hunchback regulates the temporal identity of neuroblasts in Drosophila. Here we show that hbl-1, the C. elegans hunchback ortholog, also controls temporal patterning. Furthermore, hbl-1 is a probable target of microRNA regulation through its 3'UTR. hbl-1 loss-of-function causes the precocious expression of adult seam cell fates. This phenotype is similar to loss-of-function of lin-41, a known target of the let-7 microRNA. Like lin-41 mutations, hbl-1 loss-of-function partially suppresses a let-7 mutation. The hbl-1 3'UTR is both necessary and sufficient to downregulate a reporter gene during development, and the let-7 and lin-4 microRNAs are both required for HBL-1/GFP downregulation. Multiple elements in the hbl-1 3'UTR show complementarity to regulatory microRNAs, suggesting that microRNAs directly control hbl-1. MicroRNAs may likewise function to regulate Drosophila hunchback during temporal patterning of the nervous system.

Caenorhabditis elegans let-7, a founding member of the microRNA family, is predicted to bind to six sites in the 3UTR of the mRNA of its target gene, lin-41, to downregulate LIN-41. Here, we demonstrate that wild-type let-7 microRNA binds in vitro to RNA from the lin-41 3UTR. This interaction is dependent on two conserved let-7 complementary sites (LCSs). A 27-nucleotide sequence between the LCSs is also necessary for downregulation in vivo. LCS mutations compensatory to the lesion in let-7(n2853) can partially restore lin-41 3UTR function in a let-7(n2853) background, providing the first experimental evidence for an animal miRNA binding directly to its validated target in vivo.Supplemental material is available at http://www.genesdev. org.Received October 28, 2003; revised version accepted December 8, 2003. Genes that regulate the timing of Caenorhabditis elegans post-embryonic development are called heterochronic genes. Several heterochronic genes are regulated post-transcriptionally through elements in their 3ЈUTR. This regulation is mediated by small temporal RNAs (stRNAs), like lin-4 and let-7, expressed at the first larval stage (L 1 ) and L 3 stage, respectively (Wightman et al. 1993;Feinbaum and Ambros 1999;Reinhart et al. 2000;Johnson et al. 2003). stRNAs are predicted to interact with partially complementary sequences in the 3ЈUTRs of their target genes and down-regulate gene expression through an unknown mechanism; however, the direct binding of an stRNA to its validated target has not been demonstrated experimentally in vivo.lin-4 is thought to act by antisense base pairing to seven complementary sites in the lin-14 3ЈUTR to downregulate lin-14 post-transcriptionally (Lee et al. 1993;Wightman et al. 1993). The 3ЈUTR of lin-14 is sufficient for temporal down-regulation; experiments using a lacZ reporter gene fused to the lin-14 3ЈUTR show stage-specific down-regulation (Wightman et al. 1993). The seven complementary sites in the lin-14 3ЈUTR are also conserved in Caenorhabditis briggsae, a closely related Caenorhabditis species, and are deleted in lin-14 gain-offunction mutants, demonstrating that these sites are crucial to normal lin-14 regulation (Wightman et al. 1993;Ha et al. 1996). Binding of lin-4 to lin-14 is thought to block protein synthesis or accumulation after the initiation of translation (Olsen and Ambros 1999).let-7 controls the larval-to-adult (L/A) transition and let-7 mutants display retarded terminal differentiation of seam cells . . This conservation suggests that the mechanism of gene regulation is also present in higher eukaryotes. let-7 has targets in addition to lin-41, namely, hbl-1, which has eight predicted LCSs in its 3ЈUTR .lin-4 and let-7 are the founding members of a larger family of microRNAs (miRNAs). MiRNAs are genomically encoded, untranslated RNA molecules of ∼20-25 nucleotides (nt), transcribed as precursors that can form a hairpin loop (Ambros et al. 2003). For example, the let-7 miRNA precursor RNA is ∼70 nt and is processed into a mature 22-nt molecule by th...

MicroRNAs are small approximately 22 nucleotide regulators of numerous biological processes and bind target gene messenger RNAs to control gene expression. The C. elegans microRNA let-7 and its target lin-41 were the first microRNA::target interaction to be validated in vivo. let-7 molecules form imperfect duplexes with two required let-7 complementary sites in the lin-41 3' UTR. Here, we show that base pairing at both the 5' and 3' ends of the let-7 binding site, as well as the presence of unpaired RNA residues in the predicted duplexes, are required for lin-41 downregulation. In this study, our model for microRNA::target interactions also demonstrates that the context of a microRNA binding can be critical for function, revealing an unforeseen complexity in microRNA::target interactions.

OBJECTIVE-To examine in vivo in a rodent model the potential role of AMP-activated protein kinase (AMPK) within the ventromedial hypothalamus (VMH) in glucose sensing during hypoglycemia.RESEARCH DESIGN AND METHODS-Using gene silencing technology to selectively downregulate AMPK in the VMH, a key hypothalamic glucose-sensing region, we demonstrate a key role for AMPK in the detection of hypoglycemia. In vivo hyperinsulinemic-hypoglycemic (50 mg dl Ϫ1 ) clamp studies were performed in awake, chronically catheterized Sprague-Dawley rats that had been microinjected bilaterally to the VMH with an adeno-associated viral (AAV) vector expressing a short hairpin RNA for AMPK␣. (ϳ60%) and epinephrine (ϳ40%) responses to acute hypoglycemia. Rats with VMH AMPK downregulation also required more exogenous glucose to maintain the hypoglycemia plateau and showed significant reductions in endogenous glucose production and wholebody glucose uptake. RESULTS-In comparison with control studies, VMH AMPK downregulation resulted in suppressed glucagon CONCLUSIONS-We

-In nondiabetic rodents, AMP-activated protein kinase (AMPK) plays a role in the glucose-sensing mechanism used by the ventromedial hypothalamus (VMH), a key brain region involved in the detection of hypoglycemia. However, AMPK is regulated by both hyper-and hypoglycemia, so whether AMPK plays a similar role in type 1 diabetes (T1DM) is unknown. To address this issue, we used four groups of chronically catheterized male diabetic BB rats, a rodent model of autoimmune T1DM with established insulin-requiring diabetes (40 Ϯ 4 pmol/l basal c-peptide). Two groups were subjected to 3 days of recurrent hypoglycemia (RH), while the other two groups were kept hyperglycemic [chronic hyperglycemia (CH)]. All groups subsequently underwent hyperinsulinemic hypoglycemic clamp studies on day 4 in conjunction with VMH microinjection with either saline (control) or AICAR (5-aminoimidazole-4-carboxamide) to activate AMPK. Compared with controls, local VMH application of AICAR during hypoglycemia amplified both glucagon [means Ϯ SE, area under the curve over time (AUC/t) 144 Ϯ 43 vs. 50 Ϯ 11 ng ⅐ l Ϫ1 ⅐ min Ϫ1 ; P Ͻ 0.05] and epinephrine [4.27 Ϯ 0.96 vs. 1.06 Ϯ 0.26 nmol ⅐ l Ϫ1 ⅐ min Ϫ1 ; P Ͻ 0.05] responses in RH-BB rats, and amplified the glucagon [151 Ϯ 22 vs. 85 Ϯ 22 ng ⅐ l Ϫ1 ⅐ min Ϫ1 ; P Ͻ 0.05] response in CH-BB rats. We conclude that VMH AMPK also plays a role in glucose-sensing during hypoglycemia in a rodent model of T1DM. Moreover, our data suggest that it may be possible to partially restore the hypoglycemia-specific glucagon secretory defect characteristic of T1DM through manipulation of VMH AMPK. epinephrine; glucagon; ventromedial hypothalamus; adeno-associated viral vector HYPOGLYCEMIA REMAINS THE MAJOR limiting factor to intensive insulin therapy in type 1 diabetes (T1DM) (9). Individuals with T1DM are particularly prone to hypoglycemia both because of their need for exogenous and unregulated insulin therapy and because of defects in the normal physiological counterregulatory response to acute hypoglycemia (29). Developing therapies to reduce the frequency or severity of hypoglycemia will require a greater understanding of the physiological mechanisms underlying hypoglycemia detection and the impact of T1DM on these mechanisms.Falling glucose levels are detected within discrete regions of the brain (3-5, 14, 40), and periphery (11). Of these, the ventromedial hypothalamus (VMH) is thought to play a key role in the detection and integration of hypoglycemia signals and subsequent triggering of a counterregulatory defense response (3-5, 13, 30, 31, 33, 42, 47). Emerging evidence supports a key role for the serine/threonine kinase AMPactivated protein kinase (AMPK) in the sensing of hypoglycemia within the VMH. Local activation of AMPK in the VMH with 5-aminoimidazole-4-carboxamide (AICAR) amplified the glucose counterregulatory response to hypoglycemia in normal Sprague-Dawley rats (31) and reversed impaired hormonal counterregulatory responses in normal rats exposed to recurrent hypoglycemia (RH) (30). Moreover, sele...