CrebA

DEVELOPMENTAL BIOLOGY

Embryonic

The CrebA protein is expressed in the nuclei of the embryonic salivary gland, proventriculus and stomodeum. CREBA's mRNAs are first seen at germ band extension (about 7 h into embryogenesis) in the salivary-gland placodes, and continue to be expressed in the salivary gland up to the 16th hour of development (Smolik, 1992).

The highest level of CREB-A mRNA is detected in the salivary gland primordia, initially in both presecretory cells and a subset of the duct cells (embryonic stages 9 through early 11), and later in only the secretory cells (Andrew, 1997).

CrebA transcripts are first seen at germ band extention in the salivary-gland placodes and continue to be expressed in the salivary gland up to the 16th hour of development. Low levels of CrebA mRNA are detected in the cell bodies of the brain and the optic lobe. CrebA mRNA is detected in midgut epithelial cells. CrebA is also expressed at lower levels in other tissues, including the trachea, a subset of neuroblasts, the proventriculus, the amnioserosa, the epidermis, and the foregut and its derivatives. The protein first appears in the foregut primordia by embryonic stage 6 and persists in the foregut derivatives until the end of embryogenesis. Expression in the amnioserosa begins during stage 8 and disappears during stage 13. Transient expression is observed in a subset of neuroblasts from stage 9 through stage 11 and in the proventriculus from stage 13 to 17. Tracheal expression is first detected at the time of tracheal pit formation (stage 11) and persists in the dorsal trunk tracheal cells throughout embryogenesis. The epidermal cells, which secrete the larval cuticle just before hatching, begin to express CrebA during stage 11 in a subset of cells in each segment, with accumulation of protein in all epidermal cells by stage 13. Staining is in the epithelial cell nuclei of the segmental boundaries (Rose, 1997). Protein persists for the remainder of embryogenesis (Andrew, 1997).

CREB-A mRNA is present throughout the life cycle of the fly, albeit at varying levels. The highest levels are seen during embryogenesis and in adult males. mRNA levels in third-instar larvae are approximately equivalent in the trachea, fat body and gut, but only the gut and fat body express Adh. CREB-A mRNA and protein are also found in the ovaries, an adult tissue that expresses Adh (Abel, 1992). Another study shows that although shown to bind fat-body and liver-specific regulatory elements, CrebA is not expressed in the fat body during any developmental stage (Andrew, 1997).

Adult

CrebA is found in the adult salivary gland, the columnar but not the squamous follicle cells in the ovary, and in the male seminal vesicle, anterior ejaculatory duct, and ejaculatory bulb. CrebA is initially expressed in stage 9 follicle cell nuclei as they migrate posteriorly toward and around the oocyte. In stages 10A and 10B, CrebA is expressed uniformly in the nuclei of the columnar follicle cells surrounding the oocyte. This expression pattern lasts until stage 11, when only a few nuclei expressing the CrebA protein can be seen over the reduced nurse cell chamber. By the onset of stage 12, CrebA protein is no longer detected (Rose, 1997).

Effects of mutation or deletion

In the epidermis, CrebA is required for patterning cuticular structures on both dorsal and ventral surfaces since CrebA mutant larvae have only lateral structures around the entire circumference of each segment. The most obvious defect is a weakening of the cuticle and a decrease in the overall length of homozygous mutants. Mutants average 40% the length of heterozygous sibling. Cuticular weakening is evident by the frequent 'blow-outs' or 'holes' which occur at random positions in mutants. The cuticular weakening and the decrease in larval length could be related. At all early embryonic stages, mutants are the same length as heterozygous siblings, suggesting that the length differences arise during the formation of the cuticle. Both weaker and smaller larval cuticles might be expected if cuticle protein synthesis were impaired. Alternatively, mutant larvae could be shorter because they have patterning defects (Andrew, 1997).

The dorsal hairs of CrebA mutants are most similar in size and morphology to the so-called quaternary hairs but these hairs are neither as orderly or as densely packed as the dorsal quaternary hairs of wild-type embryos. Rather, the loose irregular spacing of these hairs is more reminiscent of those seen in the dorsolateral position in wild-type embryos. CrebA ventral denticles are, however, indistinguishable from those found at a ventrolateral position in wild-type larvae. Because the morphology and arrangement of the ventral denticles and dorsal hairs in CrebA mutants accurately represent structures normally found on the lateral surface of wild-type larvae, it is believed that the altered cuticle patterns is not due to a failure to differentiate denticles or hairs, but rather arises because wild-type CrebA is required for correct patterning of the epidermis along the dorsal-ventral axis. Double mutants of CrebA and segment polarity genes give simple additive phenotypes, and do not demonstrate genetic interaction that would suggest that segment polarity genes and CrebA function in the same pathway (Andrew, 1997).

CrebA is thought to be epistatic to known dorsal/ventral patterning genes. Epistasis tests were done with the decapentaplegic gene and the spitz gene. In dpp/CrebA double mutants the entire cuticle is lateralized, while in dpp mutants the cuticle is ventralized. In spitz/CrebA double mutants narrower denticle bands are seen with some fusion of denticles between segments; however the denticles have the same morphology found in CrebA mutants alone. It is thought that near the end of both the Dpp- and Spi-signaling cascades, CrebA functions to translate the corresponding extracellular signals into changes in gene expression. The only determinant tested that shows altered expression patterns in CrebA mutants was Dsc73, a secreted protein expressed at late embryonic stages in the epidermal cells that produce denticles and hairs. There was a decrease in the levels of Dsc73 on the dorsal and ventral surfaces compared to levels of Dsc73 in lateral positions, which appear unchanged (Andrew, 1997).

The only defect observed in salivary glands is that they are 'crooked,' showing significant bends or kinks where normally there would be none (Andrew, 1997).

CrebA mutation is lethal during late embryonic development. One possible explanation for the late embryonic lethality is that the salivary gland is unable to secrete factors necessary for hatching. To test this possibility, CrebA was deliberately expressed in the salivary glands of CrebA mutants. Such expression does not rescue the embryonic lethality (Rose, 1997).

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date revised: 5 October 2005 

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