EdU (5-ethynyl-2'-deoxyuridine) labeling of Drosophila mitotic neuroblasts.

INTRODUCTION: When examining mutants that affect cell fate as a result of altered asymmetric division patterns, it is important to determine whether cells are mitotically active. Chemical labeling of newly synthesized DNA (during S-phase) by incorporation of BrdU (5-bromo-2'-deoxyuridine) is informative because this thymidine analog can be used to pulse-label dividing cells and then chased to identify the progeny of dividing cells. Such pulse-chase experiments can provide additional insight by distinguishing actively dividing cells from those that might be arrested at a mitotic checkpoint. EdU (5-ethynyl-2'-deoxyuridine) is another thymidine analog that provides a more sensitive and practical alternative to BrdU. Incorporation of EdU is detected through its reaction with an azide dye that is small enough to penetrate tissues efficiently. Visualization of EdU is rapid and does not interfere with subsequent antibody staining. The use of EdU in labeling Drosophila mitotic neuroblasts is described here.

Immunofluorescent staining of Drosophila larval brain tissue.

INTRODUCTION: The Drosophila larval brain is a well-established model for investigating the role of stem cells in development. Neuroblasts (neural stem cells) must be competent to generate many thousands of differentiated neurons through asymmetric divisions during normal development. Studies in fly neuroblasts have been instrumental in identifying how the establishment and maintenance of cell polarity influence cell fate, and they have produced a wide array of molecular cell-polarity markers. Moreover, neuroblasts and their progeny can be positively identified using a variety of cell-fate markers. This article describes procedures for the collection and processing of Drosophila larval brains for examination by immunolocalization of cell-fate and cell-polarity markers. The protocol can be used for dissecting, fixing, and staining brains from larvae at any developmental stage. The number of brains processed using this method is limited only by how many brains can be dissected in 20 min, which is the maximum amount of time dissected tissues should remain in buffer before fixation. This protocol can be used for simultaneous costaining of multiple proteins.

Multicolor fluorescence RNA in situ hybridization of Drosophila brain tissue.

INTRODUCTION: RNA in situ hybridization is a useful method for determining the transcriptional expression pattern of a gene when antibodies are not available. Using this technique, it is possible to assay the expression of multiple RNA species using distinct labels on RNA probes, or simultaneously examine RNA and protein localization within larval tissues. This protocol describes RNA in situ hybridization of Drosophila brain tissue. It utilizes a fluorophore-conjugated tyramide that is easily made in the laboratory for a fraction of the cost of the commercially produced product.

The Caenorhabditis elegans hunchback-like gene lin-57/hbl-1 controls developmental time and is regulated by microRNAs.

Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. We identified lin-57 as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 is hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback (hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3'UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs, including let-7. Indeed, we find that hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3'UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation.