sonication-facilitated-immunofluorescence-staining-late-stage

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval <em>Drosophila</em> Tissues <em>In Situ</em>

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

College of William & Mary

Wound closure following injury to the skin is a complex process involving both dermal contraction and keratinocyte migration. Murine models of wound healing are potentially useful because of the ability to determine protein function through gene manipulation. Owing to the dominant role of dermal contraction, the technical difficulties in preparing the wound site for morphologic studies, and the postnatal phenotypes altering the properties of transgenic skin, there are difficulties in assessing the epithelial contribution to wound closure in mouse skin. We describe a simple ex vivo assay utilizing explant culture that enables a quantitative assessment of the potential of mouse keratinocytes for wound epithelialization. In this assay, the behavior and properties of skin keratinocytes mimic well those that occur at the edge of skin wounds in situ, including a dependence upon connective tissue element(s), proliferation, and migration. The epithelial cell outgrowths emerging from skin explants can be studied in real-time or examined at specific time-points for markers of interest in the epithelialization process. The assay is quantitative and can successfully detect increases or decreases in epithelialization potential, and can be useful in the characterization of transgenic mouse models.

An ex vivo assay to assess the potential of skin keratinocytes for wound epithelialization.

Germ cell identity and development are controlled by autonomous cues in the germ plasm as well as by interactions between germ cells and somatic cells. Here, we investigate the formation of a germ cell-specific organelle, the spectrosome. We find that spectrosome formation is independent of germ cell-soma interactions and is autonomous to the germ cells. Furthermore, the germ plasm component nanos (nos) is essential for spectrosome formation. The role of nos in spectrosome formation is independent of its role in germ cell survival; nos mutant germ cells that are prevented from undergoing programmed cell death still fail to form spectrosomes. Thus, nos is required to regulate the formation of this germ cell-specific organelle, further supporting a role for nos in promoting germ cell identity.

nanos is required for formation of the spectrosome, a germ cell-specific organelle.

Germ cells must develop along distinct male or female paths to produce the sperm or eggs required for sexual reproduction. In both mouse and Drosophila, the sexual identity of germ cells is influenced by the sex of the surrounding somatic tissue (for example, refs 1, 2, reviewed in refs 3, 4); however, little is known about how the soma controls germline sex determination. Here we show that the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway provides a sex-specific signal from the soma to the germ line in Drosophila embryonic gonads. The somatic gonad expresses a JAK/STAT ligand, unpaired (upd), in a male-specific manner, and activates the JAK/STAT pathway in male germ cells at the time of gonad formation. Furthermore, the JAK/STAT pathway is necessary for male-specific germ cell behaviour during early gonad development, and is sufficient to activate aspects of male germ cell behaviour in female germ cells. Our findings provide direct evidence that the JAK/STAT pathway mediates a key signal from the somatic gonad that regulates male germline sexual development.

Somatic control of germline sexual development is mediated by the JAK/STAT pathway.

In many multicellular organisms, proper germ cell development is crucial for production of future generations. One critical step in this process is the decision of germ cells to develop into either sperm or eggs. Defects in germ cell sex determination can lead to infertility and germ cell tumors. However, while much is known about somatic sex determination, regulation of germ cell sex is not well understood. Recent studies with Drosophila reveal that the janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway promotes male germ cell development during early stages of gonad morphogenesis.(1) Here, this and other work suggesting that the JAK/STAT pathway acts alongside a host of other factors to regulate germ cell sex determination is discussed. Furthermore, recent insights into mouse germ cell sex determination are reviewed; revealing a number of correlations that suggest similar mechanisms may regulate aspects of both mouse and Drosophila germline sexual dimorphism.

Germ cell sex determination: piecing together a complex puzzle.

Germline stem cells (GSCs) in Drosophila are descendants of primordial germ cells (PGCs) specified during embryogenesis. The precise timing of GSC establishment in the testis has not been determined, nor is it known whether mechanisms that control GSC maintenance in the adult are involved in GSC establishment. Here, we determine that PGCs in the developing male gonad first become GSCs at the embryo to larval transition. This coincides with formation of the embryonic hub; the critical signaling center that regulates adult GSC behavior within the stem cell microenvironment (niche). We find that the Jak-STAT signaling pathway is activated in a subset of PGCs that associate with the newly-formed embryonic hub. These PGCs express GSC markers and function like GSCs, while PGCs that do not associate with the hub begin to differentiate. In the absence of Jak-STAT activation, PGCs adjacent to the hub fail to exhibit the characteristics of GSCs, while ectopic activation of the Jak-STAT pathway prevents differentiation. These findings show that stem cell formation is closely linked to development of the stem cell niche, and suggest that Jak-STAT signaling is required for initial establishment of the GSC population in developing testes.

Jak-STAT regulation of male germline stem cell establishment during Drosophila embryogenesis.

Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning approach after participation in GEP projects. They show knowledge gains on pre- and postcourse quizzes about genes and genomes and in bioinformatic analysis. Participating faculty also report professional gains, increased access to genomics-related technology, and an overall positive experience. We have found that using a genomics research project as the core of a laboratory course is rewarding for both faculty and students.

The genomics education partnership: successful integration of research into laboratory classes at a diverse group of undergraduate institutions.

There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit.

Article	Year
An ex vivo assay to assess the potential of skin keratinocytes for wound epithelialization. The Journal of investigative dermatology\| PubMed ID: 11982766	2002
nanos is required for formation of the spectrosome, a germ cell-specific organelle. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 16028275	2005
Somatic control of germline sexual development is mediated by the JAK/STAT pathway. Nature\| PubMed ID: 16049490	2005
Germ cell sex determination: piecing together a complex puzzle. Cell cycle (Georgetown, Tex.)\| PubMed ID: 16855383	2006
Jak-STAT regulation of male germline stem cell establishment during Drosophila embryogenesis. Developmental biology\| PubMed ID: 19643104	2009
The genomics education partnership: successful integration of research into laboratory classes at a diverse group of undergraduate institutions. CBE life sciences education\| PubMed ID: 20194808	2010
A course-based research experience: how benefits change with increased investment in instructional time. CBE life sciences education\| PubMed ID: 24591510	2014

Matthew Wawersik

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