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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.11.016 Byline: Thomas D. Marose (a), Calli E. Merkel (a), Andrew P. McMahon (b), Thomas J. Carroll (a) Keywords: [beta]-catenin; Wolffian duct; Ureteric bud; Precursor cell; Differentiation; CAKUT; Kidney development; Metanephros; Wnt Abstract: Differentiation is the process by which tissues/organs take on their final, physiologically functional form. This process is mediated in part by the silencing of embryonic genes and the activation of terminal, differentiation gene products. Mammalian kidney development is initiated when the Wolffian duct branches and invades the overlying metanephric mesenchyme. The newly formed epithelial bud, known as the ureteric bud, will continue to branch ultimately differentiating into the collecting duct system and ureter. Here, we show that Hoxb7-Cre mediated removal of [beta]-catenin from the mouse Wolffian duct epithelium leads to the premature expression of gene products normally associated with the differentiated kidney collecting duct system including the water channel protein, Aquaporin-3 and the tight junction protein isoform, ZO-1[alpha]+. Mutant cells fail to maintain expression of some genes associated with embryonic development, including several mediators of branching morphogenesis, which subsequently leads to kidney aplasia or hypoplasia. Reciprocally, expression of a stabilized form of [beta]-catenin appears to block differentiation of the collecting ducts. All of these defects occur in the absence of any effects on the adherens junctions. These data indicate a role for [beta]-catenin in maintaining cells of the Wolffian ducts and the duct derived ureteric bud/collecting duct system in an undifferentiated or precursor state. Author Affiliation: (a) Department of Internal Medicine (Nephrology Division) and Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-8856, USA (b) Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138, USA Article History: Received 15 June 2007; Revised 9 November 2007; Accepted 13 November 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.06.042 Byline: Fanxin Long (a)(b)(c), Kyu-Sang Joeng (a)(c), Shouhong Xuan (d), Argiris Efstratiadis (d), Andrew P. McMahon (e) Keywords: Ihh; Smo; IGF; IGF1R; Growth; Skeleton; Mouse Abstract: The insulin-like growth factors (IGFs) play a major role in regulating the systemic growth of mammals. However, it is unclear to what extent their systemic and/or local functions act in concert with other local growth factors controlling the sizes of individual organs. We have specifically addressed whether growth control of the skeleton by IGFs interacts genetically with that by Indian hedgehog (Ihh), a locally produced growth signal for the endochondral skeleton. Here, we report that disruption of both IGF and Ihh signaling resulted in additive reduction in the size of the embryonic skeleton. Thus, IGF and Ihh signaling appear to control the growth of the skeleton in parallel pathways. Author Affiliation: (a) Department of Medicine, Washington University Medical School, St. Louis, MO 63110, USA (b) Department of Molecular Biology and Pharmacology, Washington University Medical School, St. Louis, MO 63110, USA (c) Division of Biology and Biomedical Sciences, Washington University Medical School, St. Louis, MO 63110, USA (d) Department of Genetics and Development, Columbia University, New York, NY 10032, USA (e) Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA Article History: Received 23 January 2006; Revised 20 June 2006; Accepted 26 June 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.016 Byline: Mark Wijgerde, Seth Karp, Jill McMahon, Andrew P. McMahon Keywords: Noggin; Bmp4; Axial; Skeleton; Mesoderm; Patterning Abstract: The interaction between bone morphogenetic proteins (BMPs) and their antagonist, Noggin, is critical for normal development. Noggin null mice die at birth with a severely malformed skeleton that is postulated to reflect the activity of unopposed BMP signaling. However, the widespread expression and redundancy of different BMPs have made it difficult to identify a specific role for individual BMPs during mammalian skeletal morphogenesis. Here, we report the effects of modifying Bmp4 dosage on the skeletal development of Noggin mutant mice. The reduction of Bmp4 dosage results in an extensive rescue of the axial skeleton of Noggin mutant embryos. In contrast, the appendicular skeletal phenotype of Noggin mutants was unchanged. Analysis of molecular markers of somite formation and somite patterning suggests that the loss of Noggin results in the formation of small mispatterned somites. Mis-specification and growth retardation rather than cell death most likely account for the subsequent reduction or loss of axial skeletal structures. The severe Noggin phenotype correlates with Bmp4-dependent ectopic expression of Bmp4 in the paraxial mesoderm consistent with Noggin antagonizing an auto-inductive feed-forward mechanism. Thus, specific interactions between Bmp4 and Noggin in the early embryo are critical for establishment and patterning of the somite and subsequent axial skeletal morphogenesis. Author Affiliation: Department of Molecular and Cellular Biology, The Biolabs, 16 Divinity Avenue, Harvard University, Cambridge, MA 02138, USA Article History: Received 22 March 2005; Revised 30 June 2005; Accepted 17 July 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.027 Byline: Forugh Vaziri Sani (a), Kristina Hallberg (a), Brian D. Harfe (b), Andrew P. McMahon (c), Anders Linde (a), Amel Gritli-Linde (a) Keywords: Palate; Cleft palate; Epithelial-mesenchymal transformation; Fate-mapping; Sonic hedgehog Abstract: During palatogenesis, fusion of the palatine shelves is a crucial event, the failure of which results in the birth defect, cleft palate. The fate of the midline epithelial seam (MES), which develops transiently upon contact of the two palatine shelves, is still strongly debated. Three major mechanisms underlying the regression of the MES upon palatal fusion have been proposed: (1) apoptosis has been evidenced by morphological and molecular criteria; (2) epithelial-mesenchymal transformation has been suggested based on ultrastructural and lipophilic dye cell labeling observations; and (3) migration of MES cells toward the oral and nasal areas has been proposed following lipophilic dye cell labeling. To verify whether epithelial-mesenchymal transformation of MES cells takes place during murine palatal fusion, we used the Cre/lox system to genetically mark Sonic hedgehog- and Keratin-14-expressing palatal epithelial cells and to identify their fate in vivo. Our analyses provide conclusive evidence that rules out the occurrence of epithelial-mesenchymal transformation of MES cells. Author Affiliation: (a) Department of Oral Biochemistry, Sahlgrenska Academy at Goteborg University, Medicinaregatan 12F, SE-405 30 Goteborg, Sweden (b) Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, 1600 SW Archer Road, Gainesville, FL 32610, USA (c) Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA Article History: Received 12 May 2005; Revised 16 June 2005; Accepted 18 July 2005

In recent years, the Cre integrase from bacteriophage P1 has become an essential tool for conditional gene activation and/or inactivation in mouse. In an earlier report, we described a fusion protein between Cre and a mutated form of the ligand binding domain of the estrogen receptor (Cre-ER[TM]) that renders Cre activity tamoxifen (TM) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. In the current work, we have generated a transgenic mouse line in which Cre-ER[TM] is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages. We demonstrate that a single, intraperitoneal injection of TM into a pregnant mouse at 8.5 days postcoitum leads to detectable recombination in the developing embryo within 6 h of injection and efficient recombination of a reporter gene in derivatives of all three germ layers within 24 h of injection. In addition, by varying the dose of TM injected, the percentage of cells undergoing a recombination event in the embryo can be controlled. Dose-dependent excision induced by TM was also possible in diverse tissues in the adult mouse, including the central nervous system, and in cultured cells derived from the transgenic mouse line. This inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration. Key Words: Cre; Cre-ER[TM]; gene activation; inactivation; inducible; estrogen receptor; tamoxifen; 4-hydroxy-tamoxifen; mouse; bacteriophage P1.

The study of spontaneous mutations has aided the understanding of developmental processes. A large collection of spontaneous or 'classical' mouse mutations has been accumulated over many decades. One of the mutations causes the postaxial hemimelia (px) phenotype, which consists of limb patterning defects accompanied by Mullerian duct-associated sterility in both sexes. We were intrigued that both the limb and the Mullerian duct px phenotypes are similar to those caused by mutations in the gene encoding the Wnt 7a signaling molecule. In this paper, we investigate the nature of the px mutation. Morphological analysis and breeding experiments demonstrate that the px phenotype indeed results from a mutation in the Wnt 7a gene. Molecular analysis demonstrates that px results from a 515-bp deletion in the Writ 7a gene. This generates an abnormal splicing event, which ultimately produces a truncated Wnt 7a protein of half the normal size. Thus, the px mutation is predicted to be a likely null allele of the Writ 7a gene. Our results provide another interesting example of a classical mutation that disrupts an important patterning gene in development. Key Words: Writ 7a; limb/reproductive patterning; px mutation.

Epithelial-mesenchymal interactions govern the development of epidermal organs such as teeth. During the early stages of tooth development, a local ectodermal thickening which expresses several signaling molecules appears. It is believed that these in turn signal to the underlying mesenchyme triggering mesenchymal condensation and tooth development. For example, epithelially expressed Bmp4 induces Msx1 and Lef1 as well as itself in the underlying mesenchyme. In this paper we have investigated the role of four epithelial signaling molecules, Bmp2, Shh, Wnt10a, and Wnt10b, in the early inductive cascades that govern tooth development. We show that all four genes are specifically expressed in the epithelium between E11.0 and E12.0 when tooth morphogenesis is first apparent. Although Shh, Bmp2, and Wnt10b have similar, if not identical, expression patterns, each signal has a distinct molecular action on the jaw mesenchyme. Whereas Shh and Wnt10b can induce general Hedgehog and Wnt targets, Ptc and Gli for Shh and Lef1 for Wnt10b, only Bmp2 is able to induce tooth-specific expression of Msx1. Thus, there are distinct targets for all three pathways. Interestingly, both Bmp and Wnt signaling activate Lef1, making it a candidate for integrating the two distinct signaling pathways. Key Words: epithelial-mesenchymal interactions; tooth development; Wnt signaling; Shh signaling; Bmp signaling.

Writ-1 encodes a secreted signaling molecule which is required for development of the midbrain and anterior hindbrain in the mouse. Wnt-1 expression is initiated early in the development of the central nervous system in a region predicted to give rise to the midbrain. Later in development Wnt-1 expression is restricted to regions of the forebrain, midbrain, and spinal cord. Previous studies identified a 5.5-kb enhancer in the Wnt-1 locus which is sufficient to activate transcription of a reporter gene in a pattern very similar to that of the endogenous Wnt-1 gene. Here we have assessed if this enhancer is an important component of the endogenous regulatory sequences of Wnt-1 by gene targeting and by testing if it is able to express Wnt-1 in a pattern which is sufficient to rescue the phenotype of loss of Wnt-1. Our results show that the 5.5-kb enhancer is both necessary and sufficient for Wnt-1 expression in vivo.

The secreted signaling molecule GDNF is expressed in the metanephric mesenchyme and has recently been implicated as a factor necessary for development of the metanephric kidney. We have examined the effects of GDNF on mouse kidney explants. We show that GDNF increases cell proliferation in ureter tips. There is an increase in the number of ureter tips and expansion and fusion of adjacent tips and some tips appear to grow toward the source of GDNF. These events are accompanied by transcriptional upregulation of several genes localized to the tips, including its own receptor, c-ret, the transcription factor Sox9, and the signal Wnt-11. These results support a model in which GDNF supplied by the mesenchyme regulates growth and branching in the metanephric kidney through the local regulation of ureter tip-specific factors.

Studies on the mouse Splotch (Sp) mutation, a deletion in the transcription factor Pax-3, have revealed that Pax-3 is essential for normal development of the neural crest. We have investigated the defect in neural crest development using a Wnt-1::LacZ reporter construct to mark neural crest cells. Staining embryos for [Beta]-galactosidase activity at different developmental stages revealed a severe reduction in the number of neural crest cells which emigrated from the neural tube at the vagal and rostral trunk levels. At the caudal thoracic, lumbar, and sacral levels there was a complete loss of neural crest cell emigration. In contrast to previous work in culture, we saw no evidence for any delay in the onset of neural crest cell migration at anterior levels. Pax-3 is expressed in the dorsal neural tube, where the neural crest cells originate, in migrating neural crest cells, and in somitic cells along the migratory pathway. Hence, it is not clear which aspect of the Pax-3 expression accounts for the observed phenotype. We addressed this problem by transplanting neural tissue between mouse and chick embryos. Our studies indicate that the defect in the Splotch mutation is not intrinsic to the neural crest cells themselves, but appears to reflect inappropriate cell interactions either within the neural tube or between the neural tube and the somite.

Wnt-3a mutant embryos show defects caudal to the forelimb level; somites are absent, the notochord is disrupted, and the central nervous system has a pronounced dysmorphology. Previous studies revealed that the primary defects of the mutant embryos are likely to be in the process of paraxial mesoderm formation. In this study, we analyzed the phenotype of Wnt-3a mutant embryos at early somite stages (8.0 days post coitum), when somite formation is initiated. In Wnt-3a mutants, cells which have ingressed through the primitive streak do not migrate laterally but remain under the streak and form an ectopic tubular structure. Several neural-specific molecular markers, but no paraxial mesoderm markers, are expressed in this structure, suggesting that the ectopic tube is an additional neural tube. In normal embryos, Wnt-3a is expressed in the primitive ectoderm, including the cells which are fated to give rise to the paraxial mesoderm and neurectoderm, but expression is absent in migrating mesoderm cells. These results suggest that Wnt-3a signaling may play a role in regulating paraxial mesodermal fates, at the expense of neurectodermal fates, within the primitive ectoderm of the gastrulating mouse embryo.

Genes from mouse embryos were hybridized with various probes to determine the role of mouse hedgehog genes in late embryogenesis. The genes were expressed in unique locations and showed no overlap in functions. These results suggest that hedgehog genes have their own signaling activity and play a role in various morphogenetic processes within the cell. Evidence also suggests that hedgehog genes are conserved in vertebrate development.

Mice possessing a null mutation at the Wnt3 locus fail to gastrulate and die at early stages of embryogenesis (E9.5). We have generated mice with a conditional ('fioxed') allele of Wnt3 (Wnt3c) in order to determine its roles at later stages of development. We have also generated transgenic mouse lines harboring Cre recombinase driven by the Brachyury (T) promoter as a means of specifcally removing Wnt3 in the primitive streak of embryos. We observed that mice transheterozygous for the Wnt3 null and conditional alleles (Wnt3n/c) and also possessing a 'TCre' line (Tcre14) exhibited no gastrulation anomalies but surprisingly possessed severe hindlimb outgrowth defects. We subsequently found that Tcre14 exhibited robust Cre activity in the hindlimb ectoderm. In addition, we found Wnt3 to be expressed ubiquitously in the ectoderm but not the mesenchyme of the developing limb. We demonstrate that these conditional mutants do not express markers characteristic of the apical ectodermal ridge (AER) suggesting that Wnt3 plays an important role in the establishment of this tissue. We also show that Fgf10, which is also essential for the establishment of the AER, continues to be strongly expressed in the early limb mesenchyme of these mutants suggesting that Wnt3 is likely to act downstream of this molecule. We describe further molecular characterization of the mutant hindlimbs. Finally, we describe the conditional removal of [Beta]-catenin in the limb mesenchyme vs ectoderm and argue that Wnt3 signals to the distal limb ectoderm to establish the AER.

Indian hedgehog (Ihh), one of the three mammalian Hedgehog proteins, plays an essential role in endochondral bone development by regulating chondrocyte proliferation, differentiation, and osteoblast differentiation. Smoothened (Smo) is a transmembrane protein that transduces Hedgehog signals. To investigate further the regulatory functions of Indian Hedgehog (Ihh) during endochondral bone formation, we have used the CRE-LoxP approach to remove Smo activity in chondrocytes. These animals develop shorter long bone when compared to wild-type littermates. In contrast to Ihh-/- mice, chondrocyte differentiation appears to be relatively normal, and osteoblast differentiation does occur. However, like Ihh-/- mice, proliferation of chondrocytes is markedly reduced: Brdu analyses at E14.5 show that proliferation is about 50% of that of the wild type. This result suggests that Ihh directly regulates chondrocyte proliferation. By expressing either Ihh or a constitutively active Smo allele (Smo*) specifically in the cartilage using the bigenic UAS-Gal4 system, we demonstrate that proliferation of chondrocytes is significantly increased. Thus, activation of the Ihh signaling pathway is sufficient to promote chondrocyte proliferation. Taken together, the present study establishes Ihh as a key mitogen in the endochondral skeleton.