Assay for Blood-brain Barrier Integrity in <em>Drosophila melanogaster</em>

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Loyola University Chicago

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.

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".

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.

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

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.

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

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.

Identification of raw as a regulator of glial development.

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.

Jennifer Jemc

Department of Biology

Loyola University Chicago

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Jennifer Jemc

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

Loyola University Chicago

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Department of Biology