Bruce H. Reed

Integrin-Dependent Apposition of Drosophila Extraembryonic Membranes Promotes Morphogenesis and Prevents Anoikis

BACKGROUND Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure. RESULTS By using live-imaging techniques, we report intimate interactions between the amnioserosa and the yolk sac during germ band retraction and dorsal closure. These tissue interactions fail in a subset of myospheroid (mys: betaPS integrin) mutant embryos, leading to failure of germ band retraction and dorsal closure. The Drosophila homolog of mammalian basigin (EMMPRIN, CD147)-an integrin-associated transmembrane glycoprotein-is highly enriched in the extraembryonic tissues. Strong dominant genetic interactions between basigin and mys mutations cause severe defects in dorsal closure, consistent with basigin functioning together with betaPS integrin in extraembryonic membrane apposition. During normal development, JNK signaling is upregulated in the amnioserosa, as midgut closure disrupts contact with the yolk sac. Subsequently, the amnioserosal epithelium degenerates in a process that is independent of the reaper, hid, and grim cell death genes. In mys mutants that fail to establish contact between the extraembryonic membranes, the amnioserosa undergoes premature disintegration and death. CONCLUSIONS Intimate apposition of the amnioserosa and yolk sac prevents anoikis of the amnioserosa. Survival of the amnioserosa is essential for germ band retraction and dorsal closure. We hypothesize that during normal development, loss of integrin-dependent contact between the extraembryonic tissues results in JNK-dependent amnioserosal disintegration and death, thus representing an example of developmentally programmed anoikis.

Autophagy promotes caspase-dependent cell death during Drosophila development.

The relationship between autophagic cell death and apoptosis is a poorly understood aspect of programmed cell death (PCD). We have examined this relationship by studying the elimination of an extra-embryonic tissue, known as the amnioserosa (AS), during Drosophila development. The AS becomes autophagic during the final stages of embryogenesis; ultimately, however, the elimination of the AS involves caspase-dependent nuclear fragmentation, tissue dissociation, and engulfment by phagocytic macrophages. Mutants that are defective in the activation or execution of caspase-dependent PCD fail to degrade and eliminate the AS but show no abatement in AS autophagy. Sustained autophagy does not, therefore, necessarily result in cell death. Surprisingly, the down-regulation of autophagy also results in a persistent AS phenotype and reduced cell death. Conversely, up-regulation of autophagy results in caspase-dependent premature AS dissociation. These observations are consistent with the interpretation that autophagy is a prerequisite for caspase-dependent cell death in the AS.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

Modulation of Morphogenesis by Egfr during Dorsal Closure in Drosophila

During Drosophila embryogenesis the process of dorsal closure (DC) results in continuity of the embryonic epidermis, and DC is well recognized as a model system for the analysis of epithelial morphogenesis as well as wound healing. During DC the flanking lateral epidermal sheets stretch, align, and fuse along the dorsal midline, thereby sealing a hole in the epidermis occupied by an extra-embryonic tissue known as the amnioserosa (AS). Successful DC requires the regulation of cell shape change via actomyosin contractility in both the epidermis and the AS, and this involves bidirectional communication between these two tissues. We previously demonstrated that transcriptional regulation of myosin from the zipper (zip) locus in both the epidermis and the AS involves the expression of Ack family tyrosine kinases in the AS in conjunction with Dpp secreted from the epidermis. A major function of Ack in other species, however, involves the negative regulation of Egfr. We have, therefore, asked what role Egfr might play in the regulation of DC. Our studies demonstrate that Egfr is required to negatively regulate epidermal expression of dpp during DC. Interestingly, we also find that Egfr signaling in the AS is required to repress zip expression in both the AS and the epidermis, and this may be generally restrictive to the progression of morphogenesis in these tissues. Consistent with this theme of restricting morphogenesis, it has previously been shown that programmed cell death of the AS is essential for proper DC, and we show that Egfr signaling also functions to inhibit or delay AS programmed cell death. Finally, we present evidence that Ack regulates zip expression by promoting the endocytosis of Egfr in the AS. We propose that the general role of Egfr signaling during DC is that of a braking mechanism on the overall progression of DC.

Autophagy can promote but is not required for epithelial cell extrusion in the amnioserosa of the Drosophila embryo

During Drosophila embryogenesis the majority of the extra-embryonic epithelium known as the amnioserosa (AS) undergoes programmed cell death (PCD) following the completion of the morphogenetic process of dorsal closure. Approximately ten percent of AS cells, however, are eliminated during dorsal closure by extrusion from the epithelium. Using biosensors that report autophagy and caspase activity in vivo, we demonstrate that AS cell extrusion occurs in the context of elevated autophagy and caspase activation. Furthermore, we evaluate AS extrusion rates, autophagy, and caspase activation in embryos in which caspase activity or autophagy are altered by genetic manipulation. This includes using the GAL4/UAS system to drive expression of p35, reaper, dINRACT and Atg1 in the AS; we also analyze embryos lacking both maternal and zygotic expression of Atg1. Based on our results we suggest that autophagy can promote, but is not required for, epithelial extrusion and caspase activation in the amnioserosa.

Drosophila Hindsight and mammalian RREB-1 are evolutionarily conserved DNA-binding transcriptional attenuators

The Drosophila Hindsight (hnt) gene encodes a C2H2-type Zinc-finger protein, HNT, that plays multiple developmental roles including control of embryonic germ band retraction and regulation of retinal cell fate and morphogenesis. While the developmental functions of the human HNT homolog, RREB-1, are unknown, it has been shown to function as a transcriptional modulator of several tumor suppressor genes. Here we investigate HNTs functional motifs, target genes and its regulatory abilities. We show that the C-terminal region of HNT, containing the last five of its 14 Zinc fingers, binds in vitro to DNA elements very similar to those identified for RREB-1. We map HNTs in vivo binding sites on salivary gland polytene chromosomes and define, at high resolution, where HNT is bound to two target genes, hnt itself and nervy (nvy). Data from both loss-of-function and over-expression experiments show that HNT attenuates the transcription of these two targets in a tissue-specific manner. RREB-1, when expressed in Drosophila, binds to the same polytene chromosome sites as HNT, attenuates expression of the hnt and nvy genes, and rescues the germ band retraction phenotype. HNTs ninth Zinc finger has degenerated or been lost in the vertebrate lineage. We show that a HNT protein mutant for this finger can also attenuate target gene expression and rescue germ band retraction. Thus HNT and RREB-1 are functional homologs at the level of DNA binding, transcriptional regulation and developmental control.

Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.