Differentiation最新文献

Plastic pollution negatively affects ecosystems and human health globally, with single-use plastic representing the majority of marine litter in some areas. Life science laboratories prefer pristine conditions for experimental reliability and therefore make use of factory standardized single-use plastic products. This contributes to overall plastic waste in the United States and globally. Here, we investigate the potential of reusing plastic culture dishes and subsequently propose methods to mitigate single-use plastic waste in developmental biology research laboratories. We tested the efficacy of bleach and ethyl alcohol in sterilizing used dishes. We then tested the feasibility of washing and reusing plastic to culture Xenopus laevis embryos subjected to various manipulations. Cleaning and reusing laboratory plastic did not affect the development or survival of X. laevis, indicating that these cleaning methods do not adversely affect experimental outcome and can be used to sterilize plastic before reuse or recycling. Lastly, we performed a survey of various life science laboratories to estimate both waste reduction and savings associated with recycling single-use plastics. Standardization of these procedures would allow research laboratories to benefit economically while practicing environmentally conscious consumption.

Tuberin is a member of a large protein complex, Tuberous Sclerosis Complex (TSC), and acts as a sensor for nutrient status regulating protein synthesis and cell cycle progression. Mutations in the Tuberin gene, TSC2, permits the formation of tumors that can lead to developmental defects in many organ systems, including the central nervous system. Tuberin is expressed in the brain throughout development and levels of Tuberin have been found to decrease during neuronal differentiation in cell lines in vitro. Our current work investigates the levels of Tuberin at two stages of embryonic development in vivo, and we study the mRNA and protein levels during a time course using immortalized cell lines in vitro. Our results show that total Tuberin levels are tightly regulated through developmental stages in the embryonic brain. At a cell biology level, we show that Tuberin levels are higher when cells are cultured as neurospheres, and knockdown of Tuberin results in a reduction in the number of neurospheres. This functional data supports the hypothesis that Tuberin is an important regulator of stemness and the reduction of Tuberin levels might support functional differentiation in the central nervous system. Understanding how Tuberin expression is regulated throughout neural development is essential to fully comprehend the role of this protein in several developmental and neural pathologies.

The International Society of Differentiation was born from the First International Conference on Cell Differentiation conceived by D.V. and held in Nice, France in 1971. The conference also resulted in the creation of the journal of the Society named Differentiation. The Society advocates for the field of differentiation through the journal Differentiation, organizing and supporting international scientific conferences, honoring scientific achievements, and supporting trainees.

The anterior segment is a critical component of the visual system. Developing independent of the retina, the AS relies partially on cranial neural crest cells (cNCC) as its earliest progenitors. The cNCCs are thought to first adopt a periocular mesenchyme (POM) fate and subsequently target to the AS upon formation of the rudimentary retina. AS targeted POM is termed anterior segment mesenchyme (ASM). However, it remains unknown when and how the switch from cNCC to POM or POM to ASM takes place. As such, we sought to visualize the timing of these transitions and identify the regulators of this process using the zebrafish embryo model. Using two color fluorescence in situ hybridization, we tracked cNCC and ASM target gene expression from 12 to 24hpf. In doing so, we identified a tfap2a and foxC1a co-expression at 16hpf, identifying the earliest ASM to arrive at the AS. Interestingly, expression of two other key regulators of NCC, foxD3 and sox10 was not associated with early ASM. Functional analysis of tfap2a, foxD3 and sox10 revealed that tfap2a and foxD3 are both critical regulators of ASM specification and AS formation while sox10 was dispensable for either specification or development of the AS. Using genetic knockout lines, we show that in the absence of tfap2a or foxD3 function ASM cells are not specified, and subsequently the AS is malformed. Conversely, sox10 genetic mutants or CRISPR Cas9 injected embryos displayed no defects in ASM specification, migration or the AS. Lastly, using transcriptomic analysis, we show that GFP + cNCCs derived from Tg [foxD3:GFP] and Tg [foxC1b:GFP] share expression profiles consistent with ASM development whereas cNCCs isolated from Tg [sox10:GFP] exhibit expression profiles associated with vasculogenesis, muscle function and pigmentation. Taken together, we propose that the earliest stage of anterior segment mesenchyme (ASM) specification in zebrafish is approximately 16hpf and involves tfap2a/foxC1a positive cNCCs.

Human gonadal development culminating in testicular differentiation is described through analysis of histologic sections derived from 33-day to 20-week human embryos/fetuses, focusing on early development (4–8 weeks of gestation). Our study updates the comprehensive studies of Felix (1912), van Wagenen and Simpson (1965), and Juric-Lekic et al. (2013), which were published in books and thus are unsearchable via PubMed. Human gonads develop from the germinal ridge, a thickening of coelomic epithelium on the medial side of the urogenital ridge. The bilateral urogenital ridges contain elements of the mesonephric kidney, namely the mesonephric duct, mesonephric tubules, and mesonephric glomeruli. The germinal ridge, into which primordial germ cells migrate, is initially recognized as a thickening of coelomic epithelium on the urogenital ridge late in the 4th week of gestation. Subsequently, in the 5th week of gestation, a dense mesenchyme develops sub-adjacent to the epithelium of the germinal ridge, and together these elements bulge into the coelomic cavity forming bilateral longitudinal ridges attached to the urogenital ridges. During development, primordial cells migrate into the germinal ridge and subsequently into testicular cords that form within the featureless dense mesenchyme of the germinal ridge at 6–8 weeks of gestation. The initial low density of testicular cords seen at 8 weeks remodels into a dense array of testicular cords surrounded by α-actin-positive myoid cells during the second trimester. Human testicular development shares many features with that of mice being derived from 4 elements: coelomic epithelium, sub-adjacent mesenchyme, primordial germ cells, and the mesonephros.

The role of the mesonephros in testicular development was re-evaluated by growing embryonic day 11.5 (E11.5) mouse testes devoid of mesonephros for 8–21 days in vivo under the renal capsule of castrated male athymic nude mice. This method provides improved growth conditions relative to previous studies based upon short-term (4–7 days) organ culture. Meticulous controls involved wholemount examination of dissected E11.5 mouse testes as well as serial sections of dissected E11.5 mouse testes which were indeed shown to be devoid of mesonephros. As expected, grafts of E11.5 mouse testes with mesonephros attached formed seminiferous tubules and also contained mesonephric derivatives. Grafts of E11.5 mouse testes without associated mesonephros also formed seminiferous tubules and never contained mesonephric derivatives. The consistent absence of mesonephric derivatives in grafts of E11.5 mouse testes grafted alone is further proof of the complete removal of the mesonephros from the E11.5 mouse testes. The testicular tissues that developed in grafts of E11.5 mouse testes alone contained canalized seminiferous tubules composed of Sox9-positive Sertoli cells as well as GENA-positive germ cells. The seminiferous tubules were surrounded by α-actin-positive myoid cells, and the interstitial space contained 3βHSD-1-positive Leydig cells. Grafts of E11.5 GFP mouse testes into wild-type hosts developed GFP-positive vasculature indicating that E11.5 mouse testes contain vascular precursors. These results indicate that the E11.5 mouse testis contains precursor cells for Sertoli cells, Leydig cells, myoid cells and vasculature whose development and differentiation are independent of cells migrating from the E11.5 mesonephros.

We present a comprehensive description of the differentiating somatic cell types (Sertoli, Leydig, and peritubular myoid cells) of the mouse testis from embryonic day 10.5 (E10.5) to adulthood, postnatal day 60 (P60). Immunohistochemistry was used to analyze expression of: Sox9 (a Sertoli cell marker), 3βHSD-1 (a fetal Leydig cell marker), 3βHSD-6 (an adult Leydig cell marker), α-actin (a peritubular myoid cell marker), and androgen receptor (a marker of all three somatic cell types). The temporal-spatial expression of these markers was used to interrogate findings of earlier experimental studies on the origin of Sertoli, Leydig and peritubular myoid cells, as well as extend previous descriptive studies across a broader developmental period (E10.5-P60). Such comparisons demonstrate inconsistencies that require further examination and raise questions regarding conservation of developmental mechanisms across higher vertebrate species.

The mouse has been used as a model of human organogenesis with the tacit assumption that morphogenetic and molecular mechanisms in mice are translatable to human organogenesis. While many morphogenetic and molecular mechanisms are shared in mice and humans, many anatomic, morphogenetic, and molecular differences have been noted. Two critical gaps in our knowledge prevent meaningful comparisons of mouse versus human testicular development: (a) human testicular development is profoundly under-represented in the literature, and (b) an absence of a detailed day-by-day ontogeny of mouse testicular development from E11.5 to E16.5 encompassing the ambisexual stage to seminiferous cord formation. To address these deficiencies, histologic and immunohistochemical studies were pursued in comparable stages of mouse and human testicular development with a particular emphasis on Leydig, Sertoli and myoid cells through review of the literature and new observations. For example, an androgen-receptor-positive testicular medulla is present in the developing human testis but not in the developing mouse testis. The human testicular medulla and associated mesonephros were historically described as the source of Sertoli cells in seminiferous cords. Consistent with this idea, the profoundly androgen receptor (AR)-positive human testicular medulla was shown to be a zone of mesenchymal to epithelial transition and a zone from which AR-positive cells appear to migrate into the human testicular cortex. While mouse Sertoli and Leydig cells have been proposed to arise from coelomic epithelium, Sertoli (SOX9) or Leydig (HSD3B1) cell markers are absent from the immediate coelomic zone of the developing human testis, perhaps because Leydig and Sertoli cell precursors are undifferentiated when they egress from the coelomic epithelium. The origin of mouse and human myoid cells remains unclear. This study provides a detailed comparison of the early stages of testicular development in human and mouse emphasizing differences in developmental processes.

A definition of normal human fetal and early postnatal ovarian development is critical to the ability to accurately diagnose the presence or absence of functional ovarian tissue in clinical specimens. Through assembling an extensive histologic and immunohistochemical developmental ontogeny of human ovarian specimens from 8 weeks of gestation through 16 years of postnatal, we present a comprehensive immunohistochemical mapping of normal protein expression patterns in the early fetal through post-pubertal human ovary and detail a specific expression-based definition of the early stages of follicular development. Normal fetal and postnatal ovarian tissue is defined by the presence of follicular structures and characteristic immunohistochemical staining patterns, including granulosa cells expressing Forkhead Box Protein L2 (FOXL2). However, the current standard array of immunohistochemical markers poorly defines ovarian stromal tissue, and additional work is needed to identify new markers to advance our ability to accurately identify ovarian stromal components in gonadal specimens from patients with disorders of sexual differentiation.