Precise timing of synthesis of different eggshell components is relevant for the ordered assembly of the five layers and it relies on a fine regulation of gene expression (Waring,2000). The genes encoding major eggshell structural proteins are transcribed in follicle cells during stages 8–14 of oogenesis in a well-defined temporal order (Table 1) (King,1970; Spradling,1993; Waring,2000). The vitelline membrane genes are mainly expressed during mid-oogenesis stages 8–10, while the chorion genes are transcribed from stage 11 onward. The chorion synthesis, which occurs in the last 5–6 hours of oogenesis, requires both rapid production of large amounts of protein as well as fine control over the timing of gene expression. These requirements are met in two ways: (1) by amplification of the two chorion gene clusters and (2) by precise transcriptional control of the individual chorion genes.
Developmental control of eggshell gene expression relies in some instances on transcription of closely linked genes. Four vitelline membrane structural components are located on the left arm of the second chromosome. Their names refer to the map location on the polytene chromosome: VM32E, VM34C, VM26A.1, and VM26A.2 (Higgins et al.,1984; Mindrinos et al.,1985; Burke et al.,1987; Popodi et al.,1988; Gigliotti et al.,1989). While VM32E and VM34C are isolated, VM26A.1 and VM26A.2 appear to be clustered with other putative vitelline membrane genes (Popodi et al.,1988). Major chorion genes are set in two clusters, respectively, located at region 7F on the X chromosome (s36 and s38; Parks et al.,1986) and at region 66D on the third chromosome (s15, s16, s18, and s19; Spradling,1981; Griffin-Shea et al.,1982). Transcripts from genes s36 and s38 of the X-linked chorion cluster appear during early stages of chorion formation at stage 11, reach a peak at stage 12 and 13, and decline at stage 14 (Parks et al.,1986). Transcripts encoded by genes s15, s16, s18, and s19 of the third chromosome cluster accumulate at various overlapping late periods, mostly at stages 13 and 14 (Griffin-Shea et al.,1982). According to the choriogenic stages at which their corresponding genes are mainly expressed in the follicle cells, the major chorion proteins are designated as developmentally early (s38, s36), middle (s19, s16), and late (s18, s15). However, each gene has a unique mRNA accumulation profile indicating that temporal control is gene specific (Griffin-Shea et al.,1982; Parks et al.,1986). For example, already at stage 10 very low levels of s36 and s38 chorion genes can be detected by northern blot analysis (Parks and Spradling,1987). An earlier expression at stage 10 was also found for the s19 chorion gene but not for s16 (Griffin-Shea et al.,1982). In addition to these chorion genes, other genes have been identified that encode minor chorion components (Waring,2000). Among these, the Femcoat protein is specifically expressed in the follicle cells at late stages and it is required for endochorion formation (Kim et al.,2002). In a recent effort to find unknown proteins playing relevant structural or regulatory roles in eggshell biogenesis, 11 new distinct proteins have been identified as structural components of the eggshell (Fakhouri et al.,2006). Among these three are putative vitelline membrane proteins, seven are putative chorion components and one is a novel protein. The genes encoding the putative vitelline membrane proteins (CG9050, CG13992, and CG13997) map to the 26A region and are immediately adiacent to the VM26A.1 and VM26A.2 coding regions. Three of the putative chorion components (CG11381, CG14796, and CG15570) are expressed in a temporal pattern overlapping that of the early chorion genes. The proteins encoded by the CG14796 and CG15570 genes do not show any structural relationship with known proteins while the CG11381 product, a glutamine-rich protein, may correspond to the minor chorion protein s70. Two other putative chorion components (CG13083 and CG13084) are expressed in the same pattern of intermediate and late chorion genes. The two last predicted chorion proteins idientified (CG15350 and CG33962) are encoded by predicted genes mapping near the s36 and s38 early chorion genes. Finally, a small proline-rich protein was identified as putative component of the eggshell (CG13114).
The timing of eggshell protein synthesis is not always related to the final position of the proteins in the eggshell. Indeed, after their secretion the eggshell components could undergo trafficking between layers. Therefore, classifying them either as vitelline membrane or chorion proteins is not always clear. The highly dynamic state shown by some eggshell components, reflected by their trafficking between layers, appears evident by analyzing the VM32E protein (Andrenacci et al.,2001). At the time of its synthesis (stage 10), VM32E protein is not detectable in anterior and posterior follicle cells. However, it is able to diffuse in the extracellular space around the oocyte, and by stage 11 it is evenly distributed in the vitelline membrane. Moreover, as assessed by immunoelectron microscopy, at late stages of oogenesis VM32E protein is partially released from the vitelline membrane and becomes included in the endochorion layer too. A detailed functional analysis of the different VM32E domains showed that the C-terminal domain is required for this partial relocalization of the VM32E protein. Another example is provided by the early chorion protein s36, which initially localizes mainly in the vitelline membrane layer and, at late stages, it becomes distributed throughout the endochorion (Pascucci et al.,1996). Although s36 is described as a chorion protein and its mutant as producing egg chambers that lack endochorion organization (Digan et al.,1979), the mutant eggs show defects also in the vitelline membrane assembly (Cernilogar et al.,2001). This could imply that s36 protein might represent a structural component of both chorion and vitelline membrane layers. Cleavage and relocalization of eggshell components have been documented also for some products of the dec-1 locus (Hawley and Waring,1988). Stage-specific alternative RNA processing gives rise to three dec-1 transcripts, fc125, fc177, and fc106, encoding proteins with different C-terminal ends (Waring et al.,1990). The fc106 isoform is processed in the vitelline membrane giving rise to some products that remain in the vitelline membrane and other products that relocalize to the chorion (Nogueron et al.,2000). Similarly, the processing of the late isoform fc177 in the vitelline membrane results in products that move into the chorion or to the oocyte cytoplasm.