Proper cellularization of the embryo requires syntaxin1, a protein that is known to be important for membrane trafficking. Though this protein appears to be essential for the viability of many cells (Fig. ; Schulze and Bellen, 1996
), a direct role in cellularization is indicated by our analysis of the hypomorphic allele syxL266
. Ovary cells homozygous for this allele are viable and give rise to embryos that can develop normally until the moments when membrane in-growth should occur. A model for the formation of the cell membranes can be proposed that incorporates both the genetic and cytochemical information on syntaxin1
and the existing wealth of morphological descriptions of cellularization (Fullilove and Jacobson, 1971
; Sanders, 1975
; Turner and Mahowald, 1976
; Loncar and Singer, 1995
). Most recently, an electron microscopic study (Loncar and Singer, 1995
) observed vesicles that were lined up in front of the invaginating cleavage furrow between the nuclei of the syncytium. The addition of these vesicles is likely to be a major source of the membranes that advance the furrow and divide the newly forming cells. This process requires the t-SNARE syntaxin1. As in synaptic transmission, the syntaxin1 is present on the target membrane and is concentrated at sites where fusion occurs, the invaginating cleavage furrows. Syntaxin1, however, was not exclusively localized to a specific region of the furrow. This may mean that vesicles are added along its length, or it may simply reflect a lack of precision in syntaxin1 localization. This widespread distribution of syntaxin1 is similar to the situation in neurons, where syntaxin1 is not exclusively at the synapse but is also present along axons (Garcia et al., 1995
; Sesack and Snyder, 1995
Because the amount of syntaxin1 on the embryo's surface appears to increase during cellularization and because zygotically transcribed syntaxin1 contributes to this process, additional syntaxin1 must be added with the fusing vesicles. Therefore, vesicles may be able to fuse with one another, in addition to fusing with the surface membrane, and this pattern of growth has been suggested (Loncar and Singer, 1995
). Though we have not seen syntaxin1 in advance of the furrow, it may be below our detection limit. Syntaxin is known to reside on synaptic vesicles as well as on the plasma membrane at synapses (Walch-Solimena et al., 1995
Residual cellularization was observed in the syntaxin1-deficient embryos. This cellularization may have been mediated by the small amount of wild-type protein made by syxL266. Resorbtion of apical microvilli may serve as another source of membrane in normal furrow formation and may contribute to the residual cellularization that occurs outside of the defective patches. The acellular areas, however, did not have partial furrows; perhaps since microvillar resorbtion is thought to follow vesicular membrane addition, the resorbtion was blocked in these regions and could not contribute to furrow formation.
A noteworthy feature of the defect is its all or nothing nature: abrupt transitions were seen between properly cellularized and completely acellular areas. Though the patchiness of the phenotype is not yet understood, two possibilities can be suggested. First, regions of the surface that have a little syntaxin1 at the start of cellularization may be competent to receive additional syntaxin1 via vesicle fusion and thereby progress successfully at the expense of regions lacking syntaxin1. Alternatively, a failure in cellularization in a small area may expand into a larger patch because of an interdependence of neighboring cells on membrane and cytoskeletal interactions.
The onset of the phenotype coincides with the onset of cellularization. The formation of metaphase furrows in the mitotic cycle before cellularization may proceed by a different, syntaxin1-independent mechanism, e.g., the rearrangement of microvilli or other features of the surface. Alternatively, the demands placed on membrane trafficking may not be as great at the earlier stages, and the small amount of syntaxin1 present in syxL266 may be sufficient to meet the demand. Metaphase furrows are neither as deep as cleavage furrows nor are there as many of them since there are fewer nuclei at the surface during earlier cycles. Therefore, the initial phase of cellularization at cycle 14 probably represents the first time membrane trafficking is severely taxed during development, and this demand causes defects to arise when insufficient amounts of syntaxin1 are present.
The requirement for a syntaxin1 protein during cellularization is consistent with recent findings in other systems. Membrane fusions may be important for cell divisions in Arabidopsis thalliana
as well, where a distant homologue of syntaxin, KNOLLE, has been implicated in cytokinesis (Lukowitz et al., 1996
). One explanation of the apparent cell lethality of syxL371
is that syntaxin1
is required for cell divisions in many Drosophila
tissues in addition to the syncytial blastoderm. In its absence, these tissues would be unable to form. Thus, membrane addition via vesicle trafficking during cytokinesis may be essential for the formation of new cells (Rothman and Warren, 1994
). The apparent cell lethality may also reflect a requirement of syntaxin1
in constitutive membrane trafficking to the plasma membrane in all cells.
The existence of both neuronal and nonneuronal functions for the syntaxin1
gene was previously indicated by subtle abnormalities of the cuticle, gut, and trachea in zygotic null mutants (Schulze et al., 1995
) and is confirmed by our present study. In synapses, syntaxin acts with a cognate v-SNARE called VAMP or synaptobrevin. Current models of SNARE function require such an interaction to occur in the early embryo as well. Interestingly, however, the embryo and the synapse probably employ two different v-SNAREs to pair with the same syntaxin1 protein. We have previously found a neuron-specific synaptobrevin isoform (n-syb
) (DiAntonio et al., 1993
) that is the likely mediator of synaptic transmission (Sweeney et al., 1995
). We have also shown that a different, widely expressed gene (syb
) (Sudhof et al., 1989
) is present in 0–3-h embryos (Chin et al., 1993
). The use of distinct synaptobrevins may reflect the distinct regulatory control of these different trafficking events. It also indicates that the specificity of vesicle fusion cannot rely entirely on the interaction of syntaxin with synaptobrevin. This lack of specificity becomes particularly problematic if regulated neurosecretion and constitutive trafficking require the same t-SNARE in the same cell, despite the fact that their vesicles must be directed to distinct domains on the cell surface. Other proteins, perhaps in the SNARE complex, may contribute to the specificity or fine-tune it. For instance, syntaxin1 may complex with one SNAP-25 homologue in the early embryo, allowing it to bind with syb, while it complexes with another SNAP-25 in the nerve terminal, allowing it to bind n-syb.
Previous studies have indicated that docking of synaptic vesicles at appropriate sites persists in syx
mutants (Broadie et al., 1995
), and these studies have also shown that syntaxin is not localized only to active zones (Garcia et al., 1995
; Sesack et al., 1995). Our present observations suggest that a single isoform of syntaxin can mediate fusions by many different classes of transport vesicle. Though syntaxin may indeed play a role in docking and targeting, additional proteins must be invoked to support docking in its absence and to refine the specificity of targeting. On the other hand, a posttargeting role in exocytosis, such as a role in fusion itself, is consistent with our current findings and strongly implicated by the earlier genetic analysis (Broadie et al., 1995
; Schulze et al., 1995
In addition to syntaxin1, the cellularization of early Drosophila
embryos will undoubtedly involve many other proteins to mediate and direct the addition of membrane to the cell surface. Although the cytoskeletal rearrangements that accompany the membrane changes have been the object of considerable study (for reviews see Schejter and Wieschaus, 1993
; Theurkauf, 1994
), the genetic analysis of the membrane addition remains largely unexplored. Many of the components, like syntaxin1
, may be provided by both maternal contributions and zygotic transcription. Once cellularization is complete, the blastoderm must differentiate into a polarized tissue (Wodarz et al., 1995
) that selectively secretes cuticle proteins and signaling molecules from distinct domains. Thus, the formation of the cellular blastoderm may also provide a valuable genetic system in which to investigate the development of vectorial membrane trafficking.