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Motor neurons in the vertebrate spinal cord have long served as a paradigm to study the transcriptional logic of cell type specification and differentiation. At limb levels, pool-specific transcriptional signatures first restrict innervation to only one particular muscle in the periphery, and get refined, once muscle connection has been established. Accordingly, to study the transcriptional dynamics and specificity of the system, a method for establishing muscle target-specific motor neuron transcriptomes would be required.; To investigate target-specific transcriptional signatures of single motor neurons, here we combine ex-ovo retrograde axonal labeling in mid-gestation chicken embryos with manual isolation of individual fluorescent cells and Smart-seq2 single-cell RNA-sequencing. We validate our method by injecting the dorsal extensor metacarpi radialis and ventral flexor digiti quarti wing muscles and harvesting a total of 50 fluorescently labeled cells, in which we detect up to 12,000 transcribed genes. Additionally, we present visual cues and cDNA metrics predictive of sequencing success.; Our method provides a unique approach to study muscle target-specific motor neuron transcriptomes at a single-cell resolution. We anticipate that our method will provide key insights into the transcriptional logic underlying motor neuron pool specialization and proper neuromuscular circuit assembly and refinement.

Transcriptomic signatures based on cellular mRNA expression profiles can be used to categorize cell types and states. Yet whether different functional groups of genes perform better or worse in this process remains largely unexplored. Here we test the core matrisome – that is, all genes coding for structural proteins of the extracellular matrix – for its ability to delineate distinct cell types in embryonic single-cell RNA-sequencing (scRNA-seq) data. We show that even though expressed core matrisome genes correspond to less than 2% of an entire cellular transcriptome, their RNA expression levels suffice to recapitulate essential aspects of cell type-specific clustering. Notably, using scRNA-seq data from the embryonic limb, we demonstrate that core matrisome gene expression outperforms random gene subsets of similar sizes and can match and exceed the predictive power of transcription factors. While transcription factor signatures generally perform better in predicting cell types at early stages of chicken and mouse limb development, i.e., when cells are less differentiated, the information content of the core matrisome signature increases in more differentiated cells. Moreover, using cross-species analyses, we show that these cell type-specific signatures are evolutionarily conserved. Our findings suggest that each cell type produces its own unique extracellular matrix, or matreotype, which becomes progressively more refined and cell type-specific as embryonic tissues mature., Matrix Biology Plus, 10, ISSN:2590-0285

BackgroundThrough precise implementation of distinct cell type specification programs, differentially regulated in both space and time, complex patterns emerge during organogenesis. Thanks to its easy experimental accessibility, the developing chicken limb has long served as a paradigm to study vertebrate pattern formation. Through decades’ worth of research, we now have a firm grasp on the molecular mechanisms driving limb formation at the tissue-level. However, to elucidate the dynamic interplay between transcriptional cell type specification programs and pattern formation at its relevant cellular scale, we lack appropriately resolved molecular data at the genome-wide level. Here, making use of droplet-based single-cell RNA-sequencing, we catalogue the developmental emergence of distinct tissue types and their transcriptome dynamics in the distal chicken limb, the so-called autopod, at cellular resolution.ResultsUsing single-cell RNA-sequencing technology, we sequenced a total of 17,628 cells coming from three key developmental stages of chicken autopod patterning. Overall, we identified 23 cell populations with distinct transcriptional profiles. Amongst them were small, albeit essential populations like the apical ectodermal ridge, demonstrating the ability to detect even rare cell types. Moreover, we uncovered the existence of molecularly distinct sub-populations within previously defined compartments of the developing limb, some of which have important signaling functions during autopod pattern formation. Finally, we inferred gene co-expression modules that coincide with distinct tissue types across developmental time, and used them to track patterning-relevant cell populations of the forming digits.ConclusionsWe provide a comprehensive functional genomics resource to study the molecular effectors of chicken limb patterning at cellular resolution. Our single-cell transcriptomic atlas captures all major cell populations of the developing autopod, and highlights the transcriptional complexity in many of its components. Finally, integrating our data-set with other single-cell transcriptomics resources will enable researchers to assess molecular similarities in orthologous cell types across the major tetrapod clades, and provide an extensive candidate gene list to functionally test cell-type-specific drivers of limb morphological diversification.

Introduction The vertebroplasty (VP) and kyphoplasty (KP) are well known methods for the treatment of vertebral bodies fractures, due osteoporosis, primary, and metastatic tumor. The major complications are the cord and root compression, due cement leakage, epidural and subdural hematomas, and pulmonary emboli. These complications are most described in VP. In this retrospective study, the authors describe the way they find to avoid major complications with KP and a modification of de VP technique. Patients and Methods Between January 2007 and July 2014, 138 patients with 184 levels were treated in the three institutions. In 124 levels, KP was performed and in 60 levels VP. Balloon KP with pressure manometer was used. The VP were performed using de bone feelers and the cannulas of the KP set, without the insufflation of the balloon. A retrospective analysis of the cases was made and a review of the literature. Results A total of 102 patients (73.9%) were females and 36 patients (26.1%) were males. The 69% of the total of patients were single fractures and 31% were double level fractures. The etiology was 92% osteoporotic fractures, four patients (2.9%) underwent KP due multiple myeloma, five patients (3.6%) for hemangioma, and two patients (1.4%) for spinal metastasis. Pain relief was reported in all the patients with a media of VAS 8 of 10 in pre-op and VAS 2 of 10 in post-op. Neither major complications nor post-op infections were reported. Overall, 3 patients with cement leakage in the spinal canal, 14 patients with interbody disc space leakage, and 9 patients with lateral and anterior extravasations were described without symptoms. Conclusion The cement injection with the bone fillers provides more control in the cement infusion in the VP as well as in the KP, avoiding the leakage of cement in great quantity and preventing major complications.