Since their isolation in the early 1990s, members of the Hedgehog family of intercellular signaling proteins have come to be recognized as key mediators of many fundamental processes in embryonic development. Their activities are central to the growth, patterning, and morphogenesis of many different regions within the body plans of vertebrates and insects, and most likely other invertebrates. In some contexts, Hedgehog signals act as morphogens in the dose-dependent induction of distinct cell fates within a target field, in others as mitogens regulating cell proliferation or as inducing factors controlling the form of a developing organ. These diverse functions of Hedgehog proteins raise many intriguing questions about their mode of operation. How do these proteins move between or across fields of cells? How are their activities modulated and transduced? What are their intracellular targets? In this article we review some well-established paradigms of Hedgehog function inDrosophila and vertebrate development and survey the current understanding of the synthesis, modification, and transduction of Hedgehog proteins. Embryological studies over much of the last century that relied primarily on the physical manipulation of cells within the developing embryo or fragments of the embryo in culture, provided many compelling examples for the primacy of cell–cell interactions in regulating invertebrate and vertebrate development. The subsequent identification of many of the signaling factors that mediate cellular communication has led to two general conclusions. First, although there are many important signals, most of these fall into a few large families of secreted peptide factors: theWnt (Wodarz and Nusse 1998), fibroblast growth factor (Szebenyi and Fallon 1999), TGFsuperfamily (Massague and Chen 2000), plateletderived growth factor (Betsholtz et al. 2001), ephrin (Bruckner and Klein 1998), and Hedgehog families. Second, parallel studies in invertebrate and vertebrate systems have shown that although the final outcome might look quite different (e.g., a fly vs. a mouse), there is a striking conservation in the deployment of members of the same signaling families to regulate development of these seemingly quite different organisms. This review focuses on one of the most intriguing examples of this phenomenon, that of the Hedgehog family. As with many of the advances in our understanding of the genetic regulation of animal development, hedgehog (hh) genes owe their discovery to the pioneering work of Nusslein-Volhard and Wieschaus (1980). In their screen for mutations that disrupt the Drosophila larval body plan, these authors identified several that cause the duplication of denticles (spiky cuticular processes that decorate the anterior half of each body segment) and an accompanying loss of naked cuticle, characteristic of the posterior half of each segment (see Fig. 1). The ensuing appearance of a continuous lawn of denticles projecting from the larval cuticle evidently suggested the spines of a hedgehog to the discoverers, hence the origin of the name of one of these genes. Other loci identified by mutants with this phenotype included armadillo, gooseberry, and wingless (wg). In contrast, animals mutant for the aptly named naked gene showed the converse phenotype, with denticle belts replaced by naked cuticle in every segment. On the basis of these mutant phenotypes, Nusslein-Volhard and Wieschaus (1980) proposed that these so-called segment-polarity genes regulate pattern within each of the segments of the larval body, individual genes acting within distinct subregions of the emerging segmental pattern. The first important breakthrough in unraveling how segment-polarity genes act came in the mid-1980s with the cloning of two members of the class, wingless and engrailed (en). Wg was shown to be the ortholog of the vertebrate proto-oncogene int1 (subsequently renamed Wnt1 and the founder member of the Wnt family of secreted peptide factors; Rijsewijk et al. 1987), whereas the sequence of en revealed that it encodes a homeodomaincontaining transcription factor (Fjose et al. 1985; Poole et al. 1985). Intriguingly, the two genes were found to be expressed in adjacent narrow stripes of cells in each segment (Martinez Arias et al. 1988). A close spatial relationship between Wnt1 and En expression domains was also reported in the primordial midbrain and hindbrain of the vertebrate embryo (McMahon et al. 1992). AnalyWe dedicate this review to the memory of our dear friend and colleague Rosa Beddington, whose encouragement led to our initial collaboration. 3Corresponding authors. E-MAIL p.w.ingham@sheffield.ac.uk; FAX 0114-222-288. E-MAIL amcmahon@biosun.harvard.edu; FAX (617) 496-3763. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.938601.

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Hedgehog signaling in animal development: paradigms and principles.

■ Abstract Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process. The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among the death proteases. Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage via upstream proteases in an intracellular cascade. Regulation of caspase activation and activity occurs at several different levels: ( a) Zymogen gene transcription is regulated; ( b) antiapoptotic members of the Bcl-2 family and other cellular polypeptides block proximity-induced activation of certain procaspases; and ( c) certain cellular inhibitor of apoptosis proteins (cIAPs) can bind to and inhibit active caspases. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases. Collectively, these scissions disrupt survival pathways and disassemble important architectural components of the cell, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death.

Mammalian caspases: structure ,a ctivation ,s ubstrates, and functions during apoptosis

https://www.cell.com/cell/pdf/0092-8674(92)90471-N.pdf

Homeobox genes and axial patterning

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of ~21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila , at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Nuclear factor (NF)-kappaB and inhibitor of NF-kappaB kinase (IKK) proteins regulate many physiological processes, including the innate- and adaptive-immune responses, cell death and inflammation. Disruption of NF-kappaB or IKK function contributes to many human diseases, including cancer. However, the NF-kappaB and IKK pathways do not exist in isolation and there are many mechanisms that integrate their activity with other cell-signalling networks. This crosstalk constitutes a decision-making process that determines the consequences of NF-kappaB and IKK activation and, ultimately, cell fate.

Integrating cell-signalling pathways with NF-kappaB and IKK function.

The unidirectional maternofetal clearance (Kmf) of 45Ca was measured across the rat placenta over the last one-third of gestation. Kmf for 45Ca normalized to its diffusion coefficient in water (Kmf/Dw) increased 72-fold between days 15 and 22 of gestation from 3.5 +/- 0.3 to 253.1 +/- 22.0 cm/g placenta, respectively. At 15 and 18 days of gestation, Kmf/Dw for 45Ca was similar to Kmf/Dw for the paracellular marker [14C]mannitol, but at 21 and 22 days of gestation, Kmf/Dw for 45Ca was significantly higher than Kmf/Dw for [14C]mannitol, indicating that an additional route of transfer, other than diffusion, becomes available to calcium during this period. Northern hybridization analysis demonstrated that rat placental calbindin9K-to-beta-actin mRNA ratio increased 135-fold between 15 and 22 days of gestation and was temporally associated with the gestational increase in Kmf/Dw for 45Ca. In contrast, rat placental Ca(2+)-ATPase-to-beta-actin mRNA ratio increased only two- to threefold over the same gestational period and did not mirror the gestational changes in calcium clearance. These trends suggest that the expression of placental calbindin9K, but not Ca(2+)-ATPase, may be rate limiting to placental calcium transport in the rat.

Gestational changes in Ca2+ transport across rat placenta and mRNA for calbindin9K and Ca(2+)-ATPase.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2888%2980631-8

Low frequency electric and magnetic fields have different effects on the cell surface

Wnt extracellular signaling molecules have essential roles as regulators of cell proliferation, migration, differentiation, and in epithelial-mesenchymal interactions involved in tissue morphogenesis. Frizzled integral membrane proteins have been shown to function as receptors for Wnt signaling molecules. Vertebrates also produce secreted proteins related to Frizzled receptors, Frizzled-related proteins (FRPs), which contain the cysteine-rich domain of Frizzleds and appear to function as Wnt antagonists. Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions, and many Wnt signaling pathway genes are expressed in the developing tooth at these sites. Here we report the expression of one FRP gene, Mfrzb1, in the rostral mesenchyme of the mandibular primordium. Using explant cultures, we show that expression of Mfrzb1 in the mandibular mesenchyme is under the control of signals derived from the overlying epithelium. Bead implantation experiments in vitro show that FGF8 induces Mfrzb1 expression, whereas BMP4 and SHH proteins have no effect. We studied the effect of ectopic MFrzb1 protein on the developing tooth germs by transplanting explants treated with Mfrzb1 protein into renal capsules, and found it to retard tooth development. This suggests that Wnt signaling is required early in tooth germ formation and that interference with signaling via addition of an antagonist results in retarded development and formation of smaller teeth.

Inhibition of Wnt signaling by exogenous Mfrzb1 protein affects molar tooth size.

The Ectodysplasin and NFkappaB signalling pathways in odontogenesis.

Tooth autotransplantation, allotransplantation and dental implants have existed for many years, but have never been totally satisfactory. Thus, the development of new methods of tooth replacement has become desirable, and with the increasing knowledge of stem cell biology becomes a realistic possibility. Stem cell-based tissue engineering involving the recapitulation of the embryonic environment demonstrates that dental, non-dental, embryonic and adult stem cells can contribute to teeth formation in the appropriate setting. Evidence that stem cell populations may be present in human teeth provides the opportunity to consider biological tooth replacement 'new for old'.

Paul T. Sharpe

Papers

Gestational changes in Ca2+ transport across rat placenta and mRNA for calbindin9K and Ca(2+)-ATPase.

Low frequency electric and magnetic fields have different effects on the cell surface

Inhibition of Wnt signaling by exogenous Mfrzb1 protein affects molar tooth size.

The Ectodysplasin and NFkappaB signalling pathways in odontogenesis.

Regeneration of teeth using stem cell-based tissue engineering.