BIOLOGICAL OVERVIEW

The temporally ordered expression of transcription factors is one process that can determine the course of development. In this model, genes are activated or repressed according to a genetically based, hierarchical time table. Alternative splicing, regulated both temporally and spatially, can likewise be a determining factor in developmental fate. Through alternative splicing, the RNA maturation machinery of the cell can generate different messenger RNAs from identical pre-messenger RNA transcripts. Alternative splicing is the primary mechanism used by the cell to regulate the function of Broad.

Before describing BRC, and the role of alternative splicing in BRC function, a word about genetic complementation is in order. Crossing two different mutant alleles of a gene in the same organism often gives rise to one of two alternative results. If the two alleles are part of the same complementation group, then the alleles do not complement, and a mutant phenotype results. If the two alleles are part of different complementation groups, then a wild type phenotype results. A test for complementation is the classic means that geneticist use to define the concept of a gene. When two alleles do not complement then they are part of the same functional genetic unit, or gene.

Broad Complex (BR-C), now know as broad is the apt term for a very complex gene that contains up to four classical complementation groups (broad, reduced bristles on palpus (rbp), l(1)2Bc and l(1)2Bd), as well as a group of non-pupariating (npr1) alleles that do not complement these functions. The original BR-C allele, br1, is viable when homozygous in females or hemizygous in males; here, the phenotypical wings are slightly shorter and wider than in wild type. Mutations of the l(1)2Bc complementation group causes late prepupal or early pupal developmental arrest; no head or dorsal thorax form because fusion of the epidermis in the thorax and head regions is defective. npr1 alleles produce the most severe phenotypes, resulting in a failure to pupariate and imaginal discs that form swollen vesicles (Kiss, 1988).

Molecular genetics has clarified the complex nature of Broad Complex. This single gene codes for multiple protein isoforms; they share a common (core) amino terminus fused to any one of four pairs of C2H2-type zinc-finger domains (Z1, Z2, Z3 and Z4). BR-C protein products are widely distributed among all tissues examined in the late larval to prepupal stages of development. All tissues studied to date contain all BR-C isoforms. However, their relative abundance differs greatly among tissue types, suggesting that the various members (or combinations) of the BR-C family of proteins function in different developmental pathways. The messenger RNA species coding for alternative isoforms are generated by alternative splicing of a single pre-messenger RNA transcript (Bayer, 1996).

Although the BR-C transcript and protein isoforms have been detected in every tissue examined during metamorphosis (Emery, 1994 and Huet, 1993), the relative ratio of zinc-finger isoforms differs among tissues, implying that differential expression of isoforms may contribute to the diversity of ecdysone responses among tissues. For example, Z1 and Z3 are the predominant isoforms expressed in late third instar larval salivary glands, while at the same stage Z2 predominates in instar discs. The relative ratios of zinc-finger mRNA isoforms also change over time. Tested for induction in tissue culture, the Z2, Z3, and Z4 mRNA isoforms accumulate rapidly in late third instar larval discs in response to ecdysone but abruptly disappear after 6 hours of culture. In contrast, the Z1 mRNA accumulates more slowly, to become the predominant isoform expressed after 6 hours. Thus, the expression of the four zinc-finger mRNA isoforms is dynamic, changing with time in a hormonally regulated sequence through the course of metamorphosis. (Bayer, 1996).

At least some of the changes in BR-C isoform expression appear to be posttranscriptional. For example, the switch in zinc-finger isoform expression does not result from differential use of the two BR-C promoters. Each can drive synthesis of the full repertoire of zinc-finger isoforms. In addition, sequences from the intronic regions upstream of the Z1 exon and Z4 exon are present in BR-C mRNAs of the largest size class. This suggests that splicing of some mRNA forms is regulated. It is thought that transcription of the early isoforms must involve transcription through the Z1 exon, located closest to the core DNA sequence. The Z1 exon could initially be eliminated from the pool of early BR-C mRNAs by splicing (Bayer, 1996).

The switch in BR-C mRNA isoform expression seen in cultured imaginal discs is also manifest at puparium formation, as a dramatic shift occurs in isoform expression, from Z2 to Z1 (Emery, 1994). The precipitous nature of the switch in zinc-finger isoform expression at a key stage in metamorphosis suggests that alterations in the function of BR-C are critical for the temporal regulation of downstream target genes during metamorphosis (Bayer, 1996).

It was originally proposed (DiBello, 1991) that the complementation groups represent subfunctions corresponding to specific zinc-finger proteins; that Z1 mediates rbp+ functions, Z2 mediates br+ functions and Z3 mediates 2Bc+ functions. This view is now amply supported by current studies. No genetic correlation has yet been made for the newly described Z4 isoform (Bayer, 1996).

Genetic and transgenic analysis of BR-C regulation of downstream genes shows that, in at least three cases the one isoform provides a regulatory function opposite that of the other isoforms. In one case the Z1 isoform activates a gene and the Z2 protein represses it. Thus a switch from predominant Z2 to Z1 isoform expression may mediate the precise temporal control of gene expression. In another case, expression of a salivary gland specific gene Sgs4 Z3 acts as a repressive isoform, while Z1 activates. However, since all isoforms can bind Sgs4 regulatory regions (von Kalm, 1994) it is suggested that Z1 and Z3 may compete for control of the Sgs4 regulatory element. For Dopa decarboxylase (Andres, 1993 and Hodgetts, 1995), Z2 transgene activates transcription, while either Z1 or Z4 represses this gene. Thus the primary role of BR-C appears to be the restriction of temporal expression of downstream genes to discrete developmental periods (Bayer, 1996).

Another example of the importance of alternative splicing in regulation of cell fate is the spatial variation in splicing of Ultrabithorax mRNA. Splicing factors are pivotal to the success of this process. These factors can recognize specific sequences in RNA, and based on sequence recognition, specifically remove unwanted introns and splice together the desired exons. In RNAs subjected to alternative splicing (both BR-C and UBX provide good examples), different introns and exons are removed depending on particular tissue types. The result is different tissue specific splice variants, a total of six different protein isozymes coded for by the different splice variants of UBX. The UBX splice variants differ in the distance between the homeodomain and a domain responsible for interaction with Extradenticle, required as a coactivator on UBX target genes. Different UBX isozymes function effectively with EXD on different target genes depending on the distance between the homeodomain and the interactive domain (Johnson, 1995).

In the case of UBX and BR-C it seems that cell fate depends primarily on the nucleoplasmic environment provided by splicing proteins, and not on the ordered expression of transcription factors. In the case of UBX, the pivotal role played by splicing is spatial, while in the case of BR-C, the pivotal role of splicing on cell fate determination is both spatial and temporal. The importance of alternative splicing in cell fate determination is a radical shift in paradigm for those that think that temporally ordered expression of transcription factors is the key determiner of cell fate.

Temporal patterns of broad isoform expression during the development of neuronal lineages in Drosophila

During the development of the CNS of Drosophila, neuronal stem cells, the neuroblasts (NBs), first generate a set of highly diverse neurons, the primary neurons that mature to control larval behavior, and then more homogeneous sets of neurons that show delayed maturation and are primarily used in the adult. These latter, 'secondary' neurons show a complex pattern of expression of broad, which encodes a transcription factor usually associated with metamorphosis, where it acts as a key regulator in the transitions from larva and pupa. The Broad-Z3 (Br-Z3) isoform appears transiently in most central neurons during embryogenesis, but persists in a subset of these cells through most of larval growth. Some of the latter are embryonic-born secondary neurons, whose development is arrested until the start of metamorphosis. However, the vast bulk of the secondary neurons are generated during larval growth and bromodeoxyuridine incorporation shows that they begin expressing Br-Z3 about 7 hours after their birth, approximately the time that they have finished outgrowth to their initial targets. By the start of metamorphosis, the oldest secondary neurons have turned off Br-Z3 expression, while the remainder, with the exception of the very youngest, maintain Br-Z3 while they are interacting with potential partners in preparation for neurite elaboration. That Br-Z3 may be involved in early sprouting is suggested by ectopically expressing this isoform in remodeling primary neurons, which do not normally express Br-Z3. These cells now sprout into ectopic locations. The expression of Br-Z3 is transient and seen in all interneurons, but two other isoforms, Br-Z4 and Br-Z1, show a more selective expression. Analysis of MARCM clones shows that the Br-Z4 isoform is expressed by neurons in virtually all lineages, but only in those cells born during a window during the transition from the second to the third larval instar. Br-Z4 expression is then maintained in this temporal cohort of cells into the adult. These data show the potential for diverse functions of Broad within the developing CNS. The Br-Z3 isoform appears in all interneurons, but not motoneurons, when they first begin to interact with potential targets. Its function during this early sorting phase needs to be defined. Two other Broad isoforms, by contrast, are stably expressed in cohorts of neurons in all lineages and are the first examples of persisting molecular 'time-stamps' for Drosophila postembryonic neurons (Zhou, 2009).

In Drosophila and other insects with complete metamorphosis, broad expression is associated with the initial phase of metamorphosis - the transition of the larva to the pupa. The broad isoforms play critical roles in this transition and are needed for the proper 'read out' of ecdysone-coordinated molecular events. In broad null mutants (for example, br^npr3) the premetamorphic growth and development of larval and imaginal tissues appear normal, but metamorphosis is blocked in all tissues before puparium formation. In br^npr3 mutants, the larval CNS appears to be normal before the wandering stage, although its subsequent metamorphosis is disrupted (Zhou, 2009).

While the classic role for broad is in the regulation of metamorphosis, the gene is known to have a wider role in development. It is involved in chorion formation during oogenesis in adult Drosophila, and this study shows that Br-Z3 expression is initiated in the CNS of the late embryo and that Br-Z1 and Br-Z4 continue to be expressed in the adult. The origins of the four isoforms of broad are ancient, extending back into the Crustacea (Spokony, 2007), much earlier than the evolution of complete metamorphosis in insects. Functions typified by its role in the nervous system may represent an ancestral function for broad, and its role in metamorphosis a more recently derived function for this gene (Zhou, 2009).

In the developing vertebrate CNS, and especially the spinal cord, the initial set of neurons that set-up the basic architecture of the CNS are often called primary neurons. They are typically large and readily identifiable and may be later replaced by a larger flood of secondary neurons. In metamorphic species, like frogs, the primary neurons are ascribed to function in the larval tadpole while secondary neurons are generated in preparation for metamorphosis to the adult frog. An analogous concept of primary and secondary neurons is becoming established for Drosophila development. Similar to that seen in metamorphic vertebrates, the term primary neuron is typically associated with cells born during the first phase of neurogenesis in the embryo and secondary neuron for the cells born during the later, larval phase. The larval-born cells arrest their development soon after their birth and wait until metamorphosis before maturing into functional neurons. However, BrdU feeding experiments show that some embryonic-born neurons also arrest without showing any branching, a condition identical to that seen in the neurons that are born in the larva. Moreover, it appears that these arrested, embryonic neurons show a sustained expression of Br-Z3, a feature characteristic of the secondary neurons born during the larval phase of neurogenesis (Zhou, 2009).

In light of the latter observations, it is worth contemplating the definition of primary and secondary neurons. There are three potential uses of this designation/terminology. One would be to use it to denote embryonic versus postembryonic born neurons. A second would be to use it to refer to the functional status of a neuron, where embryonic neurons that are fully differentiated, functional components of a network could be termed primary neurons and neurons that had initiated neurite growth but 'stalled' their development prior to establishing pre- and postsynaptic specializations would be secondary neurons. A third use would be to refer to the developmental transition within lineages where a neural precursor switches from producing diverse progeny at each division to generating homogenous 'blocks' of cell types (Zhou, 2009).

There is a striking difference between the mechanisms of specification of neuronal identities of early and late-born neurons in insects. For a given NB, the characteristics of the first few progeny can be strikingly diverse and are based on birth-order of the parent GMC. The determination of birth-order is based on unique molecular identities that are bestowed on successive GMCs through an intrinsic, temporal program of transcription factor production by the NB and the stable passage of that expression to the GMC during cell division. A series of loss-of-function and gain-of-function studies confirmed that the presence of these transcription factors is responsible for the diversity of neuronal phenotypes within a given lineage and underlies the great diversity of early neuronal types seen across the lineages. This intrinsic progression, however, appears to stop after the expression of castor, and remaining neurons, both embryonic and postembryonic, express the transcription factor gene. During the postembryonic neurogenic period, the NBs typically produce groups of neurons with similar characteristics. This is most evident during the postembryonic phase of neurogenesis in moths and flies, where neurons are produced in discrete blocks of similar cells, rather than as unique individuals. The expression of Chinmo is required for establishing the identities of an early-born block of cells in the mushroom bodies and antennal lobes, among other lineages. This study reports that the expression of Broad-Z4 apparently provides a molecular marker for the next block of postembryonic neurons. Unlike the early embryonic identity factors, though, Broad is not expressed first in the NB and then passed into this cohort of cells. Rather, it appears in neurons some time after their birth, likely in response to hormonal conditions experienced by them or their parent GMC (Zhou, 2009).

Clonal studies on Drosophila embryos suggest that the generation of neuron classes does not abruptly begin with the postembryonic neurons. Although the embryonic lineages are remarkable for the diversity of their early-born neurons, the later-born cells (farther from the neuropil) in many lineages show clustered cell bodies and similar neurite trajectories, indicating that they may constitute a discrete neuronal type. This study proposes that these later cells, along with the post-embryonic neurons, should be called the secondary neurons and the term primary neurons should be confined to the initial neurons that are produced during embryogenesis during the progression from Hunchback to Castor. Typically, the first four GMCs produced by a given NB would account for its primary neurons, although, in some lineages, like NB 7-1, the expression of a factor may be prolonged to give two GMCs that produce similar properties. In the ventral CNS, the primary neurons would account for the majority of the neurons present at hatching. Most of the early-born secondary neurons are also functional and contribute to larval behavior, while others show the arrested development described in this study (Zhou, 2009).

Uncoupling of the terms primary and secondary from embryonic versus postembryonic origins circumvents problems with using these terms in a comparative context. In more basal insects, like grasshoppers, that have direct development, all of the neurons in the ventral CNS are made during embryogenesis, and, hence, would be considered as primary neurons if time of birth was the deciding criterion. Similarly, within the insects with complete metamorphosis, neurogenic arrest occurs at different points in the lineage depending on whether you are making a reduced nervous system for a relatively simple larva like a fly maggot, or a complex CNS that is a feature of the hunting larvae of ground beetles. Basing the designation of primary and secondary on the mechanisms that generate cellular diversity, rather than on the stage at their birth or their functional status within the network allows for homologues to have the same designation across these diverse taxa (Zhou, 2009).

In Drosophila, most primary neurons express low to moderate levels of Br-Z3 during late embryogenesis. Their embryonic expression was not characterized in detail, and these neurons do not then re-express Br-Z3 (or any other Broad isoform) when they undergo remodeling at the end of larval life. The secondary interneurons also transiently express Br-Z3 during their early development. The results from feeding larvae on a BrdU diet from hatching indicate that these embryonic secondary neurons account for many of the strong Br-Z3+ cells seen at hatching (Zhou, 2009).

The main focus of the paper has been on the expression of Broad in the neurons born during the larval phase of neurogenesis. BrdU incorporation was used to measure the latency to the onset of Br-Z3 expression in the secondary neurons. For larvae at 96 h AEL, the BrdU pulse length needed to see labeling in Br-Z3+ neurons was 11 h, and defines the latency from the end of DNA synthesis in a GMC to the appearance of Br-Z3 in its daughters. At 96 h AEL (72 h post-hatching), the average lifetime of a GMC is about 7 h and its G2 phase lasts about 4 h. Hence, a young neuron starts expressing Br-Z3 about 7 h after its birth, which is about 14 h after the birth of its parent GMC. The arrested embryonic NBs resume dividing early in the second instar larva, and GMCs are seen associated with them by 60 h AEL (36 h post-hatching). In this study, Br-Z3 started to appear in the clusters of neurons by about 72 h AEL, a timing consistent with a 14 h latency between the birth of a GMC and the subsequent appearance of Br-Z3 in its daughter cells. Consequently, it is assumed that this latency is likely similar throughout the period of postembryonic neurogenesis (Zhou, 2009).

Although the secondary neurons may be relatively uniform in the timing of the onset of Br-Z3 expression, they differed in how long this expression was maintained. One pattern was shown by the embryonic and oldest postembryonic secondary cells. These showed initial Br-Z3 expression but then lost it by the start of wandering. They correspond to those cells characterized as being Broad negative and Chinmo+ at pupariation. The other pattern was shown by later born neurons (whose GMCs are born after about 72 h AEL) and these show the persistence of Br-Z3 through pupariation but then lose it early in adult differentiation (Zhou, 2009).

The distinction between the Chinmo+ and Broad+ groups of secondary neurons is set up by the postembryonic actions of Seven-up and Castor. For many lineages, removal of Seven-up resulted in the loss of Broad+ neurons in favor of the larger Chinmo+ cells. A similar result was seen in some of the lineages by the prolonged expression of Castor. It is interesting that both Seven-up and Castor have roles in temporal identity in the embryo. It could be that Br-Z3 expression in the embryonic secondary neurons may likewise be the result of the embryonic interplay of these two genes (Zhou, 2009).

The appearance of Br-Z3 in thoracic interneurons about 7 h after their birth coincides with the loss of expression of Notch in the membrane of these young neurons. Notch is prominent in the membranes of newborn neurons during pathfinding but then rapidly disappears. Consequently, the appearance of Br-Z3 is correlated with a new neuron arriving at its initial target. The best data on the behavior of growth cones in this post-pathfinding stage are for in-growing retinal axons in the first optic neuropil in Drosophila. After arriving at their initial location these axons undergo a 'sorting phase', during which their growth cones spread filopodia laterally to select their cartridge partners. Interestingly, photoreceptors are the only sensory neurons that express Br-Z3 and they do so during this early sorting phase (Zhou, 2009).

Expression of Br-Z3 in the developing medulla interneurons may also be correlated with the sorting of potential partners. When Br-Z3 first appears in the medulla in the mid third instar, it is confined to the most medial columns of neurons. Through time, more lateral columns progressively acquire Br-Z3 expression, and all outer medulla neurons are expressing this isoform by 14 h APF. The neurons of the most medial columns interact with the first photoreceptors growing in from the posterior border of the eye, and as axons grow in from the subsequent rows of ommatidia over the next 2 days, these interact with the neurons of progressively more lateral columns. The wave of Br-Z3 expression across the medulla therefore matches the wave of afferent in-growth into this structure. Br-Z3 expression in the medulla remains prominent through 27 h APF after which it fades. Throughout this time the axon terminals of photoreceptors R7 and R8 are refining their initial positions in the layers of the medulla (Zhou, 2009).

The Br-Z3 expression in thoracic interneurons suggests that there may be a similar 'sorting phase' within the developing central neuropils. The exact role of Br-Z3 in this sorting process is still being explored, but the ectopic expression of Br-Z3 in the LNv neurons shows that the presence of Br-Z3 during initial outgrowth results in excessive sprouting. If the expression of Br-Z3 in the secondary neurons marks a sorting phase, then groups of these neurons may vary in the time when the sorting is completed. Notably, the early, Chinmo+ secondary neurons lose Br-Z3 expression by the start of wandering. This early loss may mean an early commitment to initial targets, and they may serve a pioneer function in organizing the structure of the nascent adult neuropils. The bulk of secondary neurons, however, retain BR-Z3 expression until neurogenesis is almost complete in the central brain and the thorax. The last-born cells in these regions show only a very brief Br-Z3 expression as they are inserting into a neuropil that is already highly sorted (Zhou, 2009).

Br-Z3 expression in the larval γ neurons of the mushroom body is notable because it is the rare case of functioning neurons that continue to express Br-Z3. The significance of this expression is not known. This may represent a unique function of Br-Z3 in larval mushroom body neurons. Alternatively, it may indicate that a 'sorting phase' may continue even in mature neurons. γ neurons are continually added through the early larval stages and the older γ neurons may have to continue to sort and rewire as the γ class swells in number. Besides the mushroom body γ neurons, it was found that a few mature-looking neurons in the brain and thorax of newly hatched larvae were also BR-Z3 positive. The identity of these neurons is not known, but as proposed for the mushroom body γ neurons, they may also have some unique plasticity demands as the larva grows (Zhou, 2009).

A final puzzle involving Br-Z3 is its absence from motoneurons. In the growing larva, the axons from the lineage 15 motoneurons extend to the leg imaginal disc where they end without obvious elaborations. The subsequent interactions of their sprouting axon terminals with nascent muscles may require a very different set of processes from those used by interneurons as they sort out central targets (Zhou, 2009).

The BTB domain transcription factor gene chinmo was the first temporal specifying gene found for the postembryonic neurogenic period. Its presence in early-born neurons in both mushroom body and antennal lobe projection neuron lineages establishes early neuronal phenotypes in these lineages. Just prior to metamorphosis, the secondary neuron lineages can be divided into older, Chinmo+ and younger, Broad+ neurons, the latter being due to the Br-Z3 isoform. Both Chinmo and Br-Z3 disappear early in metamorphosis, though, and it is not known how their transient expression is then turned into persisting markers of temporal identity. Other Broad isoforms, notably Br-Z1 and Br-Z4, were found to provide permanent molecular time-stamps of birthdates (Zhou, 2009).

Prior to metamorphosis, the Br-Z4+ neurons were always found clustered within a given lineage, a pattern suggesting that they arose from consecutively born GMCs. Br-Z4+ neurons were found in all of the thoracic lineages, except for the very small motor lineage generated by NB 24. Although brain lineages were not examined in detail, the broad pattern of Br-Z4 expression shows that this isoform is also expressed in most brain lineages. Thoracic lineages that made two classes of interneurons had roughly twice the number of Br-Z4+ neurons as lineages that produced only one class of interneuron. The secondary neurons generated by a NB can be divided into two hemilineages, each composed of the collective Notch+ or Notch- daughters arising from the division of the series of GMCs. Studies blocking programmed cell death showed that in lineages with one bundle, one of the daughters consistently dies and only one hemilineage survives. The lineages with both hemilineages had roughly twice the number of neurons compared with those that retain a single hemilineage. This pattern is consistent with a thoracic NB typically generating 13 to 16 GMCs whose daughters will express Br-Z4, but then cell death reducing the number by half in those that have a single hemilineage. Some lineages may deviate from this pattern; for example, lineage 0 has more Br-Z4+ neurons than would be expected for a lineage with a single surviving daughter, and atypically small lineages, such as lineages 2 and 15, have reduced numbers of Br-Z4+ cells (Zhou, 2009).

MARCM clones induced at 24 and 54 h AEL contained their complete complement of Br-Z4+ cells. Those induced at 76 h contained only a few Br-Z4+ neurons, and most Br-Z4+ cells were outside of the clone boundaries. The presence of about six Br-Z4+ neurons within the latter clones suggests that only three to six GMCs with the Br-Z4 fate were left to be born at the time of the clone induction. Given that a NB produces about one GMC per hour at this time in larval life, it is estimated that the GMCs that generate the Br-Z4 cells are born from about 66 to about 80 h AEL (that is, from about 18 h into the second instar until about 8 h of the third larval stage). The analysis of MARCM clones in the adult shows that neurons born during this period in the larva continue to express Br-Z4 after metamorphosis. Br-Z4 expression in the adult therefore provides a molecular marker for the neurons whose GMCs were born during this window of larval life. Br-Z1 is co-localized in a subgroup of the Br-Z4 neurons, but it is not known if this expression defines a specific temporal subset of the Br-Z4 neurons. Within the postembryonic lineages, neuronal fate is clearly related to the time of birth. How Br-Z4 and Br-Z1 participate in these fate decisions remains to be examined (Zhou, 2009).

Broad differs from the embryonic temporal specification genes in that neither Br-Z4 nor Br-Z1 is expressed in the NBs or parent GMCs. Br-Z4 expression only becomes evident towards the start of metamorphosis, well after the neurons are born. This suggests a mechanistic difference in the way in which primary versus secondary neurons are 'time-stamped'. The cells that will express Br-Z4 at the outset of metamorphosis are included in the Br-Z3+ group and not in the Chinmo+ group. Based on its timing, the Chinmo/Broad-Z3 boundary likely marks the start of production of the Br-Z4 neurons. Interestingly, Chinmo and Br-Z3 then disappear early in metamorphosis, whereas Br-Z4 persists as a permanent temporal marker for these neurons. This is the first reported example of a marker for temporal identity that persists into the adult (Zhou, 2009).

A second potential difference from the embryonic pattern is in the signals that control expression of the timing genes. In the embryo, the switch from one timing gene to the next is intrinsically controlled within each lineage. In the postembryonic phase of neurogenesis, by contrast, extrinsic factors appear to play a role in determining the timing of switching from one cell type to another. The switching on and off of GMCs that will make Br-Z4+ neurons coincides with two 'milestones' in Drosophila larval development. The GMCs with the Br-Z4 fate start to be made late in the second instar, around the time larvae achieve the threshold size for metamorphosis and become committed to entering the terminal larval stage. They stop being made at the time in the third instar when the larva achieves its 'critical weight' sufficient to carry it through to pupariation and metamorphosis. Both of these milestones represent endocrine-generated decisions. These extrinsic signals may be responsible for the expression of Br-Z4 and the specification of a distinct subtype of neuron, but this remains to be examined (Zhou, 2009).

Dynamic feedback circuits function as a switch for shaping a maturation-inducing steroid pulse in Drosophila

Steroid hormones trigger the onset of sexual maturation in animals by initiating genetic response programs that are determined by steroid pulse frequency, amplitude and duration. Although steroid pulses coordinate growth and timing of maturation during development, the mechanisms generating these pulses are not known. This study shows that the ecdysone steroid pulse that drives the juvenile-adult transition in Drosophila is determined by feedback circuits in the prothoracic gland (PG), the major steroid-producing tissue of insect larvae. These circuits coordinate the activation and repression of hormone synthesis, the two key parameters determining pulse shape (amplitude and duration). Ecdysone has a positive-feedback effect on the PG, rapidly amplifying its own synthesis to trigger pupariation as the onset of maturation. During the prepupal stage, a negative-feedback signal ensures the decline in ecdysone levels required to produce a temporal steroid pulse that drives developmental progression to adulthood. The feedback circuits rely on a developmental switch in the expression of Broad isoforms that transcriptionally activate or silence components in the ecdysone biosynthetic pathway. Remarkably, this study shows that the same well-defined genetic program that stimulates a systemic downstream response to ecdysone is also utilized upstream to set the duration and amplitude of the ecdysone pulse. Activation of this switch-like mechanism ensures a rapid, self-limiting PG response that functions in producing steroid oscillations that can guide the decision to terminate growth and promote maturation (Moeller, 2013).

Although extensive studies have made it clear that transition to the adult stage in insects requires a high-level pulse of ecdysone, the mechanism that shapes the pulse, by determining its duration and amplitude, has remained unclear. These experiments show that the maturation-inducing pulse that coordinates the juvenile-adult transition in Drosophila is generated by ecdysone feedback control of PG steroidogenic activity. At the end of the third larval instar, ecdysone acts through EcR in a feed-forward circuit to produce the high-level pulse that triggers pupariation in response to PTTH. This illustrates an EcR-dependent positive feedback operating downstream of PTTH to generate a sustained output in terms of biosynthesis in response to neuropeptide signaling (Moeller, 2013).

The feed-forward loop described in this study provides an explanation for a number of previous observations. These studies have indicated that ecdysone can modulate PG steroidogenic activity and that PG cells undergo autonomous activation under long-term culture conditions. Interestingly, autonomous activation is prevented by juvenile hormone (JH), which inhibits br expression. During the last larval instar of holometabolous insects, a drop in JH levels eventually leads to the production of a high-level ecdysone pulse that triggers metamorphosis, although the mechanism underlying this is poorly understood. Since the decline of JH is permissive for br expression, the fact that Br promotes PG steroidogenic activity is likely to explain how the drop in JH results in the production of a high-level ecdysone pulse initiating metamorphosis. Thus, the data provide a link between JH and ecdysone that might explain how the presence of JH prevents metamorphosis (Moeller, 2013).

Observations clearly show that positive feedback is required for the transcriptional upregulation of phantom (phm), disembodied (dib) and shadow (sad), all of which encode enzymes that act at late steps in the ecdysone biosynthetic pathway. By contrast, EcR and Br activity are not necessary for the normal activity of spookier (spok), which is involved in an earlier step in the pathway and whose transcription is regulated by Molting defective, a factor that is not involved in the regulation of the other identified biosynthetic enzymes. In addition, in contrast to the other ecdysone biosynthetic enzymes, Spok is also likely to be regulated at the level of translation and phosphorylation in response to PTTH signaling. Furthermore, expression of PTTH receptor-encoding torso is not EcR and Br dependent, consistent with levels of torso not being synchronized with the ecdysone peaks. Together with the results demonstrating that the feedback is required downstream of Ras in the PG, this shows that the feed-forward loop functions downstream of PTTH to amplify the signal and not for endowing the PG with competence to respond to PTTH (Moeller, 2013).

The findings raise an important issue that challenges the classical view that ecdysone released from the PG is converted to its more active metabolite 20-hydroxyecdysone (20E) in peripheral target tissues, where it interacts with EcR. Although 20E may travel back and inform the PG, a more direct route would be that ecdysone produced by the PG acts on the gland itself or that the PG produces small amounts of 20E that control the activity of the gland. Consistent with these possibilities, reduced expression of shade, which encodes the enzyme that converts ecdysone to 20E, in the PG leads to a developmental arrest in the larval stages and all three Drosophila EcR isoforms can induce transcription in response to ecdysone. Interestingly, recent reports have demonstrated the essential function of E75, DHR3 (Hr46 – FlyBase), βFTZ-F1 and DHR4 in regulating the production of ecdysone in the PG. Although nitric oxide and PTTH regulate the activity of some of these factors, these signals alone are unlikely to explain the regulation of their function in the PG. Based on the results, an obvious possibility is that EcR controls the expression of these classical ecdysone-inducible genes in the PG. Extensive studies on these ecdysone target genes have led to the elucidation of an early response network for steroid hormone action and the molecular characterization of the genetic architecture underlying the cellular responses to steroids. Surprisingly, this study shows that this genetic program that guides the downstream cellular decisions in response to regulatory ecdysone pulses is utilized upstream to shape the pulse by setting its duration and amplitude. Thus, the same genetic components are used for coordinating the production and reception of the steroid signals that drive directional developmental progression (Moeller, 2013).

Previous experiments demonstrated that ecdysone, produced by the PG, induces an inactivation enzyme responsible for clearance of circulating ecdysone (Rewitz, 2010). This study shows that termination of the pulse requires negative feedback that represses PG steroid production activity in coordination with peripheral clearance. How does ecdysone stimulate and repress biosynthesis in the PG through EcR? The results show that EcR induces different Br isoforms, forming circuits that either increase or inhibit the activity of the biosynthetic pathway by regulating the levels of the enzymatic components. Br is required specifically for the juvenile-adulti transition and is expressed during the last instar. This study shows that the appearance of Br in the PG requires EcR and correlates with the ecdysone peak. The positive effect of EcR on ecdysone biosynthesis is mediated largely through Br-Z4, which has previously been shown to induce transcription of Niemann-Pick type C-1a (Npc1a), which encodes a key cellular component required in the PG for the delivery of cholesterol as a substrate for steroid synthesis. Together, this suggests that ecdysone-mediated positive feedback coordinates increased substrate delivery with upregulation of the biosynthetic machinery in order to produce the maturation-inducing ecdysone pulse. Conversely, the Br-Z1 isoform inhibits ecdysone synthesis, forming a negative feedback that is important for the decline of the ecdysone titer during the prepupal stage. Thus, the temporal control of these circuits relies on a dynamic switch in the PG from Br-Z4 to Br-Z1. A similar switch has been found in the imaginal discs, where Br-Z4 rapidly accumulates in response to ecdysone and then disappears several hours later when Br-Z1 is upregulated. It has been suggested that the switch from Br-Z4 to Br-Z1 is regulated at the level of alternative splicing of br transcripts. The data suggest that the switch is a hard-wired genetic timing mechanism rather than being dependent on ecdysone concentrations. This switching might also occur at the enhancer level through competition of binding to overlapping Br-Z1/Z4 regulatory sites, as was found in the phm promoter. Importantly, coupling a negative with a positive feedback through a common regulatory site ensures a self-limiting response by preventing 'run away' synthesis that would otherwise result from positive-feedback amplification alone (Moeller, 2013).

In conclusion, this study shows that the maturation-inducing ecdysone pulse is shaped by an autonomous feed-forward and feedback circuitry within the endocrine tissue that modulates the rate of hormone synthesis. The coupling of these feedback circuits ensures rapid, self-limiting hormone production that translates neuropeptide signaling into a regulatory steroid pulse which functions as a switch to drive developmental progression (Moeller, 2013).

GENE STRUCTURE

Mapping of 31 cDNA clones indicates that approximately 100 kb of the genome is devoted to the synthesis of many BR-C RNAs. The initial molecular analysis by DiBello (1991) revealed that BR-C encodes a family of DNA-binding proteins with an amino-terminal core region linked by alternative splicing to one of three pairs (Z1, Z2 and Z3) of C2H2 zinc-finger domains. Since that time, an additional zinc-finger sequence has been found (Z4), which encodes a pair of zinc-finger motifs similar to the other three. Z1 shares a start site with Z2, located 55 kb upstream of the DNA coding for the core sequence. For Z2, this start site is used alternatively with another start site shared with Z3 and Z4 as well as Z1, located only 10 kb from the shared core sequence. The four different zinc-finger sequences characterizing Z1, Z4, Z2 and Z3 are located on the other side of the core sequence in the order given. Each of the zinc-finger pairs shows a high degree of similarity among the four BR-C protein isoforms. The 68% similarity between Z1 and Z4 domains is unusually high and suggests a similar specificity in DNA binding (Bayer, 1996).

Using a Z1 specific probe, 4.4-kb mRNA species are detected plus a faint, diffuse band in the 10-kb size range. In contrast, the Z2-Z3 and Z4 specific probes do not detect the 4.4 kb transcript. Each hybridizes instead to the three large RNA size classes of 10, 9 and 7 kb forms. The predominant Z2 and Z3 transcripts are the 9 and 7 kb forms, while the predominant z4 transcript is the 7 kb form (Bayer, 1996).

PROTEIN STRUCTURE

Amino Acids - 877 (Z4 transcript)

Structural Domains

Sequence analyses of cDNA clones show that the BR-C encodes a family of related proteins characterized by a common core amino-terminal domain fused to alternate carboxy domains each containing a pair of zinc fingers. Most proteins also contain domains rich in distinctive amino acids located between the common core and C-terminal zinc finger regions (DiBello, 1991).

The J element is a novel DNA sequence involved in the regulated expression of class II major histocompatibility complex genes. DPA, a J element binding protein, contains 688 amino acid residues, including 11 zinc finger motifs of the C2H2 type in the C-terminal region, that are Krüppel-like in the conservation of the H/C link sequence connecting them. The H/C motif is a stretch of seven amino acids connecting the final histindine of one finger to the first cysteine of the next finger. The 160 N-terminal amino acids in the nonfinger region of clone 18 are highly homologous with similar regions of several other human, mouse, and Drosophila sequences, defining a subfamily of Krüppel-like zinc finger proteins termed TAB (tramtrack [ttk]-associated box). This N-terminal region shares sequence homology with Drosophila proteins Tramtrack, Broad Complex and Kelch, a structural component of ring canals. It has been suggested that the TAB is a protein-protein interaction domain (Sugarawa, 1994).

broad: Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

date revised: 1 March 2024

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