No requirement for localized Nudel protein expression in Drosophila embryonic axis determination

Abstract

Drosophila embryonic dorsal-ventral polarity is defined by a maternally encoded signal transduction pathway. Gastrulation Defective, Snake, and Easter comprise a serine protease cascade that operates in the perivitelline space to generate active ligand for the Toll receptor, which resides in the embryonic membrane. Toll is activated only on the ventral side of the embryo. Spatial regulation of this pathway is initiated by the ventrally restricted expression of the sulfotransferase Pipe in the follicular epithelium that surrounds the developing oocyte. Pipe is thought to modify a target molecule that is secreted and localized within the ventral region of the egg and future embryo, where it influences the activity of the pathway such that active Toll ligand is produced only ventrally. A potential substrate for Pipe is encoded by nudel, which is expressed throughout the follicle cell layer and encodes a large, multi-functional secreted protein that contains a serine protease domain as well as other structural features characteristic of extracellular matrix proteins. A previous mosaic analysis suggested that the protease domain of Nudel is not a target for Pipe activity as its expression is not required in pipe-expressing cells, but failed to rule out such a role for other functional domains of the protein. To investigate this possibility, we carried out a mosaic analysis of additional nudel alleles, including some that affect the entire protein. Our analysis demonstrated that proteolytically processed segments of Nudel are secreted into the perivitelline space and stably localized, as would be expected for the target of Pipe, However, we found no requirement for nudel to be expressed in ventral, pipe-expressing follicle cells, thereby eliminating Nudel as an essential substrate of Pipe sulfotransferase activity.

Figure 1

Large clones of cells homozygous for Class I ndl alleles can be generated in the follicle cell layer. Follicle cell clones homozygous for the following mutations were generated by e22c-Gal4/UAS-FLP-mediated site-specific recombination in the follicle cell layer: (A and B) Df(3L)03844; (C and D) ndl¹⁰; (E and F) ndl²; (G and H) ndl⁵; (I and J) ndl¹²; (K and L) ndl¹⁴; (M and N) ndl¹⁵; (O and P) pipe². Staining with DAPI (A, C, E, G, I, K, M and O) shows the position of nurse cell and follicle cell nuclei. The absence of GFP-associated fluorescence from regions of the follicle layer in panels (F, H, J, L, N and P) indicates the position of homozygous mutant cells. Note that mutant follicle clones of cells homozygous for Df(3L)03844 and ndl¹⁰ could not be generated, while cells mutant for the class II ndl allele, ndl², for the Class I alleles ndl⁵, ndl¹², ndl¹⁴ and ndl¹⁵, and for the pipe allele, pip², were readily obtained.

Figure 2

N- and C-terminal segments of the Nudel protein maintain their localization in the perivitelline space following secretion from follicle cells. e22c-Gal4/UAS-FLP-mediated site-specific recombination was used to generate follicle cell clones homozygous for the ndl alleles ndl² (A–F), ndl¹² (G–L), and ndl¹⁴ (M-R). Clone-bearing follicles were visualized with DAPI to illuminate nuclei (A, D, G, J, M and P), with GFP to identify wild-type, fluorescent follicle cells (B, E, H, K, N and Q), and with antibodies specific for determinants within the N-terminal (C, I and O), and C-terminal (F, L and R) regions of Nudel protein. Nudel protein, detected by both antibodies was present throughout the perivitelline space of egg chambers containing ndl² mutant clones (C and F), consistent with the fact that the ndl² allele is a missense mutations that does not affect the level of Nudel protein produced. In contrast, in the cases of follicles carrying cells mutant for ndl¹² and ndl¹⁴, which lead to dramatic decreases in the amount of Nudel protein produced and secreted, epitopes detected using both the N- and C-terminal specific antisera were only detected in the perivitelline space adjacent to the position of wild-type cells (Compare panels H and I, K and L, N and O, and Q and R), indicating that Nudel protein does not diffuse away from its site of secretion within the perivitelline space. (N and C) refer to stainings using antibodies specific to the amino- and carboxy-terminal fragments of Nudel.

Figure 3

Expression of Nudel protein in ventral follicle cells is not necessary for the establishment of dorsal/ventral polarity in progeny embryos. (A and C) Completely dorsalized (D0) embryo produced by e22c-Gal4/ UAS-FLP-mediated generation of pip² (A) and ndl² (C) homozygous mutant cells, respectively, in the follicle cell layers of the egg chambers that generated these eggs. In (B and D–G), the technique of Nilson and Schüpbach (1998) was used to generate marked mutant follicle cells whose position in the follicle layer could be determined by the transparent appearance of the chorion imprints that they left on the egg, in comparison to the opaque appearance of the chorion imprints made by wild-type cells. (B) A ventral clone of pip² homozygous mutant cells led to the production of a partially dorsalized embryo as indicated by the tail-up phenotype and narrowed ventral denticle bands. In contrast, ventral follicle cells homozygous for the class II ndl allele, ndl² (C and D), and for the Class I ndl alleles, ndl⁵, ndl¹² and ndl¹⁴ (E–G) did not perturb DV patterning in the progeny embryos. For each of the mutations tested, the number of dorsalized embryos/total number of eggs exhibiting a ventral dec-1 clone are as follows: pipe² (95/169), ndl² (0/51), ndl⁵ (0/76), ndl¹² (1/71), ndl¹⁴ (3/58). The small number of embryos scored as dorsalized in the mosaic analyses of ndl¹² and ndl¹⁴ included three embryos exhibiting a tail-up phenotype, a defect arising from a perturbation in gastrulation that is occasionally seen in embryo collections independent of alterations in DV patterning, and one embryo from an egg carrying a very large mutant clone that likely resulted in insufficient Nudel protease activity to support DV axis formation.