2 A and Fig. causes external to the epidermis. In contrast, lack of VAB-10B prospects to increased epidermal thickness during embryonic morphogenesis when epidermal cells switch shape. We suggest that this isoform protects cells against tension that builds up within the epidermis. embryos elongate more than fourfold along their anteriorCposterior axis in the absence of cell division. This process mainly depends on the epidermis surrounding the embryo and the underlying body wall musculature (Chin-Sang and Chisholm, 2000). The initial phase of elongation, which corresponds to a twofold increase in embryonic length, is usually driven by the contraction of circumferentially oriented actin microfilaments (MFs) causing changes of epidermal cell designs (Priess and Hirsh, 1986). Mutations affecting proteins that anchor MFs (-catenin, -catenin, E-cadherin) or that organize the MF bundles and regulate their contractions (rho kinase, myosin light chain, spectrins) disrupt embryonic elongation (for review observe Chin-Sang hSPRY2 and Chisholm, 2000). The subsequent phase of elongation requires the activity of muscle mass cells, which assemble sarcomeres at the plasma membrane facing epidermal cells during the initial phase (Hresko et al., 1994). Genetic analysis has recognized most components involved in building a functional sarcomere (Williams and Waterston, 1994; Mackinnon et al., 2002). Mutations in the corresponding genes generally prevent embryonic elongation beyond the twofold stage, resulting in a terminal phenotype termed Pat, for paralyzed at twofold (Williams and Waterston, 1994). The molecular mechanism underlying the mechanical coupling between muscle mass and epidermal cells is usually poorly comprehended. In corresponds to the spectraplakin locus, a recently acknowledged plakin subfamily defined by vertebrate and and by loci (Roper et al., 2002). The functions of BPAG1 and microtubule actin cross-linking factor (MACF) 1 during morphogenesis, if any, have not yet been explained (Fuchs and Karakesisoglou, 2001; Leung et al., 2002). Shot is known to form complexes with integrins and play a function very similar to that of plectin/BPAG1-e in vertebrate epidermal cells (Gregory and Brown, 1998; Prokop et al., 1998; Strumpf and Volk, 1998). However, besides its role in controlling actin ML349 remodeling in tracheal cells, its function during morphogenesis has not been fully investigated (Lee and Kolodziej, 2002a). We show that in encodes several protein isoforms related either to plectin and BPAG1-e, or to MACF and BPAG1-a, with distinct functions in the epidermis. Our work shows that molecules initially explained for their role in protecting cells against mechanical stress are essential for epithelial and embryonic morphogenesis. We suggest that spectraplakins safeguard epidermal cells against external causes exerted by muscle tissue, and against internal forces resulting from cell shape changes occurring in the epidermis. Results Identification of valleles Previously, we performed a ML349 genetic screen to identify loci required for embryonic morphogenesis, and reported that embryos homozygous for the chromosomal deficiency arrest with severe morphogenic defects ML349 (Labouesse, 1997). We could identify an embryonic lethal mutation, mutant phenotype (Fig. 1 B), and then found that is usually allelic to the viable mutation allele (Fig. 1 C). Finally, after cloning using a molecular approach (Fig. 1 E; see Materials and methods). Open in a separate window Physique 1. mutants display elongation and body morphology defects. Differential interference contrast micrographs of terminal-stage ML349 mutants. (A) Wild-type twofold embryo (mid-embryogenesis). (B) = 149) elongated 2.5-fold like this embryo, and occasionally hatched to generate kinked and paralyzed larvae, whereas 21% of those raised at 25C looked like embryos. (D) = ML349 135) resembled embryos. (E) Arrested L1 = 403, could hatch), and (F) hatching L1 is usually a spectraplakin locus that generates two unique units of plakins We molecularly recognized by a positional cloning strategy (see Materials and methods). is usually a complex locus that generates two distinct units of isoforms by option splicing of a common 5 region to two distinct 3 regions. The locus spans a region previously predicted to contain three genes (ZKexons (dark gray) spliced either to (medium gray) or to (light gray) exons (Fig. 2 A and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200302151/DC1); these isoforms will subsequently be referred to as and and exons (Fig. 2 A and Fig. S1). We did not.