Ellen K. LeMosy

Integrin alpha chains exhibit distinct temporal and spatial localization patterns in epithelial cells of the Drosophila ovary

Integrins are heterodimeric transmembrane receptors that modulate cell adhesion, migration, and signaling. Multiple integrin chains contribute to development and morphogenesis of a given tissue. Here, we analyze the expression of Drosophila integrin alpha chains in the ovarian follicular epithelium, a model for tissue morphogenesis and cell migration. We find expression throughout development of the beta chain, βPS. Alpha chains, however, exhibit both spatial and temporal expression differences. αPS1 and αPS2 integrins are detected during early and mid‐oogenesis on apical, lateral, and basal membranes with the βPS chain, whereas αPS3‐family integrins (αPS3, αPS4, αPS5) are expressed in anterior cells late in oogenesis. Surprisingly, we find that αPS3‐family integrins are dispensable for dorsal appendage morphogenesis but play a role in the final length of the egg, suggesting redundant functions of integrins in a simple tissue. We also demonstrate roles for αPS3βPS integrin in border cell migration and in stretch cells. Developmental Dynamics 237:3927–3939, 2008.

Spatially dependent activation of the patterning protease, Easter

The dorsoventral axis of the Drosophila embryo is established by the activating cleavage of a signaling ligand by a serine protease, Easter, only on the ventral side of the embryo. Easter is the final protease in a serine protease cascade in which initial reaction steps appear not to be ventrally restricted, but where Easter activity is promoted ventrally through the action of a spatial cue at an unknown step in the pathway. Here, biochemical studies demonstrate that this spatial control occurs at or above the level of Easter zymogen activation, rather than through direct promotion of Easters catalytic activity against the signaling ligand.

Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane.

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. Here we show that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. Our results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell.

Activation of Snake in a serine protease cascade that defines the dorsoventral axis is atypical and pipe-independent in Drosophila embryos.

During Drosophila embryogenesis, establishment of ventral and lateral cell fates requires spatial regulation of an extracellular serine protease cascade composed of Nudel, Gastrulation Defective (GD), Snake, and Easter. Pipe, a sulfotransferase expressed ventrally during oogenesis, sulfates secreted targets that somehow confer positive spatial input to this cascade. Nudel and GD activation are pipe‐independent, while Easter activation requires pipe. The effect of pipe on Snake activation has been unknown. Here we show that Snake activation is cascade‐dependent but pipe‐independent. These findings support a conclusion that Snakes activation of Easter is the first spatially regulated step in the dorsoventral protease cascade.

Mutagenesis of the cysteine-rich clip domain in the Drosophila patterning protease, Snake

A common motif found in invertebrate serine proteases involved in immunity and development is the clip domain, proposed to regulate catalytic activity or protein-protein interactions within proteolytic cascades. Snake functions in a cascade that patterns the Drosophila embryo, and provides an accessible model for exploring the structural requirements for clip domain function. We tested Snake zymogens bearing charged-to-alanine mutations in the clip domain for their ability to rescue embryos lacking endogenous Snake and for their interactions by S2 cell co-transfection with upstream Gastrulation Defective and downstream Easter in the protease cascade. Of 13 single and multiple substitutions, one double mutant in a predicted protruding region exhibited a severe defect in embryonic rescue but showed only minimal defects in the co-transfection assay. We discuss implications of these and other results for potential biological roles of the Snake clip domain and for use of the in vitro assay in predicting protease behavior.

Pattern Formation: The Eggshell Holds the Cue

Recent data indicate that Torsolike, a spatial cue for patterning terminal structures of a Drosophila embryo, is stably anchored in the fruitfly eggshell; an as yet unidentified factor is required for the high activity of Torsolike at the embryo termini.

Early Craniofacial Defects in Zebrafish That Have Reduced Function of a Wnt-Interacting Extracellular Matrix Protein, Tinagl1

Objective Tinagl1 has a weak genetic association with craniosynostosis, but its functions in cartilage and bone development are unknown. Knockdown of Tinagl1 in zebrafish embryos allowed an initial characterization of its potential effects on craniofacial cartilage development and a test of whether these effects could involve Wnt signaling. Results Tinagl1 knockdown resulted in dose-dependent reductions and defects in ventral pharyngeal arch cartilages as well as the ethmoid plate, a zebrafish correlate to the palate. These defects could be correlated to reduced numbers of cranial neural crest cells in the pharyngeal arches and could be reproduced with comanipulation of Tinagl1 and Wnt3a by morpholino-based knockdown. Conclusions These results suggest that Tinagl1 is required early in the proliferation or migration of cranial neural crest cells and that its effects are mediated via Wnt3a signaling. Because Wnt3a is among the Wnts that contribute to nonsyndromic cleft lip and cleft palate in mouse and man, further investigation of Tinagl1 may help to elucidate mechanisms underlying these disorders.

GASTRULATION DEFECTIVE PROTEASE INTERACTS WITH ANIONIC COMPONENTS OF THE DROSOPHILA OVARY EXTRACELLULAR MATRIX

The Drosophila proteases Gastrulation Defective and Snake function in embryonic polarity establishment and bind heparin, a surrogate for anionic species present in the extracellular matrix. Here we demonstrate binding of GD, but not Snake, to anionic species that appear to be tightly associated with a highly purified eggshell matrix.