To be or not to be: orb, the fusome and oocyte specification in Drosophila.

In the fruit fly Drosophila melanogaster, two cells in a cyst of 16 interconnected cells have the potential to become the oocyte, but only one of these will assume an oocyte fate as the cysts transition through regions 2a and 2b of the germarium. The mechanism of specification depends on a polarized microtubule network, a dynein dependent Egl:BicD mRNA cargo complex, a special membranous structure called the fusome and its associated proteins, and the translational regulator orb. In this work, we have investigated the role of orb and the fusome in oocyte specification. We show here that specification is a stepwise process. Initially, orb mRNAs accumulate in the two pro-oocytes in close association with the fusome. This association is accompanied by the activation of the orb autoregulatory loop, generating high levels of Orb. Subsequently, orb mRNAs become enriched in only one of the pro-oocytes, the presumptive oocyte, and this is followed, with a delay, by Orb localization to the oocyte. We find that fusome association of orb mRNAs is essential for oocyte specification in the germarium, is mediated by the orb 3' UTR, and requires Orb protein. We also show that the microtubule minus end binding protein Patronin functions downstream of orb in oocyte specification. Finally, in contrast to a previously proposed model for oocyte selection, we find that the choice of which pro-oocyte becomes the oocyte does not seem to be predetermined by the amount of fusome material in these two cells, but instead depends upon a competition for orb gene products.

Yield of Brain Magnetic Resonance Imaging in Epilepsy Diagnosis from 1998 to 2020: A Large Retrospective Cohort Study.

BACKGROUND: We aimed to find the clinical significance of brain abnormalities on magnetic resonance imaging (MRI) in epilepsy and the lateralization of these findings with electroencephalogram (EEG). METHODS: We retrospectively analyzed the results of all EEGs and brain MRIs of 600 consecutive epilepsy patients from 1998 to 2020. RESULTS: Data were available for 563 cases (267 females). Ninety percent of the patients were 18 years old or younger. A total of 345 patients (61.3%) had focal epilepsy, 180 (32%), generalized, and 38 (6.7%), inconclusive. In 187 (33.2%), the first MRI was abnormal and in 81 (out of 108 repeated MRI), the second was pathological. The most frequent brain abnormalities were cortical dysplasia in 41 (18.1%), other structural abnormalities in 25 (11%), various phacomatoses in 23 (10.1%), and mesial temporal sclerosis in 17 (7.5%). Among 226 patients with abnormal MRI, 171 (75.6%) had focal epilepsy when compared with 36 (15.9%) with generalized epilepsy (p <0.001). In 121 patients (53.5%), the result of the abnormal MRI contributed significantly to the understanding of the epilepsy etiology. The side of abnormality was lateralized to the EEG focus in 120 cases (53%); in 10/15 cases with infantile spasms (66%), MRI was significantly abnormal. In 33, in whom the first MRI was normal, a second MRI revealed a significant abnormality. CONCLUSION: Brain MRI is an important tool in epilepsy diagnosis, mainly in focal seizures and infantile spasms. A repeat MRI is mandatory in intractable focal cases to improve the yield of this test.

Concomitant diagnosis of endometrial and breast cancer - does the sequence matters?

OBJECTIVE: To examine whether patients with both breast cancer (BC) and endometrial cancer (EC) have different features of disease, and whether the sequence of appearance of these tumors is correlated with a more aggressive course. METHODS: A retrospective, multi-center observational cohort study of patients treated in two tertiary medical centers between 2014 and 2020. Files of patients who had a co-diagnosis of BC and EC were reviewed and clinical, epidemiological, pathological and genetic characteristics were collected. RESULTS: 67 patients with a co-diagnosis of both malignances were divided into two groups according to primary tumor diagnosis: BC first group (43/67, 64%) and EC first group (24/67, 36%). The time interval between diagnosis of malignancies was significantly longer in the BC first group (mean 144.5 months vs. 67 months, p < 0.05). BRCA mutations were found in higher numbers in the BC first group (27.5% vs. 9.5%, p = 0.18). A significantly higher number of patients in the BC first group had uterine serous carcinoma (USC) histology (44% vs. 12.5%, p < 0.05). This was independent of tamoxifen usage among patients (OR 0.65, 95% CI 0.17-2.49). CONCLUSIONS: In patients suffering from both BC and EC, the sequence of occurrence of malignancies has relevance: When EC presents as a second primary tumor, it tends to present in a more aggressive form, independent of previous tamoxifen use. The time interval between the diagnosis of malignancies was significantly longer in this group, offering an opportunity to improve preventive measures to decrease the likelihood of a subsequent lethal second cancer.

Size scaling in collective cell growth.

Size is a fundamental feature of living entities and is intimately tied to their function. Scaling laws, which can be traced to D'Arcy Thompson and Julian Huxley, have emerged as a powerful tool for studying regulation of the growth dynamics of organisms and their constituent parts. Yet, throughout the 20th century, as scaling laws were established for single cells, quantitative studies of the coordinated growth of multicellular structures have lagged, largely owing to technical challenges associated with imaging and image processing. Here, we present a supervised learning approach for quantifying the growth dynamics of germline cysts during oogenesis. Our analysis uncovers growth patterns induced by the groupwise developmental dynamics among connected cells, and differential growth rates of their organelles. We also identify inter-organelle volumetric scaling laws, finding that nurse cell growth is linear over several orders of magnitude. Our approach leverages the ever-increasing quantity and quality of imaging data, and is readily amenable for studies of collective cell growth in other developmental contexts, including early mammalian embryogenesis and germline development.

Development of migrating tendon-bone attachments involves replacement of progenitor populations.

Tendon-bone attachment sites, called entheses, are essential for musculoskeletal function. They are formed embryonically by Sox9+ progenitors and continue to develop postnatally, utilizing Gli1 lineage cells. Despite their importance, we lack information on the transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and the Gli1 lineage. Here, by performing a series of lineage tracing experiments in mice, we identify the onset of Gli1 lineage contribution to different entheses. We show that Gli1 expression is regulated embryonically by SHH signaling, whereas postnatally it is maintained by IHH signaling. During bone elongation, some entheses migrate along the bone shaft, whereas others remain stationary. Interestingly, in stationary entheses Sox9+ cells differentiate into the Gli1 lineage, but in migrating entheses this lineage is replaced by Gli1 lineage. These Gli1+ progenitors are defined embryonically to occupy the different domains of the mature enthesis. Overall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple embryonic tissue, whereas another population contributes to its maturation. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.

The Proprioceptive System Masterminds Spinal Alignment: Insight into the Mechanism of Scoliosis.

Maintaining posture requires tight regulation of the position and orientation of numerous spinal components. Yet, surprisingly little is known about this regulatory mechanism, whose failure may result in spinal deformity as in adolescent idiopathic scoliosis. Here, we use genetic mouse models to demonstrate the involvement of proprioception in regulating spine alignment. Null mutants for Runx3 transcription factor, which lack TrkC neurons connecting between proprioceptive mechanoreceptors and spinal cord, developed peripubertal scoliosis not preceded by vertebral dysplasia or muscle asymmetry. Deletion of Runx3 in the peripheral nervous system or specifically in peripheral sensory neurons, or of enhancer elements driving Runx3 expression in proprioceptive neurons, induced a similar phenotype. Egr3 knockout mice, lacking muscle spindles, but not Golgi tendon organs, displayed a less severe phenotype, suggesting that both receptor types may be required for this regulatory mechanism. These findings uncover a central role for the proprioceptive system in maintaining spinal alignment.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries.

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.

Cell lineage analysis of a mouse tumor.

Revealing the lineage relations among cancer cells can shed light on tumor growth patterns and metastasis formation, yet cell lineages have been difficult to come by in the absence of a suitable method. We previously developed a method for reconstructing cell lineage trees from genomic variability caused by somatic mutations. Here, we apply the method to cancer and reconstruct, for the first time, a lineage tree of neoplastic and adjacent normal cells obtained by laser microdissection from tissue sections of a mouse lymphoma. Analysis of the reconstructed tree reveals that the tumor initiated from a single founder cell, approximately 5 months before diagnosis, that the tumor grew in a physically coherent manner, and that the average number of cell divisions accumulated in cancerous cells was almost twice than in adjacent normal lung epithelial cells but slightly less than the expected figure for normal B lymphocytes. The cells were also genotyped at the TP53 locus, and neoplastic cells were found to share a common mutation, which was most likely present in a heterozygous state. Our work shows that the ability to obtain data regarding the physical appearance, precise anatomic position, genotypic profile, and lineage position of single cells may be useful for investigating cancer development, progression, and interaction with the microenvironment.

Reconstruction of cell lineage trees in mice.

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.