Raw regulates glial population of the eye imaginal disc.

Glia are critical for proper development, support, and function of the nervous system. The Drosophila eye has proven an excellent model for gaining significant insight into the molecular mechanisms regulating glial development and function. Recent studies have demonstrated that Raw is required in glia of the central and peripheral nervous systems; however, the function of Raw in glia of the developing eye has not been explored. These studies demonstrate that raw knockdown results in a reduction in the number of glia in the third instar eye imaginal disc and reduced glial spreading across the field of differentiating photoreceptor neurons. Expression of a raw enhancer trap reveals that raw is expressed in eye disc glia. Exploration of the mechanism by which raw knockdown results in glial reduction reveals that Raw is required for glial proliferation and migration into the eye disc. In addition, Raw negatively regulates Jun N-terminal kinase (JNK) signaling in glia of the developing eye and increased JNK signaling results in a reduction in the number of glia populating the eye disc, similar to that observed upon raw knockdown. Thus, Raw functions as a critical regulator of glial population of the eye imaginal disc by regulating glial proliferation and migration and inhibiting JNK signaling.

Broad-complex, tramtrack, and bric-à-brac (BTB) proteins: Critical regulators of development.

Broad-complex, Tramtrack, and Bric-à-brac/poxvirus and zinc finger (BTB/POZ) family proteins are a diverse family of proteins that are characterized by the presence of a common protein-protein interaction domain, known as the BTB domain. BTB proteins have been identified in poxviruses and many eukaryotes, and have diverse functions, ranging from transcriptional regulation and chromatin remodeling to protein degradation and cytoskeletal regulation. Specificity of function is determined in part by additional domains present in BTB family proteins, as well as by interaction partners. Studies of BTB proteins in Drosophila and mammalian systems have revealed the importance of these genes in multiple developmental contexts, as well as in cancer and neurological and musculoskeletal diseases. In this review, we discuss the functions of BTB/POZ proteins during development with an emphasis on BTB-zinc finger (BTB-ZF) proteins, which play critical roles in transcriptional regulation and chromatin remodeling.

Onset of atonal expression in Drosophila retinal progenitors involves redundant and synergistic contributions of Ey/Pax6 and So binding sites within two distant enhancers.

Proneural transcription factors drive the generation of specialized neurons during nervous system development, and their dynamic expression pattern is critical to their function. The activation of the proneural gene atonal (ato) in the Drosophila eye disc epithelium represents a critical step in the transition from retinal progenitor cell to developing photoreceptor neuron. We show here that the onset of ato transcription depends on two distant enhancers that function differently in subsets of retinal progenitor cells. A detailed analysis of the crosstalk between these enhancers identifies a critical role for three binding sites for the Retinal Determination factors Eyeless (Ey) and Sine oculis (So). We show how these sites interact to induce ato expression in distinct regions of the eye field and confirm them to be occupied by endogenous Ey and So proteins in vivo. Our study suggests that Ey and So operate differently through the same 3' cis-regulatory sites in distinct populations of retinal progenitors.

raw Functions through JNK signaling and cadherin-based adhesion to regulate Drosophila gonad morphogenesis.

To form a gonad, germ cells (GCs) and somatic gonadal precursor cells (SGPs) must migrate to the correct location in the developing embryo and establish the cell-cell interactions necessary to create proper gonad architecture. During gonad morphogenesis, SGPs send out cellular extensions to ensheath the individual GCs and promote their development. We have identified mutations in the raw gene that result in a failure of the SGPs to ensheath the GCs, leading to defects in GC development. Using genetic analysis and gene expression studies, we find that Raw negatively regulates JNK signaling during gonad morphogenesis, and increased JNK signaling is sufficient to cause ensheathment defects. In particular, Raw functions upstream of the Drosophila Jun-related transcription factor to regulate its subcellular localization. Since JNK signaling regulates cell adhesion during the morphogenesis of many tissues, we examined the relationship between raw and the genes encoding Drosophila E-cadherin and ß-catenin, which function together in cell adhesion. We find that loss of DE-cadherin strongly enhances the raw mutant gonad phenotype, while increasing DE-cadherin function rescues this phenotype. Further, loss of raw results in mislocalization of ß-catenin away from the cell surface. Therefore, cadherin-based cell adhesion, likely at the level of ß-catenin, is a primary mechanism by which Raw regulates germline-soma interaction.

Tracking Follicle Cell Development.

Somatic follicle cells are critical support cells for Drosophila oogenesis, as they provide signals and molecules needed to produce a mature egg. Throughout this process, the follicle cells differentiate into multiple subpopulations and transition between three different cell cycle programs to support nurse cell and oocyte development. The follicle cells are mitotic in early egg chamber development, as they cover the germline cyst. In mid-oogenesis, follicle cells switch from mitosis to endocycling, increasing their ploidy from 2C to 16C. Finally, in late oogenesis, cells transition from endocycling to gene amplification, increasing the copy number of a small subset of genes, including the genes encoding proteins required for egg maturation. In order to explore the genetic regulation of these cell cycle switches and follicle cell development and specification, clonal analysis and the GAL4/UAS system are used frequently to reduce or increase expression of genes of interest. These genetic approaches combined with immunohistochemistry and in situ hybridization are powerful tools for characterizing the mechanisms regulating follicle cell development and the mitosis/endocycle and endocycle/gene amplification transitions. This chapter describes the genetic tools available to manipulate gene expression in follicle cells, as well as the methods and reagents that can be utilized to explore gene expression throughout follicle cell development.

A genetic screen for mutations affecting gonad formation in Drosophila reveals a role for the slit/robo pathway.

Organogenesis is a complex process requiring multiple cell types to associate with one another through correct cell contacts and in the correct location to achieve proper organ morphology and function. To better understand the mechanisms underlying gonad formation, we performed a mutagenesis screen in Drosophila and identified twenty-four genes required for gonadogenesis. These genes affect all different aspects of gonad formation and provide a framework for understanding the molecular mechanisms that control these processes. We find that gonad formation is regulated by multiple, independent pathways; some of these regulate the key cell adhesion molecule DE-cadherin, while others act through distinct mechanisms. In addition, we discover that the Slit/Roundabout pathway, best known for its role in regulating axonal guidance, is essential for proper gonad formation. Our findings shed light on the complexities of gonadogenesis and the genetic regulation required for proper organ formation.

Somatic gonadal cells: the supporting cast for the germline.

Cell-cell signaling and adhesion are critical for establishing tissue architecture during development and for maintaining tissue architecture and function in the adult. Defects in adhesion and signaling can result in mislocalization of cells, uncontrolled proliferation and improper differentiation, leading to tissue overgrowth, tumor formation, and cancer metastasis. An important example is found in the germline. Germ cells that are not incorporated into the gonad exhibit a greater propensity for forming germ cell tumors, and defects in germline development can reduce fertility. While much attention is given to germ cells, their development into functional gametes depends upon somatic gonadal cells. The study of model organisms has provided great insights into how somatic gonadal cells are specified, the molecular mechanisms that regulate gonad morphogenesis, and the role of germline-soma communication in the establishment and maintenance of the germline stem cell niche. This work will be discussed in the context of Drosophila melanogaster.

Identification of raw as a regulator of glial development.

Glial cells perform numerous functions to support neuron development and function, including axon wrapping, formation of the blood brain barrier, and enhancement of synaptic transmission. We have identified a novel gene, raw, which functions in glia of the central and peripheral nervous systems in Drosophila. Reducing Raw levels in glia results in morphological defects in the brain and ventral nerve cord, as well as defects in neuron function, as revealed by decreased locomotion in crawling assays. Examination of the number of glia along peripheral nerves reveals a reduction in glial number upon raw knockdown. The reduced number of glia along peripheral nerves occurs as a result of decreased glial proliferation. As Raw has been shown to negatively regulate Jun N-terminal kinase (JNK) signaling in other developmental contexts, we examined the expression of a JNK reporter and the downstream JNK target, matrix metalloproteinase 1 (mmp1), and found that raw knockdown results in increased reporter activity and Mmp1 levels. These results are consistent with previous studies showing increased Mmp levels lead to nerve cord defects similar to those observed upon raw knockdown. In addition, knockdown of puckered, a negative feedback regulator of JNK signaling, also causes a decrease in glial number. Thus, our studies have resulted in the identification of a new regulator of gliogenesis, and demonstrate that increased JNK signaling negatively impacts glial development.

Regulation of Gonad Morphogenesis in Drosophila melanogaster by BTB Family Transcription Factors.

During embryogenesis, primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) migrate and coalesce to form the early gonad. A failure of the PGCs and SGPs to form a gonad with the proper architecture not only affects germ cell development, but can also lead to infertility. Therefore, it is critical to identify the molecular mechanisms that function within both the PGCs and SGPs to promote gonad morphogenesis. We have characterized the phenotypes of two genes, longitudinals lacking (lola) and ribbon (rib), that are required for the coalescence and compaction of the embryonic gonad in Drosophila melanogaster. rib and lola are expressed in the SGPs of the developing gonad, and genetic interaction analysis suggests these proteins cooperate to regulate gonad development. Both genes encode proteins with DNA binding motifs and a conserved protein-protein interaction domain, known as the Broad complex, Tramtrack, Bric-à-brac (BTB) domain. Through molecular modeling and yeast-two hybrid studies, we demonstrate that Rib and Lola homo- and heterodimerize via their BTB domains. In addition, analysis of the colocalization of Rib and Lola with marks of transcriptional activation and repression on polytene chromosomes reveals that Rib and Lola colocalize with both repressive and activating marks and with each other. While previous studies have identified Rib and Lola targets in other tissues, we find that Rib and Lola are likely to function via different downstream targets in the gonad. These results suggest that Rib and Lola act as dual-function transcription factors to cooperatively regulate embryonic gonad morphogenesis.

Sorting Out Identities: An Educational Primer for Use with "Novel Tools for Genetic Manipulation of Follicle Stem Cells in the Drosophila Ovary Reveal an Integrin-Dependent Transition from Quiescence to Proliferation".

Organisms are made up of thousands of different cell types that must migrate, proliferate, and interact with each other to yield functional organ systems and ultimately a viable organism. A characteristic that distinguishes one cell type from another is the set of genes that it expresses. An article by Hartman et al. in the April 2015 issue of GENETICS identified methods to uniquely identify different cell populations during oogenesis, providing valuable tools for future studies. This Primer article provides background information on the Drosophila ovary as a system in which to study stem cell regulation, mechanisms for regulating gene expression, and the techniques used by Hartman et al. to identify specific cell populations and study their function.