Ecdysone (Molecular Biology)

Pulses of the steroid hormone ecdysone coordinate postembryonic development in insects. In recent years, significant progress has been made in understanding the molecular mechanisms of ecdysone action in the fruit fly, Drosophila melanogaster (see Drosophila). Ecdysone, bound to its receptor protein, directly activates primary-response genes. A subset of these genes encodes transcription factors that induce large batteries of secondary-response target genes. Dynamic changes in ecdysone titer, combined with cross-regulatory interactions among these transcription factors, determine the timing and order of regulatory gene expression during the onset of metamorphosis. Furthermore, connections are now being made between these genetic regulatory hierarchies and the biological responses they direct. Studies of ecdysone action in Drosophila have provided a foundation for understanding the molecular mechanisms of steroid hormone action during development.

1. Ecdysone Pulses During Drosophila Development

At specific times during development, and in response to environmental cues, neurons in the insect central nervous system release prothoracicotropic hormone (PTTH). This neuropeptide signals the adjacent ring gland to secrete the steroid hormone ecdysone into the hemolymph of the circulatory system (1). Some peripheral tissues, most notably the larval fat body, modify ecdysone into its biologically active form, 20-hydroxyecdysone. This hormone can then directly regulate gene expression through its interaction with the ecdysone receptor. Although insect endocrinologists use the term "ecdysone" to refer to the inactive form of the hormone, this word is synonymous with 20-hydroxyecdysone in the molecular biology literature.


Ecdysone pulses direct Drosophila through its life cycle, with peak hormone titers signaling the major postembryonic developmental transitions (Fig. 1). Larval development progresses through three instars, each punctuated by ecdysone-triggered molting of the cuticle. A high-titer ecdysone pulse at the end of the third instar triggers puparium formation and the onset of prepupal development. This is followed, 10-12 hours after puparium formation, by another ecdysone pulse that initiates the 3.5 days of pupal development, followed by eclosion of the adult fly (Fig. 1). Most larval tissues are destroyed during the early stages of metamorphosis and are replaced by adult tissues that develop from clusters of imaginal progenitor cells. The crawling larva thus undergoes a complete transformation, resulting in the formation of a highly motile, reproductively active adult fly. Remarkably, this transformation occurs in apparent response to a single steroid hormone, ecdysone. One focus of current research is understanding how the systemic hormonal signal is refined into the appropriate stage- and tissue-specific developmental pathways that direct this transformation.

Figure 1. The ecdysteroid titer profile during Drosophila development. The composite ecdysteroid titer is depicted, in 20-hydroxyecdysone equivalents from whole-body homogenates (44). Each of the developmental stages of the Drosophila life cycle are also shown, below a time scale in days. Reprinted with permission from Thummel (45). 

The ecdysteroid titer profile during Drosophila development. The composite ecdysteroid titer is depicted, in 20-hydroxyecdysone equivalents from whole-body homogenates (44). Each of the developmental stages of the Drosophila life cycle are also shown, below a time scale in days. Reprinted with permission from Thummel (45).

2. Ecdysone Regulation of Primary-Response Genes

Ecdysone manifests its effects on development by activating its receptor, a heterodimer of two members of the nuclear receptor superfamily: EcR and USP (see Hormone Receptors) (2, 3). Interestingly, at least two of the three protein isoforms encoded by the EcR gene are expressed in a tissue-restricted manner that can contribute to the specificity of developmental responses to ecdysone (4). EcR-B1 is expressed primarily in larval tissues that are fated to die, whereas EcR-A is expressed in developing adult structures and tissues. Both high-affinity DNA binding and ecdysone binding require EcR heterodimerization with USP, a homologue of vertebrate retinoid X receptor (see Retinoic Acids) (5, 6). The ecdysone/EcR/USP complex can then bind DNA and activate the transcription of target genes (see Hormone Response Elements). It is interesting to note that a Drosophila orphan receptor, DHR38, a homologue of the vertebrate NGFI-B receptor, can also heterodimerize with USP (7). This raises the possibility that ecdysone signaling may be modified through distinct heterodimer combinations of receptors, similar to the interactions that have been demonstrated in vertebrate organisms.

Many genes are induced directly by the ecdysone-receptor complex, presumably reflecting their immediate requirement following ecdysone pulses (8). Some of these genes, like IMP-E1 (9) and Fbp-1 (10), are induced by ecdysone in a restricted tissue-specific manner, whereas others, like hsp23 (11) and Eip28 / 29 (12), are expressed in multiple ecdysone target tissues.

A subset of primary-response genes encode transcription factors that function at the top of ecdysone-triggered genetic regulatory hierarchies. Most of these genes were defined as puffs that are induced directly by ecdysone in the larval salivary gland polytene chromosomes . Detailed studies by Michael Ashburner and co-workers demonstrated that these so-called early puffs appeared to encode regulatory proteins that both repress their own activity and induce large sets of secondary-response late puffs (13). The protein products of the late puffs were, in turn, thought to play a more direct role in specifying appropriate biological responses to the hormone.

Four early puff genes have been described at the molecular level. One of these, E63-1, encodes a calcium binding protein related to calmodulin (see Calcium Signaling), providing the possibility of cross-regulation between hormone and calcium signaling pathways (14). The remaining three genes each encode multiple transcription factor isoforms. The Broad-Complex (BR-C) encodes more than a dozen proteins, each of which carries one of four possible pairs of zinc fingers (15, 16). The E74 early gene contains nested promoters that direct the synthesis of two related proteins, E74A and E74B, that share an identical C-terminal ETS domain that binds DNA (17). Similarly, the E75 early gene contains nested promoters that direct the synthesis of three orphan members of the nuclear receptor superfamily, designated E75A, E75B, and E75C (18). Curiously, three other orphan receptor genes, DHR3, E78, and bFTZ-F1, correspond to ecdysone-regulated puffs in the polytene chromosomes, suggesting that this family of transcription factors may play an important role in ecdysone signaling during development (19-21).

Ecdysone dose-response studies have revealed that each early promoter is activated at a specific critical threshold hormone concentration. Combined with ecdysone titer measurements, these dose-response studies have led to a model for the timing of early gene expression during the onset of metamorphosis (22). The EcR, BR-C, and E74B promoters are most sensitive to ecdysone, and are induced in mid-third instar larvae in apparent response to a low-titer hormone pulse (Figs. 2 and 3). The high-titer late larval ecdysone pulse then represses some of these messenger RNAs, such as EcR and E74B, as it induces the less-sensitive promoters, including E74A, E75A, E75B, and DHR3 (Figs. 2 and 3) (22, 23). The ecdysone titer drops during mid-prepupal development, allowing bFTZ-F1 to be expressed. This gene is repressed by ecdysone and thus depends on the decrease in hormone titer for its induction (24) (Fig. 3). The rapid rise in ecdysone titer in late prepupae then recapitulates the larval pattern of early gene expression, with a burst of EcR and E74B expression preceding that of E74A and E75A (Fig. 2). In addition to these dose-dependent responses to ecdysone pulses, many early genes also cross-regulate their activity, ensuring that they will be expressed at the correct time and for the appropriate duration (see text below).

Figure 2. Temporal patterns of ecdysone-regulated gene expression during the onset of metamorphosis. A schematic representation of the ecdysone pulses is shown at the top, with the magnitude of each pulse represented by the width of the stippled bar. Developmental time proceeds from left to right, with the major ecdysone-triggered transitions marked by dotted lines. The dotted line on the left represents the second-to-third instar larval molt, and the dotted line on the right represents head eversion and the prepupal-pupal transition. Dark grey bars show the timing and duration of primary-response regulatory gene transcription, the light grey bar represents bFTZ-F1 transcription, and the black bars represent secondary-response gene transcription.

Temporal patterns of ecdysone-regulated gene expression during the onset of metamorphosis. A schematic representation of the ecdysone pulses is shown at the top, with the magnitude of each pulse represented by the width of the stippled bar. Developmental time proceeds from left to right, with the major ecdysone-triggered transitions marked by dotted lines. The dotted line on the left represents the second-to-third instar larval molt, and the dotted line on the right represents head eversion and the prepupal-pupal transition. Dark grey bars show the timing and duration of primary-response regulatory gene transcription, the light grey bar represents bFTZ-F1 transcription, and the black bars represent secondary-response gene transcription.

Figure 3. Multiple ecdysone-triggered regulatory hierarchies direct the onset of Drosophila metamorphosis. This figure summarizes regulatory interactions discussed in the text. Bars represent repressive effects, and arrows represent inductive effects.

Multiple ecdysone-triggered regulatory hierarchies direct the onset of Drosophila metamorphosis. This figure summarizes regulatory interactions discussed in the text. Bars represent repressive effects, and arrows represent inductive effects.

3. Regulation of Ecdysone Secondary-Response Genes

Molecular and genetic studies have demonstrated that these stage-specific patterns of primary-response regulatory gene expression are transduced into corresponding patterns of secondary-response gene activity. Two classes of secondary-response genes have been studied in the larval salivary glands: the glue genes (25, 26) and the L71 genes from the 71E late puff (27). The glue genes are induced in mid-third instar salivary glands and encode a polypeptide glue that is used by the animal to affix itself to a solid surface for pupariation. In contrast, the L71 genes are of unknown function and are induced at puparium formation, as the glue genes are repressed (Fig. 2).

Both BR-C and E74B are required for proper glue gene induction, defining a mid-third instar regulatory hierarchy in the salivary glands (Fig. 3) (28). BR-C binding sites in the glue gene enhancers are required for proper induction, indicating that this is a direct regulatory link (29). The tissue specificity of this response is directed by the Fork head homeodomain transcription factor that binds adjacent to the BR-C in glue gene enhancers (30, 31).

The L71 late genes also depend on BR-C and E74 for their proper induction in late third instar larval salivary glands (Fig. 3). E74B maintains the L71 late genes in a repressed configuration during third instar larval development. The high-titer late larval ecdysone pulse then shuts off the E74B repressor and induces the E74A activator, which, together with the BR-C, directly induces L71 transcription (Fig. 3) (32-34). These studies provide molecular support for Ashburner’s proposal that early puff proteins direct the induction of late puff genes. In addition, they establish an important paradigm for understanding the mechanisms of hormone signaling in vertebrates, in which regulatory hierarchies are more difficult to define.

4. Cross-Regulation Among Ecdysone-Induced Transcription Factors

In addition to their roles in regulating ecdysone secondary-response genes, the ecdysone-induced transcription factors also regulate one another. BR-C is required for maximal ecdysone-induced early gene transcription in late third instar larvae (Fig. 3) (35). In this sense, BR-C is functioning as a competence factor that facilitates the subsequent regulatory response to ecdysone. Similarly, the early induction of EcR by a mid-third instar hormone pulse allows for increased levels of ecdysone receptor in preparation for the high hormone titer at the end of larval development (4).

Evidence has also been obtained for cross-regulatory interactions among the ecdysone-regulated orphan receptor genes. DHR3, which is induced by ecdysone in newly formed prepupae, is an inducer of bFTZ-F1 expression in mid-prepupae (Fig. 3) (36, 37). Furthermore, this induction can be inhibited by heterodimerization of DHR3 with E75B (37). E75B protein levels normally decay during early prepupal development, thus determining the time at which DHR3 can activate bFTZ-F1 (Fig. 2). bFTZ-F1, in turn, appears to be a critical competence factor for maximal reinduction of the early genes (Fig. 3) (24). Furthermore, bFTZ-F1 is sufficient to induce the stage-specific E93 early gene in the salivary glands of late prepupae (38). The DHR3, E75B, and bFTZ-F1 orphan receptors thus provide a regulatory link between the late larval and prepupal responses to ecdysone. Their stage-specific expression confirms that a critical developmental stage has been achieved and that the next response to the hormone should be distinct from the last response. These receptors thus provide a mechanism by which a hormonal signal can be refined into stage-specific developmental pathways.

5. From Gene Regulation to Biological Responses

Studies are currently under way that attempt to link ecdysone regulatory hierarchies with their appropriate biological responses during metamorphosis. As mentioned in the text above, most larval tissues are destroyed during prepupal and early pupal development. Recent studies have revealed that the destruction of the larval midgut and salivary gland occurs as stage-specific programmed cell death responses that display many of the hallmark features of apoptosis (39). Furthermore, the Drosophila death inducer genes reaper and head involution defective are coordinately induced in these tissues immediately preceding their destruction. A current focus of research is aimed at understanding how these death inducers are regulated by ecdysone at the appropriate time and place during metamorphosis. Specific neurons in the central nervous system also undergo programmed cell death in newly eclosed adults, in response to a decrease in ecdysone titer (40). Interestingly, these neurons can be distinguished by their high expression of the EcR-A isoform, suggesting that this receptor may play a key role in directing the death of these neurons.

As the larval tissues are being destroyed, the adult fly is being constructed. During prepupal development, the imaginal discs evert and elongate to form rudiments of the adult appendages. BR-C and E74 both play a critical role in this process (41, 42). Studies have also shown that bFTZ-F1 is sufficient to induce the pupal cuticle genes in mid-prepupal imaginal discs, providing a mechanism for the stage specificity of pupal cuticle deposition (Fig. 3) (43). It seems likely that future studies will provide more links between the ecdysone regulatory hierarchies and the biological responses to ecdysone during the onset of metamorphosis.

Next post:

Previous post: