The formation of the apical ectodermal ridge (AER) is critical for the distal outgrowth and patterning of the vertebrate limb. Recent work in the chick has demonstrated that interplay between the Wnt and Fgf signaling pathways is essential in the limb mesenchyme and ectoderm in the establishment and perhaps the maintenance of the AER. In the mouse, whereas a role for Fgfs for AER establishment and function has been clearly demonstrated, the role of Wnt/␤-catenin signaling, although known to be important, is obscure. In this study, we demonstrate that Wnt3, which is expressed ubiquitously throughout the limb ectoderm, is essential for normal limb development and plays a critical role in the establishment of the AER. We also show that the conditional removal of ␤-catenin in the ventral ectodermal cells is sufficient to elicit the mutant limb phenotype. In addition, removing ␤-catenin after the induction of the ridge results in the disappearance of the AER, demonstrating the requirement for continued ␤-catenin signaling for the maintenance of this structure. Finally, we demonstrate that Wnt/␤-catenin signaling lies upstream of the Bmp signaling pathway in establishment of the AER and regulation of the dorsoventral polarity of the limb. The formation of the apical ectodermal ridge (AER) has long been known to play a critical role in the distal outgrowth and patterning of the vertebrate limb. Classical experiments performed by Saunders (1948) demonstrated that surgical removal of this tissue shortly after its formation results in severe truncations of the entire limb, whereas removal at progressively later stages in development allows outgrowth of the more distal elements in a progressive fashion. Given the critical role that the AER plays in limb development, a major focus within the limb field has been to identify molecules that are involved in its establishment and maintenance. The result of this effort has been the discovery that several signaling pathways interact in the establishment of the AER. For example, recent work in the chick has demonstrated that cooperation between the Wnt/␤-catenin and Fgf signaling pathways is essential in establishing the AER. Briefly, the prevailing model is as follows: Wnt/␤-catenin signaling in the limb mesenchyme appears to be required to activate Fgf10 expression in the same tissue (Kawakami et al. 2001). Mesenchymally derived Fgf10 then regulates expression of Wnt3a in the overlying surface ectoderm and later in the subset of ectodermal cells that is destined to give rise to the AER (Kengaku et al. 1997(Kengaku et al. , 1998. Wnt3a signaling is then thought to act through the ␤-catenin pathway to activate the expression of Fgf8 in these pre-AER cells (Kengaku et al. 1998). Fgf8 signaling to the mesenchyme maintains Fgf10 expression, presumably through the mesenchymal Wnt/␤-catenin pathway, thereby completing a regulatory circuit that is critical for maintenance of the AER (see Kawakami et al. 2001).In the mouse, genetic evidence supports the involvement of both Fgf and Wnt/␤-c...

The Drosophila porcupine gene is required for secretion of wingless and other Wnt proteins, and sporadic mutations in its unique human ortholog, PORCN , cause a pleiotropic X-linked dominant disorder, focal dermal hypoplasia (FDH, also known as Goltz syndrome). We generated a conditional allele of the X-linked mouse Porcn gene and analyzed its requirement in Wnt signaling and embryonic development. We find that Porcn -deficient cells exhibit a cell-autonomous defect in Wnt ligand secretion but remain responsive to exogenous Wnts. Consistent with the female-specific inheritance pattern of FDH, Porcn hemizygous male embryos arrest during early embryogenesis and fail to generate mesoderm, a phenotype previously associated with loss of Wnt activity. Heterozygous Porcn mutant females exhibit a spectrum of limb, skin, and body patterning abnormalities resembling those observed in human patients with FDH. Many of these defects are recapitulated by ectoderm-specific deletion of Porcn , substantiating a long-standing hypothesis regarding the etiology of human FDH and extending previous studies that have focused on downstream elements of Wnt signaling, such as β-catenin. Conditional deletion of Porcn thus provides an experimental model of FDH, as well as a valuable tool to probe Wnt ligand function in vivo.

AMP-activated protein kinase (AMPK) has been identified as a regulator of gene transcription, increasing mitochondrial proteins of oxidative metabolism as well as hexokinase expression in skeletal muscle. In mice, muscle-specific knockout of LKB1, a component of the upstream kinase of AMPK, prevents contraction- and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced activation of AMPK in skeletal muscle, and the increase in hexokinase II protein that is normally observed with chronic AICAR activation of AMPK. Since previous reports show a cAMP response element in the promoter region of the hexokinase II gene, we hypothesized that the cAMP-response element (CRE) binding protein (CREB) family of transcription factors could be targets of AMPK. Using radioisotopic kinase assays, we found that recombinant and rat liver and muscle AMPK phosphorylated CREB1 at the same site as cAMP-dependent protein kinase (PKA). AMPK was also found to phosphorylate activating transcription factor 1 (ATF1), CRE modulator (CREM), and CREB-like 2 (CREBL2), but not ATF2. Treatment of HEK-293 cells stably transfected with a CREB-driven luciferase reporter with AICAR increased luciferase activity approximately threefold over a 24-h time course. This increase was blocked with compound C, an AMPK inhibitor. In addition, AICAR-induced activation of AMPK in incubated rat epitrochlearis muscles resulted in an increase in both phospho-acetyl-CoA carboxylase and phospho-CREB. We conclude that CREB and related proteins are direct downstream targets for AMPK and are therefore likely involved in mediating some effects of AMPK on expression of genes having a CRE in their promoters.

Thomson DM, Porter BB, Tall JH, Kim H-J, Barrow JR, Winder WW. Skeletal muscle and heart LKB1 deficiency causes decreased voluntary running and reduced muscle mitochondrial marker enzyme expression in mice. Am J Physiol Endocrinol Metab 292: E196 -E202, 2007. First published August 22, 2006; doi:10.1152/ajpendo.00366.2006.-LKB1 has been identified as a component of the major upstream kinase of AMP-activated protein kinase (AMPK) in skeletal muscle. To investigate the roles of LKB1 in skeletal muscle, we used muscle-specific LKB1 knockout (MLKB1KO) mice that exhibit low expression of LKB1 in heart and skeletal muscle, but not in other tissues. The importance of LKB1 in muscle physiology was demonstrated by the observation that electrical stimulation of the muscle in situ increased AMPK phosphorylation and activity in the wild-type (WT) but not in the muscle-specific LKB1KO mice. Likewise, phosphorylation of acetyl-CoA carboxylase (ACC) was markedly attenuated in the KO mice. The LKB1KO mice had difficulty running on the treadmill and exhibited marked reduction in distance run in voluntary running wheels over a 3-wk period (5.9 Ϯ 0.9 km/day for WT vs. 1.7 Ϯ 0.7 km/day for MLKB1KO mice). The MLKB1KO mice anesthetized at rest exhibited significantly decreased phospho-AMPK and phospho-ACC compared with WT mice. KO mice exhibited lower levels of mitochondrial protein expression in the red and white regions of the quadriceps. These observations, along with previous observations from other laboratories, clearly demonstrate that LKB1 is the major upstream kinase in skeletal muscle and that it is essential for maintaining mitochondrial marker proteins in skeletal muscle. These data provide evidence for a critical role of LKB1 in muscle physiology, one of which is maintaining basal levels of mitochondrial oxidative enzymes. Capacity for voluntary running is compromised with muscle and heart LKB1 deficiency. adenosine 3Ј-cyclic monophosphate-activated protein kinase; muscle specific LKB1 knockout mouse; muscle mitochondria; citrate synthase AMP-ACTIVATED PROTEIN KINASE (AMPK) is a major regulator of skeletal muscle energy metabolism (3,5,27,29). It is activated in response to exercise and muscle contraction and other conditions of metabolic stress when AMP concentration increases (12,19,26,30). When active, AMPK works to restore cellular energy balance by promoting ATP-generating processes such as fatty acid oxidation and glucose uptake, while inhibiting anabolic processes, such as protein synthesis, that consume ATP (3-5, 14, 27-29). In addition to its role in maintaining energy homeostasis during exercise, AMPK is also thought to play an important role in many adaptations to chronic exercise such as elevations in protein levels of GLUT4, hexokinase II, and mitochondrial proteins (2,7,9,25,31,34). AMPK is a heterotrimer composed of a catalytic ␣-subunit and regulatory ␤-and ␥-subunits. Binding of AMP to the ␥-subunit of AMPK promotes phosphorylation at Thr 172 on its ␣-subunit, which is requisite for its activity. Several...

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