We used a transient-expression assay to identify two estrogen response elements (EREs) associated with the major chicken vitellogenin gene (VTGII). Each element was characterized by its ability to confer estrogen responsiveness when cloned in either orientation next to a chimeric reporter gene consisting of the herpes simplex virus thymidine kinase promoter and the chloramphenicol acetyl transferase-coding region. Deletion analyses indicated that sequences necessary for the distal ERE resided within the region from -626 to -613 (nucleotide positions relative to the VTGII start site) whereas those necessary for the proximal ERE were within the region from -358 to -335. These distal and proximal elements contain, respectively, a perfect copy and an imperfect copy of the 13-base-pair sequence that is an essential feature of the EREs associated with two frog vitellogenin genes. These chicken VTGH EREs mapped near regions that were restructured at the chromatin level when the endogenous VTGII gene was expressed in the liver in response to estradiol. These data suggest a model for the tissue-specific expression of this estrogen-responsive gene.

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The magnitude of the expression of five yolk protein genes in the avian liver in response to exogenous estradiol is shown to be developmentally regulated. Though each of these yolk protein genes gains the capacity to respond to estradiol during embryonic development, we demonstrate that maximal responses for the different genes are achieved at distinct ages between 1 and 6 weeks after hatching. This observation prompted us to look for possible correlations between yolk protein gene expression and changes in the expression of estrogen receptors that might also occur after hatching. We discovered that indeed the maximal level of nuclear estrogen receptors (assayed following the administration of estradiol) increases progressively over this same period of development from "1000 receptors per cell at 1 week after hatching to %3500 receptors per cell at 6 weeks after hatching. The latter number represents the fully mature state, as comparable levels of receptors are present in the livers of egg-laying hens. Thus, though increases in the expression of estrogen receptors during embryonic liver development have previously been reported, our results indicate that the changes that occur after hatching are quantitatively far more significant to the developmental program for this transcription factor.The vitellogenic response in the avian liver provides an excellent model system in which to study the molecular mechanisms that underlie the developmental, steroid, and tissuespecific regulation of gene expression. In normal development three vitellogenin genes and an apolipoprotein gene, which collectively account for the bulk of the yolk proteins, are expressed exclusively in the livers of hens during periods of egg laying. The expression of this set of yolk genes is dependent on the elevated level of circulating estrogen that accompanies egg laying and is thus coordinated with the complementary production of egg white proteins in the oviduct. The complete set of yolk genes can be induced in the livers of both males and females, however, by simply injecting estradiol. This de novo induction is effected at the transcriptional level (ref. 1; unpublished results) and is accentuated by an increase in yolk message stability in the presence of estradiol (2). The capacity of these genes to respond to estradiol is demonstrable by day 9 of embryonic development (ref. 3; unpublished results) and thus reflects an early tissue-specific commitment event (4).The fact that there is an extended developmental period during which the vitellogenin and apolipoprotein genes have the potential to respond to estradiol but are transcriptionally silent in their normal state has afforded the opportunity to examine in detail the kinetics of accumulation of yolk protein (5)(6)(7)(8) and RNA (9-11) following primary and secondary injections of estradiol. Studies of this sort have revealed that birds retain a "memory" of their primary response, as evidenced by the fact that secondary presentations of estradiol elicit the more rapid accumulation of...

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