let-7 RNA is first detected in late third instar (L3) larvae (Pasquinelli, 2000) around the time a pulse of the steroid hormone 20-hydroxyecdysone (ecdysone) triggers puparium formation (PF) and the onset of metamorphosis. To explore the potential influence of ecdysone on let-7 RNA expression, the profile of let-7 expression was determined during a period from late third larval instar, through prepupal development (a period marked by the sequential expression of ecdysone-inducible genes), to the adult stage. let-7 RNA is first detected at low levels around 4 h before PF. This stage coincides with a short period of high ecdysone titer that triggers PF. let-7 RNA remains at low levels during prepupal development, and then rapidly accumulates to high levels throughout pupal development, reaching a maximum of expression during the second day of pupal life. This rise in let-7 RNA parallels a prolonged pulse of high-level ecdysone secretion. This correlation between the profile of let-7 expression and the time course of changes in ecdysone titer suggests that ecdysone could induce let-7 transcription (Sempere, 2002).
The tissue-specificity of let-7 expression was determined in prepupae and adult animals in order to correlate expression domains with function. Tanning prepupae (0-4 h after PF) were dissected to obtain a variety of tissues: brains, imaginal discs, fat bodies, salivary glands, and Malphigian tubules. Levels of let-7 RNA in these tissues were quantified by Northern analysis. let-7 RNA was detected in all tissues examined, though at relatively higher levels in fat bodies and imaginal discs. The widespread expression of let-7 RNA suggests that let-7 could mediate diverse metamorphic processes, such as the terminal differentiation of imaginal discs and apoptosis of salivary glands and fat bodies. Levels of let-7 RNA in adult ovaries and carcasses (tissues remaining after ovary dissection) were quantified by Northern analysis. let-7 is expressed in both somatic and gonadal tissues in the adult (Sempere, 2002).
The Drosophila let-7-Complex (let-7-C) is a polycistronic locus encoding three ancient microRNAs: let-7, miR-100, and fly lin-4 (miR-125). The let-7-C locus is principally expressed in the pupal and adult neuromusculature. let-7-C knockout flies appear normal externally but display defects in adult behaviors (e.g., flight, motility, and fertility) as well as clear juvenile features in their neuromusculature. The function of let-7-C to ensure the appropriate remodeling of the abdominal neuromusculature during the larval-to-adult transition is carried out predominantly by let-7 alone. This heterochronic role of let-7 is likely just one of the ways in which let-7-C promotes adult behavior (Sokol, 2008).
Mutations in heterochronic genes in Caenorhabditis elegans cause cells in particular lineages to express their stage-specific fates earlier or later than normal. Detailed analysis of these genes has revealed a regulatory pathway of heterochronic genes that specifies the timing of cellular development in diverse cell types and thereby ensures a coordinated schedule of developmental events throughout the worm. The existence of the heterochronic gene pathway in worms and the conservation of some of its components through animal evolution suggest that functionally analogous pathways could also coordinate developmental timing in higher organisms. Two of these highly conserved components of the heterochronic pathway, let-7 and lin-4, are microRNAs (miRNAs), a class of small RNAs that post-transcriptionally modulate the expression of target transcripts. The sequences and developmentally regulated expression profiles of let-7 and lin-4 are conserved among diverse bilaterians. For example, Drosophila let-7 and miR-125 (fly lin-4) are robustly up-regulated during metamorphosis, as is another highly conserved miRNA, miR-100. All three of these ancient miRNAs are encoded in a 1-kb region of the Drosophila genome, and their clustered organization has been conserved and duplicated in vertebrates. These findings suggest that miR-100, let-7, and miR-125 coordinately control gene expression to regulate developmental timing in animals. To test this hypothesis, the roles of miR-100, let-7, and miR-125 were analyzed in Drosophila; these miRNAs are required for normal adult behavior, suggesting roles in neural development and/or function. let-7 in particular is required for remodeling of the fly neuromusculature during the larval-to-adult transition, confirming that a general developmental timing function of let-7 has been evolutionarily conserved from worms to flies (Sokol, 2008).
The clustered organization of Drosophila miR-100, let-7, and miR-125 suggests that these miRNAs are co-transcribed as a single polycistronic transcript. To test this hypothesis, cDNAs generated from genomic regions between miR-100 and let-7 and between let-7 and miR-125 were isolated using 5' and 3' rapid amplification of cDNA ends (RACE). This analysis identified two overlapping cDNA fragments that corresponded to a 2435-nucleotide (nt) primary transcript that encoded the ~70-nt hairpin sequences of miR-100, let-7, and miR-125, and was comprised of three exons that spanned 17,400 kb of genomic DNA. It is concluded that miR-100, let-7, and miR-125 are cotranscribed from a single locus, which is referred to as the let-7-Complex (let-7-C) since let-7 was the first of these miRNAs identified in Drosophila. It is inferred that the miR-100, let-7, and miR-125 clusters in the genomes of other animals also represent single polycistronic loci. It should be noted that cotranscribed let-7-C miRNAs may not always be coexpressed, given that post-transcriptional processing of mature miRNAs from primary transcripts can be subject to developmental regulation (Sokol, 2008).
To investigate whether let-7-C miRNAs collectively regulate developmental timing in Drosophila, two independent let-7-C knockout strains, let-7-CKO1 and let-7-CGKI, were generated. Both strains lack expression of the mature processed forms of miR-100, let-7, and miR-125. To test whether the activity of each of the let-7-C miRNAs is required for let-7-C function, the phenotypes of three different let-7-C derivative strains were analyzed in which the expression of miR-100, let-7, or miR-125 had been eliminated individually. These singly mutant strains are referred to as miR-100δ, let-7δ, and miR-125δ, respectively. miR-100δ mutants functioned normally in all behavioral assays, indicating that miR-100 was not solely responsible for any of the identified let-7-C functions. None of the single mutant strains displayed strong male fertility or climbing defects, suggesting that for normal male fertility and climbing behavior, the combinatorial action of any two let-7-C microRNAs could suffice. In contrast, let-7δ and miR-125δ mutants displayed severely reduced spontaneous locomotion as well as partial defects in flight. The normal climbing and nearly normal flight of let-7δ and miR-125δ mutants suggested that their severely impaired spontaneous locomotory activity was not simply the consequence of physically or metabolically impaired mobility, but rather likely reflected a behavioral deficit of neurological origin. Finally, let-7δ mutants alone displayed moderately severe defects in female fertility and oviposition, indicating that let-7 is required for an essential function to promote female reproduction (Sokol, 2008).
To identify the specific place where let-7-C miRNAs may function to promote adult behavior, the spatiotemporal expression pattern of the let-7-C locus was examined. The let-7-CGKI strain, in which the yeast transcriptional activator Gal4 had been inserted into the let-7-C locus, was used to drive expression of Gal4-dependent transgenes encoding membrane-bound or nuclear forms of GFP. A UAS-let-7-C transgene placed under the control of the let-7-C::Gal4 insertion restored miR-100, let-7, and miR-125 expression as well as climbing activity to let-7-CKO1/GKI mutants. Three characteristics of the let-7-C::Gal4 expression pattern are outlined below. Initially, let-7-C::Gal4 was expressed in neurons throughout the adult brain and ventral nerve cord, and this adult CNS expression was the culmination of a dramatic expansion in the spatial expression pattern of let-7-C::Gal4 that occurred in the CNS during the first half of metamorphosis. Second, let-7-C::Gal4 was expressed in neurons that innervated structures throughout the adult, including sensory organs in the head, flight muscles in the thorax, and the alimentary tract, the male and female reproductive tracts, and the male and female genitalia in the abdomen. It is noted that let-7-C::Gal4 was very densely expressed in the posterior tip of the adult abdominal ganglion, as well as in motoneurons that projected posteriorly and innervated two distinct sets of abdominal muscles, the dorsal internal oblique muscles (DIOM) and the dorsal muscles (DM). The DIOMs are remnants of the larval body wall that persist through metamorphosis (presumably to function in the process of eclosion) and in the wild type are fated to die within 12 h of eclosion. In contrast, the DMs are the adult body-wall muscles and are derived from larval myoblasts that undergo myogenesis during metamorphosis. Third, let-7-C::Gal4 was not only expressed in motoneurons but in muscle cells as well, including the DIOMs and DMs. Taken together, the expression of let-7-C::Gal4 in pupal and adult neurons and muscles is consistent with the hypothesis that the behavioral phenotypes of let-7-C mutant adults are the consequence of defects in the metamorphosis of the neuromusculature (Sokol, 2008).
To test whether let-7-C miRNAs play a role in specifying the configuration of the adult neuromusculature, the abdominal muscle system of let-7-CKO1/GKI mutants was examined, since let-7-C is expressed in abdominal motoneurons and muscles. Two very clear and highly penetrant defects were found. (1) the DIOMs that ordinarily decay during post-eclosion maturation of wild-type flies failed to disappear in older let-7-CKO1/GKI mutants. (2) The DMs of let-7-CKO1/GKI mutant adults were clearly smaller than those in age-matched wild-type controls. Restoration of let-7-C expression rescued both the DIOM and DM phenotype. To test whether let-7-CKO1/GKI mutant muscle phenotypes were the consequence of defects apparent prior to the onset of metamorphosis, the musculature and myoblasts of let-7-CKO1/GKI larvae were examined, and it was found that both appeared normal. It is therefore concluded that the abdominal muscle system of let-7-CKO1/GKI mutant adults failed to complete its larval-to-adult remodeling, displaying both persistent pupal as well as immature adult characteristics. This is interpreted as a heterochronic phenotype, since let-7-CKO1/GKI mutant adults exhibited both juvenile features (e.g., muscle system morphology) as well as mature adult traits (e.g., external appearance) at the same time (Sokol, 2008).
To test whether let-7-C affects the remodeling of other internal tissues, the morphogenesis of the CNS during metamorphosis in let-7-CKO1/GKI mutants was examined and found that at a gross level, CNS development appeared to have proceeded normally. To examine the results of nervous system remodeling in finer detail, focus was placed on the morphology of motoneurons that innervate the DIOMs or the DMs. DIOMs are innervated by DIOM motoneurons, which also degenerate after eclosion. The DIOMs and their DIOM motoneurons, however, are triggered to die at different times and therefore may be controlled by independent signals. Interestingly, it was found that the neuromuscular junctions (NMJs) connecting DIOMs and their innervating motoneurons failed to decay in let-7-CKO1/GKI mutant adults, indicating that the DIOMs and DIOM motoneurons persisted together. These data suggest that let-7-C functions to coordinate the fates of DIOMs and DIOM motoneurons. Similarly, the reduced size of let-7-CKO1/GKI mutant DMs was reflected in clear defects in let-7-CKO1/GKI mutant DM NMJs, which were either completely absent, shorter in length than wild-type NMJs, or devoid of boutons, appearing as long, thin processes along the length of the DM. It is concluded that the heterochronic abdominal muscle defect was reflected in a corollary nervous system defect, supporting the hypothesis that disruption of neuromusculature remodeling could underlie at least some of the let-7-CKO1/GKI mutant behavioral phenotypes (Sokol, 2008).
The striking similarity between let-7-CKO1/GKI mutant phenotypes and the phenotypes associated with manual denervation of abdominal muscles prior to metamorphosis was noted. In both cases, adult DM muscles fail to grow to wild-type width, contain fewer nuclei, and display aberrant NMJs. However, the let-7-C mutation and denervation differ in at least one respect: their effect on the male-specific muscle of Lawrence (MOL). MOLs are present in let-7-CKO1/GKI adult males but absent in manually denervated adult males. The overall similarity between the effects of genetic depletion of let-7-C and muscle denervation during metamorphosis supports the hypothesis that let-7-C is required to regulate an interaction between muscles and motoneurons during neuromusculature remodeling (Sokol, 2008).
To test whether the activities of miR-100, let-7, or miR-125 are required individually for neuromusculature remodeling, the abdominal muscle pattern as well as DM NMJs were examined in miR-100δ, let-7δ, and miR-125δ single mutants. Two-day-old miR-100δ and miR-125δ males retained none of the six DIOMs, while let-7δ males retained 61% ± 28.5% of DIOMs. Although the frequency of complete DIOM retention is lower in let-7δ mutants compared to let-7-CKO1/GKI mutants, it is noted that 83% ± 25% of let-7δ mutant DIOMs had arrested at some stage in the process of degeneration. With respect to both the DM and DM NMJ phenotype, it was similarly found that miR-100δ and miR-125δ mutants appeared normal, whereas let-7δ mutants phenocopied let-7-CKO1/GKI mutants. miR-100δ and miR-125δ DMs were 18.3 ± 0.8 µm and 16.8 ± 0.8 µm in width, respectively, while let-7δ DMs were 12 ± 1.8 µm in length. Similarly, miR-100δ and miR-125δ NMJs were 45.9 ± 10.2 µm and 55.1 ± 13.3 µm in length, respectively, while let-7δ NMJs were 12.5 ± 5 µm in length. For the sake of consistency, all the morphological data quantified in this study were collected from adult males. However, let-7-CKO1/GKI and let-7δ mutant females exhibited DM and DIOM phenotypes identical to their male siblings, suggesting that the reduced egg-laying displayed by let-7δ mutant females might be a consequence of defects in their abdominal neuromusculature. From these data, it is concluded that the activity of let-7 alone was predominantly responsible for let-7-C-dependent remodeling of the abdominal neuromusculature, and therefore that a heterochronic let-7 role in regulating developmental transitions had been evolutionarily conserved from worms to flies (Sokol, 2008).
The functional dissection of Drosophila let-7-C presented in this study indicates that let-7-C is required for adult behavior and that defects in neuromusculature remodeling correlate with some aspects of this requirement. The perdurance of juvenile features in adult Drosophila let-7 mutants is analogous to the reiteration of larval cell fates in adult Caenorhabditis elegans let-7 mutants (Reinhart et al. 2000), confirming the suggestion by Pasquinelli et al. in 2000 that let-7 might control developmental transitions in diverse bilateria. Future work in flies should extend this analysis to identify the relevant mRNA targets that Drosophila let-7 regulates in its heterochronic role and to examine how this heterochronic function is integrated into the more general requirements of the let-7-C locus in promoting adult behavior. For the most part, the set of targets predicted for Drosophila let-7 are distinct from those predicted for C. elegans let-7. Unpublished observations indicate that one of Drosophila let-7's targets is the transcription factor abrupt, although it was also found that ectopic expression of abrupt in a let-7-C::Gal4-driven pattern is not sufficient to recapitulate the let-7δ phenotype. The conservation of the genomic clustering as well as neuronal expression of let-7, mir-125, and mir-100 from flies to vertebrates suggests that let-7-C loci could function in neuromuscular and/or neuronal remodeling in mammals. Future work on let-7-C should reveal how its diverse effects on temporal cell fates, developmental timing, and neuronal remodeling are related (Sokol, 2008).
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date revised: 15 February 2009
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