analysis of 3D nuclear morphology reveals new insights into growth plate organization in mice

The shape of the nucleus is tightly associated with cell morphology, the mechanical environment, and differentiation and transcriptional states. Yet, imaging of nuclei in three dimensions while preserving the spatial context of the tissue has been highly challenging. Here, using the embryonic tibial growth plate as a model for cell differentiation, we study nuclear morphology by imaging cleared samples by light-sheet fluorescence microscopy. Next, we quickly segmented tens of thousands of nuclei using several open-source tools including machine learning. Finally, segmented nuclei underwent morphometric analysis and 3D spatial reconstruction using newly designed algorithms. Our method revealed differences in nuclear morphology between chondrocytes at different differentiation stages. Additionally, we identified different morphological patterns in opposing growth plates, such as gradients of volume and surface area, as well as features characteristic of specific growth plate zones, such as sphericity and orientation. Altogether, this work supports a link between nuclear morphology and cell differentiation. Moreover, it demonstrates the suitability of our approach for studying the relationships between nuclear morphology and organ development.\n\nAuthor summaryThere has been a growing interest in the relationship between nuclear morphology and its regulation of gene expression. However, to study global patterns of nuclear morphology within a tissue we must address the problem of acquiring and analyzing multiscale data, ranging from the tissue level through to subcellular resolution. We have established a new pipeline that enables acquisition and segmentation of hundreds of thousands of nuclei at a resolution that allows quantitative analysis. Moreover we have developed new algorithms that allow superimposing morphological aspects of hundreds of thousands of nuclei onto a visual representation of the entire tissue, allowing us to study nuclear morphology at an organ level. Using mouse growth plates as a model for the relationship between nuclear morphology and tissue differentiation, we show that nuclei change different aspects of their morphology during chondrocyte differentiation. Growth plates are usually described generically in the literature, suggesting they lack unique characteristics. We challenge this dogma by showing that morphological features such as volume distribute differently in opposing growth plates. Altogether, this work highlights the possible role of nuclear shape in the regulation of cell differentiation and demonstrates that our approach enables the study of nuclear morphology patterns within a tissue.

developmental biology↗

bioRxiv2018

Bone Morphology is Regulated Modularly by Global and Regional Genetic Programs

During skeletogenesis, a variety of protrusions of different shapes and sizes develop on the surfaces of long bones. These superstructures provide stable anchoring sites for ligaments and tendons during the assembly of the musculoskeletal system. Despite their importance, the mechanism by which superstructures are patterned and ultimately give rise to the unique morphology of each long bone is far from understood. In this work, we provide further evidence that long bones form modularly from Sox9+ cells, which contribute to their substructure, and from Sox9+/Scx+ progenitors that give rise to superstructures. Moreover, we identify components of the genetic program that controls the patterning of Sox9+/Scx+ progenitors and show that this program includes both global and regional regulatory modules.\n\nUsing light sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Additionally, by combining literature-based evidence and comparative transcriptomic analysis of different Sox9+/Scx+ progenitor populations, we identified genes potentially involved in patterning of bone superstructures. We present evidence indicating that Gli3 is a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work coordinately. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning that further supports the notion that long bone development is a modular process.

developmental biology↗

bioRxiv2018

On the Development of Sesamoid Bones

Sesamoid bones are a special group of small auxiliary bones that form in proximity to joints and contribute to their stability and function. Sesamoid bones display high degree of variability in size, location, penetrance and anatomical connection to the main skeleton across vertebrate species. Therefore, providing a comprehensive developmental model or classification system for sesamoid bones is challenging. Here, we examine the developmental mechanisms of three anatomically different sesamoid bones, namely patella, lateral fabella and digit sesamoids. Through a comprehensive comparative analysis at the cellular, molecular and mechanical levels, we demonstrate that all three types of sesamoid bones originated from Sox9+/Scx+ progenitors under the regulation of TGF{beta} and independent of mechanical stimuli from muscles. We show that BMP4 was necessary specifically for differentiation of patella but not of lateral fabella or digit sesamoids, whereas BMP2 regulated the growth of all examined sesamoids. Next, we show that whereas patella and digit sesamoids initially formed in juxtaposition to long bones, the lateral fabella formed independently at a distance. Finally, we provide evidence suggesting that while patella detached from the femur by formation of a synovial joint, digit sesamoids detached from the phalanx by a fibrocartilage joint. Collectively, these findings highlight both common and divergent molecular and mechanical features of sesamoid bone development, thereby advancing our understanding of their evolutionary plasticity.

developmental biology↗

bioRxiv2017

Development of migrating entheses involves replacement of progenitor populations

Attachment sites of tendons to bones, called entheses, are essential for proper musculoskeletal function. They are formed embryonically by Sox9+ progenitors and undergo a developmental process that continues into the postnatal period and involves Gli1 lineage cells. During bone elongation, some entheses maintain their relative positions by actively migrating along the bone shaft, while others, located at the bones extremities, remain stationary. Despite their importance, we lack information on the developmental transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and Gli1 lineage cells. Here, by performing a series of lineage tracing experiments, we identify the onset of Gli1 lineage contribution to different entheses during embryogenesis. We show that Gli1 expression is regulated by SHH signaling during embryonic development, whereas postnatally it is maintained by IHH signaling. Interestingly, we found that unlike in stationary entheses, where Sox9+ cells differentiate into the Gli1 lineage, in migrating entheses the Sox9 lineage is replaced by Gli1 lineage and do not contribute to the mature enthesis. Moreover, we show that these Gli1+ progenitors are pre-specified embryonically to form the different cellular domains of the mature enthesis.\n\nOverall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple, embryonic tissue, whereas another population is responsible for its maturation into a complex structure during its migration. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.

developmental biology↗