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Tissue morphogenesis relies on repeated use of dynamic behaviors at the levels of intracellular structures, individual cells, and cell groups. Rapidly accumulating live imaging datasets make it increasingly important to formalize and automate the task of mapping recurrent dynamic behaviors (motifs), as it is done in speech recognition and other data mining applications. Here, we present a \"template-based search\" approach for accurate mapping of sub- to multi-cellular morphogenetic motifs using a time series data mining framework. We formulated the task of motif mapping as a subsequence matching problem and solved it using dynamic time warping, while relying on high throughput graph-theoretic algorithms for efficient exploration of the search space. This formulation allows our algorithm to accurately identify the complete duration of each instance and automatically label different stages throughout its progress, such as cell cycle phases during cell division. To illustrate our approach, we mapped cell intercalations during germband extension in the early Drosophila embryo. Our framework enabled statistical analysis of intercalary cell behaviors in wild-type and mutant embryos, comparison of temporal dynamics in contracting and growing junctions in different genotypes, and the identification of a novel mode of iterative cell intercalation. Our formulation of tissue morphogenesis using time series opens new avenues for systematic decomposition of tissue morphogenesis.

The activity of epiphyseal growth plates, which drives long bone elongation, depends on extensive changes in chondrocyte size and shape during differentiation. Here, we develop a pipeline called 3D Morphometric Analysis for Phenotypic significance (3D MAPs), which combines light-sheet microscopy, segmentation algorithms and 3D morphometric analysis to characterize morphogenetic cellular behaviors while maintaining the spatial context of the growth plate. Using 3D MAPs, we create a 3D image database of hundreds of thousands of chondrocytes. Analysis reveals broad repertoire of morphological changes, growth strategies and cell organizations during differentiation. Moreover, identifying a reduction in Smad 1/5/9 activity together with multiple abnormalities in cell growth, shape and organization provides an explanation for the shortening of Gdf5 KO tibias. Overall, our findings provide insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover cellular mechanisms that may regulate this process. Inability to image large numbers of growth plate chondrocytes while retaining their spatial context during analysis has hindered the study of bone development. Here, the authors present a pipeline called 3D MAPs and use it to uncover morphogenic behaviors and growth strategies in normal bones as well as  aberrations in Gdf5 KO bones.

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. In this work, 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 ordered and disordered cell stacks, and small, disorganized clusters located in the outer edges. Finally, correlation between the temporal dynamics of the ratios between clusters and columns and between bone elongation and expansion suggests that clusters may promote expansion, whereas columns support elongation. Overall, our findings support the idea that modulations of division plane rotation of proliferating chondrocytes determines the formation of either clusters or columns, a multifunctional design that regulates morphogenesis throughout pre- and postnatal bone growth. Broadly, this work provides a new understanding of the cellular mechanisms underlying growth plate activity and bone elongation during development. As we develop, the long bones in our arms and legs must grow bigger and stronger to support our weight and movements. The width and length of these bones increase rapidly while in the womb, but after birth, they lengthen more quickly than they widen. Both expansion and extension occur at the growth plates, two narrow zones located at each bone’s ends and which host cells that can divide and increase in size. Traditionally, bone lengthening has been understood resulting from these ‘chondrocytes’ expanding in size after having organized themselves into columns that run parallel to the long axis of the bone. This is possible due to newly born cells performing a complex 90-degree rotation that results in this characteristic organization in column stacks. How bones widen, however, is less well-understood. To shed light on these mechanisms, Rubin, Agrawal et al. took advantage of recent technologies that allowed them to track the spatial organization of cells in 3D during development. Their experiments showed that, in mice, chondrocytes in the growth plate were rarely organized in columns before birth, with most cells not performing a 90-degree rotation of their division plane. This led to most clusters growing perpendicularly to the long axis of the bone, resulting in bone widening. After birth, however, most chondrocytes successfully completed the rotation, establishing columns running parallel to the long axis; fewer clusters contributing to the widening of the bone were present. Taken together, these results suggest that controlling the rotation of the division plane in chondrocytes helps create different growth strategies before and after birth. They also indicate that elongation in the womb may not require chondrocytes to be systematically organized in columns. Overall, the findings by Rubin, Agrawal et al. point to new mechanisms underpinning bone growth, which could be important to investigate further in both health and disease.

One of the major challenges that developing organs face is scaling, that is, the adjustment of physical proportions during the massive increase in size. Although organ scaling is fundamental for development and function, little is known about the mechanisms that regulate it. Bone superstructures are projections that typically serve for tendon and ligament insertion or articulation and, therefore, their position along the bone is crucial for musculoskeletal functionality. As bones are rigid structures that elongate only from their ends, it is unclear how superstructure positions are regulated during growth to end up in the right locations. Here, we document the process of longitudinal scaling in developing mouse long bones and uncover the mechanism that regulates it. To that end, we performed a computational analysis of hundreds of three-dimensional micro-CT images, using a newly developed method for recovering the morphogenetic sequence of developing bones. Strikingly, analysis revealed that the relative position of all superstructures along the bone is highly preserved during more than a 5-fold increase in length, indicating isometric scaling. It has been suggested that during development, bone superstructures are continuously reconstructed and relocated along the shaft, a process known as drift. Surprisingly, our results showed that most superstructures did not drift at all. Instead, we identified a novel mechanism for bone scaling, whereby each bone exhibits a specific and unique balance between proximal and distal growth rates, which accurately maintains the relative position of its superstructures. Moreover, we show mathematically that this mechanism minimizes the cumulative drift of all superstructures, thereby optimizing the scaling process. Our study reveals a general mechanism for the scaling of developing bones. More broadly, these findings suggest an evolutionary mechanism that facilitates variability in bone morphology by controlling the activity of individual epiphyseal plates.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.

Coughing is a respiratory behavior that plays a crucial role in protecting the respiratory system. Here we show that the nucleus of the solitary tract (NTS) in mice contains heterogenous neuronal populations that differentially control breathing. Within these subtypes, activation of tachykinin 1 (Tac1)-expressing neurons triggers specific respiratory behaviors that, as revealed by our detailed characterization, are cough-like behaviors. Chemogenetic silencing or genetic ablation of Tac1 neurons inhibits cough-like behaviors induced by tussive challenges. These Tac1 neurons receive synaptic inputs from the bronchopulmonary chemosensory and mechanosensory neurons in the vagal ganglion and coordinate medullary regions to control distinct aspects of cough-like defensive behaviors. We propose that these Tac1 neurons in the NTS are a key component of the airway–vagal–brain neural circuit that controls cough-like defensive behaviors in mice and that they coordinate the downstream modular circuits to elicit the sequential motor pattern of forceful expiratory responses. Gannot et al. show that Tac1 neurons in the NTS mediate an airway–vagal–brain pathway that is crucial for coughing in mice. These neurons receive direct vagal sensory inputs and coordinate downstream circuits to control coughing.

This dissertation, situated in the field of applied microeconomics, delves into the intricate dynamics between institutional changes, social movements, and microeconomic outcomes across different sectors. Through three comprehensive essays, it uncovers the nuanced impacts of educational policy adjustments, political activism, and healthcare sector consolidation on individual and institutional behavior, employing a variety of empirical methodologies to address these complex interactions.The first essay investigates the repercussions of modifying teacher testing standards on the pipeline of new educators. By leveraging a quasi-experimental setup precipitated by changes in the minimum passing scores for teacher training programs, this analysis provides a detailed examination of how increased testing standards influence enrollment and graduation rates across educational institutions. The findings reveal a pronounced decline in both metrics, with the most significant impacts observed in academically weaker institutions. This suggests that the elevated standards may exceed the optimal threshold for filtering candidates, inadvertently exacerbating shortages in the teaching workforce. Moreover, the study attributes a substantial portion of recent reductions in new teacher graduations to these augmented standards, challenging existing paradigms around educational qualification and workforce readiness.The second essay shifts the focus to the societal and economic ramifications of the Arab Spring protests, particularly concerning Egyptian women’s exposure to domestic violence. By adopting a triple difference analytical approach and exploiting regional variations in protest intensity, this research offers novel insights into the transformative power of political engagement on gender dynamics. The empirical evidence suggests a significant reduction in the likelihood of women experiencing various forms of intimate partner violence in areas with heightened protest activities. This decline is correlated with an increase in women’s decision-making autonomy, suggesting a redefinition of gender roles spurred by visible participation in public and political spheres. However, the study notes that these positive outcomes are not accompanied by shifts in societal attitudes towards domestic violence or changes in labor market participation, indicating a complex interplay between visibility, empowerment, and structural change.The third essay examines the labor market implications of hospital mergers within local commuting zones, focusing on the shifts in labor demand and employment composition post-merger. Utilizing causal inference techniques to isolate the effects of mergers from other market dynamics, the analysis challenges conventional metrics such as the Herfindahl-Hirschman Index for predicting labor demand shifts in the healthcare sector. Results demonstrate a concentrated increase in labor demand within merged entities, primarily for specialized and higher-skilled positions, underscoring the localized and sector-specific nature of employment responses to organizational consolidation.Collectively, these essays contribute to a deeper understanding of how policy decisions, social movements, and institutional transformations influence microeconomic indicators and individual behaviors. By employing rigorous empirical methodologies to dissect these relationships, this dissertation not only advances academic discourse in applied microeconomics but also offers pertinent insights for policymakers and stakeholders across the educational, social, and healthcare domains. Through its multifaceted exploration of teacher qualification standards, women’s rights and visibility in political unrest, and labor market dynamics within healthcare consolidation, the research underscores the importance of considering broader socio-economic contexts and the unintended consequences of policy and institutional changes.

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.

One of the major challenges that developing organs face is scaling, that is, the adjustment of physical proportions during the massive increase in size. Although organ scaling is fundamental for development and function, little is known about the mechanisms that regulate it. Bone superstructures are projections that typically serve for tendon and ligament insertion or articulation and, therefore, their position along the bone is crucial for musculoskeletal functionality. As bones are rigid structures that elongate only from their ends, it is unclear how superstructure positions are regulated during growth to end up in the right locations. Here, we document the process of longitudinal scaling in developing mouse long bones and uncover the mechanism that regulates it. To that end, we performed a computational analysis of hundreds of three-dimensional micro-CT images, using a newly developed method for recovering the morphogenetic sequence of developing bones. Strikingly, analysis revealed that the relative position of all superstructures along the bone is highly preserved during more than a 5-fold increase in length, indicating isometric scaling. It has been suggested that during development, bone superstructures are continuously reconstructed and relocated along the shaft, a process known as drift. Surprisingly, our results showed that most superstructures did not drift at all. Instead, we identified a novel mechanism for bone scaling, whereby each bone exhibits a specific and unique balance between proximal and distal growth rates, which accurately maintains the relative position of its superstructures. Moreover, we show mathematically that this mechanism minimizes the cumulative drift of all superstructures, thereby optimizing the scaling process. Our study reveals a general mechanism for the scaling of developing bones. More broadly, these findings suggest an evolutionary mechanism that facilitates variability in bone morphology by controlling the activity of individual epiphyseal plates.