Instituto de Neurociencias de Alicante

Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, and glands. The recent cloning of two genes responsible for these syndromes has led to the identification of a novel TNF family ligand, ectodysplasin, and TNF receptor, edar. This has indicated a developmental regulatory role for TNFs for the first time. Our in situ hybridization analysis of the expression of ectodysplasin (encoded by the Tabby gene) and edar (encoded by the downless gene) during mouse tooth morphogenesis showed that they are expressed in complementary patterns exclusively in ectodermal tissue layer. Edar was expressed reiteratively in signaling centers regulating key steps in morphogenesis. The analysis of the effects of eight signaling molecules in the TGF␤, FGF, Hh, Wnt, and EGF families in tooth explant cultures revealed that the expression of edar was induced by activin␤A, whereas Wnt6 induced ectodysplasin expression. Moreover, ectodysplasin expression was downregulated in branchial arch epithelium and in tooth germs of Lef1 mutant mice, suggesting that signaling by ectodysplasin is regulated by LEF-1-mediated Wnt signals. The analysis of the signaling centers in tooth germs of Tabby mice (ectodysplasin null mutants) indicated that in the absence of ectodysplasin the signaling centers were small. However, no downstream targets of ectodysplasin signaling were identified among several genes expressed in the signaling centers. We conclude that ectodysplasin functions as a planar signal between ectodermal compartments and regulates the function, but not the induction, of epithelial signaling centers. This TNF signaling is tightly associated with epithelial-mesenchymal interactions and with other signaling pathways regulating organogenesis. We suggest that activin signaling from mesenchyme induces the expression of the TNF receptor edar in the epithelial signaling centers, thus making them responsive to Wnt-induced ectodysplasin from the nearby ectoderm. This is the first demonstration of integration of the Wnt, activin, and TNF signaling pathways.

Members of the LEF-1/TCF family of transcription factors have been implicated in the transduction of Wnt signals. However, targeted gene inactivations of Lef1, Tcf1, or Tcf4 in the mouse do not produce phenotypes that mimic any known Wnt mutation. Here we show that null mutations in both Lef1 and Tcf1, which are expressed in an overlapping pattern in the early mouse embryo, cause a severe defect in the differentiation of paraxial mesoderm and lead to the formation of additional neural tubes, phenotypes identical to those reported for Wnt3a-deficient mice. In addition, Lef1 −/− Tcf1 −/− embryos have defects in the formation of the placenta and in the development of limb buds, which fail both to express Fgf8 and to form an apical ectodermal ridge. Together, these data provide evidence for a redundant role of LEF-1 and TCF-1 in Wnt signaling during mouse development.

Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formation of organs that depend on inductive interactions between epithelial and mesenchymal tissues. In previous tissue recombination experiments with normal and Lef1 −/− tooth germs, we found that the effect of LEF1 expression in the epithelium is tissue nonautonomous and transferred to the subjacent mesenchyme. Here we examine the molecular basis for LEF1 function and find that the epithelium of the developmentally arrested Lef1 −/− tooth rudiments fails to express Fgf4, Shh, and Bmp4, but not Wnt10a. We identify the Fgf4 gene as a direct transcriptional target for LEF1 and show that beads soaked with recombinant FGF4 protein can fully overcome the developmental arrest of Lef1 −/− tooth germs. In addition, we find that FGF4 beads induce rapidly the expression of Fgf3 in dental mesenchyme and that both epithelial and mesenchymal FGF proteins induce the delayed expression of Shh in the epithelium. Taken together, these data indicate that a single target of LEF1 can account for the function of LEF1 in tooth development and for a relay of a Wnt signal reception to a cascade of FGF signaling activities, allowing for a sequential and reciprocal communication between epithelium and mesenchyme.

Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. Here, we examine the molecular basis for this presumed hierarchy and show that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1-␤-catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1mediated regulation of Dll1.

Members of the LEF-1͞TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with ␤-catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 were previously found to show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, were shown to result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, we genetically examine the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a. Analysis of the expression of Brachyury in Lef1 ؊/؊ Tcf1 ؊/؊ mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicate that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. We also show that the expression of an activated form of LEF-1, containing the ␤-catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation. Galceran et al. PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8673 DEVELOPMENTAL BIOLOGY

CCS embryonic stem (ES) cells possessing two mutant alleles ( ry 1 r Ϫ / ry 1 r Ϫ ) for the skeletal muscle ryanodine receptor (RyR) have been produced and injected subcutaneously into severely compromised immunodeficient mice to produce teratocarcinomas in which Ry 1 R expression is absent. Several primary fibroblast cell lines were isolated and subcloned from one of these tumors that contain the knockout mutation in both alleles and exhibit a doubling time of 18-24 h, are not contact growth inhibited, do not exhibit drastic morphological change upon serum reduction, and possess the normal complement of chromosomes. Four of these fibroblast clones were infected with a retrovirus containing the cDNA encoding myoD and a puromycin selection marker. Several (1-2 g/ml) puromycin-resistant subclones from each initial cell line were expanded and examined for their ability to express myoD and to form multinucleated myotubes that express desmin and myosin upon removal of mitogens. One of these clones (1B5 cells) was selected on this basis for further study. These cells, upon withdrawal of mitogens for 5-7 d, were shown by Western blot analysis to express key triadic proteins, including skeletal triadin, calsequestrin, 1. Abbreviations used in this paper : BLM, bilayer lipid membrane; ␣ 1-DHPR, ␣ -1 subunit of L-type voltage-dependent Ca 2 ϩ channel dihydropyridine receptor; e-c, excitation-contraction; ES, embryonic stem; FKBP12, FK506 binding protein, 12 kD; G418, neomyocin antibiotic analogue; HIHS, heat-inactivated horse serum; P o , open probability; Ry 1 R, Ry 2 R, and Ry 3 R, skeletal isoform of ryanodine receptor, cardiac isoform of ryanodine receptor, and brain isoform of ryanodine receptor; SCID, severely compromised immunodeficient; SERCA, sarco(endo)plasmic reticulum Ca 2 ϩ -ATPase; SR, sarcoplasmic reticulum.

The purpose of this paper is to analyze our present knowledge of the early ecdysone puff locus 2B5. We have cloned and mapped several mutants from the region in order to discover the role played by this locus in the induction of the other early ecdysone ptis and in the late induction. We present a model that could explain the way 2B5 products operate within the sequence of events that follows the ecdysone rise in titre at the end of the third instar.

The Broad-Complex (BR-C) of D. melanogaster, mapping at the 2B5 early ecdysone puff, mediates ecdysone-induced processes. We present here the transcriptional analysis of the locus in both wild type and representative mutants. Two well defined regions are transcribed, only one of which has a pattern consistent with the proposed 2B5 located BR-C function. The 2B5 region is actively transcribed in early third instar larvae before ecdysone levels increase. Ecdysone switches this early pattern to a complex late type which requires the presence of functional BR-C product. Therefore, BR-C self-regulates its own ecdysone-induced transcription. The effect of 2B5 mutations on transcription at several intermolt, early and late puffs is also described. Null mutations at the 2B5 locus prevents ecdysone inducible transcription. This evidence supports the fact that 2B5 codes for an ecdysone-dependent transcriptional regulator.