No abstract

The secreted protein Sonic hedgehog (Shh) exerts many of its patterning effects through a combination of short- and long-range signalling. Three distinct mechanisms, which are not necessarily mutually exclusive, have been proposed to account for the long-range effects of Shh: simple diffusion of Shh, a relay mechanism in which Shh activates secondary signals, and direct delivery of Shh through cytoplasmic extensions, termed cytonemes. Although there is much data (using soluble recombinant Shh (ShhN)) to support the simple diffusion model of long-range Shh signalling, there has been little evidence to date for a native form of Shh that is freely diffusible and not membrane-associated. Here we provide evidence for a freely diffusible form of Shh (s-ShhNp) that is cholesterol modified, multimeric and biologically potent. We further demonstrate that the availability of s-ShhNp is regulated by two functional antagonists of the Shh pathway, Patched (Ptc) and Hedgehog-interacting protein (Hip). Finally, we show a gradient of s-ShhNp across the anterior-posterior axis of the chick limb, demonstrating the physiological relevance of s-ShhNp.

Examples of the palladium‐catalyzed coupling of organotin compounds with carbon electrophiles were first reported in 1977 by Kosugi, Shimizu, and Migita. The first study by Stille appeared in 1978. The early work of Beletskaya, using “ligandless” catalysts in cross‐coupling reactions, also often employed organostannanes. In recognition of Stille's comprehensive synthetic and mechanistic studies, this coupling is now referred to as the Stille reaction. R 1 is typically an unsaturated moiety (e.g., vinyl, aryl, heteroaryl, alkynyl, allyl) or less often an alkyl group, and R 2 , the nontransferable ligand, is almost always butyl or methyl. Electrophiles participating in the coupling include halides (almost always bromides or iodides) and sulfonates (most often used are the triflates). Other leaving groups have been used in special cases. The Stille reaction belongs to the larger family of palladium‐ and nickel‐catalyzed cross‐coupling reactions which features, e.g., organomagnesium, organozinc, organoboron, and organosilicon reagents. Organotin reagents are air‐ and moisture‐stable organometallics, and can be conveniently purified and stored. Since they do not react with most common functional groups, the use of protecting groups is almost always unnecessary in conjunction with the Stille reaction. This is a very unusual and attractive feature for an organometallic process. Also, the reaction is often neither air nor moisture sensitive. In some cases, water and oxygen have actually been shown to promote the coupling. Although the reaction as initially described by Stille is often carried out under rather drastic conditions (temperatures of ≥100° are not uncommon), newly developed ligand 11 and the addition of copper(I) salts have solved some of the problems associated with low reactivity. The utility and mildness of the Stille reaction are demonstrated by its frequent use in the final stages of complex natural product syntheses. This chapter attempts a critical and comprehensive coverage of the reaction scope. Mechanistic description of the reaction is rather brief, and the reader is referred to the pertinent literature for a more detailed analysis. All of the relevant literature is covered up to the end of 1994. The reaction was reviewed by Stille in 1986, and by Mitchell in 1992; a rather comprehensive account by Farina and Roth has appeared more recently. Developments that occurred in 1995, as this work was in progress, and that were deemed important were incorporated as much as possible in this review.

No abstract

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2 Tg ). The predicted protein encoded by Rspo2Tg neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2Tg/Tg mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2Tg/Tg lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2Tg and Lrp6 -alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.

In most cells, the ubiquitously expressed Na+/H+exchanger isoform 1 (NHE1) is thought to be a primary regulator of pH homeostasis, cell volume regulation, and the proliferative response to growth factor stimulation. To study the function of NHE1 during embryogenesis when these cellular processes are very active, we targeted the Nhe1 gene by replacing the sequence encoding transmembrane domains 6 and 7 with the neomycin resistance gene. NHE activity assays on isolated acinar cells indicated that the targeted allele is functionally null. Although the absence of NHE1 is compatible with embryogenesis, Nhe1 homozygous mutants (−/−) exhibit a decreased rate of postnatal growth that is first evident at 2 wk of age. At this time, Nhe1 −/− animals also begin to exhibit ataxia and epileptic-like seizures. Approximately 67% of the −/− mutants die before weaning. Postmortem examinations frequently revealed an accumulation of a waxy particulate material inside the ears, around the eyes and chin, and on the ventral surface of the paws. Histological analysis of adult tissues revealed a thickening of the lamina propria and a slightly atrophic glandular mucosa in the stomach.

In this report we describe the developmental expression and function of Sp8, a member of the Sp family of zinc finger transcription factors, and provide evidence that the legless transgene insertional mutant is a hypomorphic allele of the Sp8 gene. Sp8 is expressed during embryogenesis in the forming apical ectodermal ridge (AER), restricted regions of the central nervous system, and tail bud. Targeted deletion of the Sp8 gene gives a striking phenotype, with severe truncation of both forelimbs and hindlimbs, absent tail, as well as defects in anterior and posterior neuropore closure leading to exencephaly and spina bifida. Outgrowth of the limb depends on formation of the AER, a signaling center that forms at the limb bud apex. In Sp8 mutants, the AER precursor cells are induced and initially express multiple appropriate marker genes, but expression of these genes is not maintained and progression to a mature AER is blocked. These observations indicate that Sp8 functions downstream of Wnt3, Fgf10, and Bmpr1a in the signaling cascade that mediates AER formation.apical ectodermal ridge ͉ exencephaly ͉ spina bifida ͉ zinc finger ͉ legless

Because of the complexity derived from the existence of various phosphoinositide 3-kinase (PI3K) isoforms and their differential roles in cancers, development of PI3K inhibitors with differential pharmacologic and pharmacokinetic profiles would allow best exploration in different indications, combinations, and dosing regimens. Here, we report BAY 80-6946, a highly selective and potent pan-class I PI3K inhibitor with subnanomolar IC 50 s against PI3Ka and PI3Kd. BAY 80-6946 exhibited preferential inhibition (about 10-fold) of AKT phosphorylation by PI3Ka compared with PI3Kb in cells. BAY 80-6946 showed superior antitumor activity (>40-fold) in PIK3CA mutant and/or HER2 overexpression as compared with HER2-negative and wild-type PIK3CA breast cancer cell lines. In addition, BAY 80-6946 revealed potent activity to induce apoptosis in a subset of tumor cells with aberrant activation of PI3K as a single agent. In vivo, single intravenous administration of BAY 80-6946 exhibited higher exposure and prolonged inhibition of pAKT levels in tumors versus plasma. BAY 80-6946 is efficacious in tumors with activated PI3K when dosed either continuously or intermittently. Thus, BAY 80-6946 induced 100% complete tumor regression when dosed as a single agent every second day in rats bearing HER2-amplified and PIK3CA-mutated KPL4 breast tumors. In combination with paclitaxel, weekly dosing of BAY 80-6946 is sufficient to reach sustained response in all animals bearing patient-derived non-small cell lung cancer xenografts, despite a short plasma elimination half-life (1 hour) in mice. Thus, BAY 80-6946 is a promising agent with differential pharmacologic and pharmacokinetic properties for the treatment of PI3K-dependent human tumors.