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Mammalian organs exhibit distinct physiology, disease susceptibility, and injury responses between the sexes. In the mouse kidney, sexually dimorphic gene activity maps predominantly to proximal tubule (PT) segments. Bulk RNA sequencing (RNA-seq) data demonstrated that sex differences were established from 4 and 8 weeks after birth under gonadal control. Hormone injection studies and genetic removal of androgen and estrogen receptors demonstrated androgen receptor (AR)-mediated regulation of gene activity in PT cells as the regulatory mechanism. Interestingly, caloric restriction feminizes the male kidney. Single-nuclear multiomic analysis identified putative cis-regulatory regions and cooperating factors mediating PT responses to AR activity in the mouse kidney. In the human kidney, a limited set of genes showed conserved sex-linked regulation, whereas analysis of the mouse liver underscored organ-specific differences in the regulation of sexually dimorphic gene expression. These findings raise interesting questions on the evolution, physiological significance, disease, and metabolic linkage of sexually dimorphic gene activity.

Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states.

Analysis of kidney disease-causing genes and pathology resulting from systemic diseases highlight the importance of the kidney's filtering system, the renal corpuscles. To elucidate the developmental processes that establish the renal corpuscle, we performed single-nucleus droplet-based sequencing of the human fetal kidney. This enabled the identification of nephron, interstitial, and vascular cell types that together generate the renal corpuscles. Trajectory analysis identified transient developmental gene expression, predicting precursors or mature podocytes express FBLN2, BMP4, or NTN4, in conjunction with recruitment, differentiation, and modeling of vascular and mesangial cell types into a functional filter. In vitro studies provide evidence that these factors exhibit angiogenic or mesangial recruiting and inductive properties consistent with a key organizing role for podocyte precursors in kidney development. Together these studies define a spatiotemporal developmental program for the primary filtration unit of the human kidney and provide novel insights into cell interactions regulating co-assembly of constituent cell types.

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a-/-Wnt5b-/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry.

The urothelium is a multilayered epithelium that serves as a barrier between the urinary tract and blood, preventing the exchange of water and toxic substances. It consists of superficial cells specialized for synthesis and transport of uroplakins that assemble into a tough apical plaque, one or more layers of intermediate cells, and keratin 5-expressing basal cells (K5-BCs), which are considered to be progenitors in the urothelium and other specialized epithelia. Fate mapping, however, reveals that intermediate cells rather than K5-BCs are progenitors in the adult regenerating urothelium, that P cells, a transient population, are progenitors in the embryo, and that retinoids are critical in P cells and intermediate cells, respectively, for their specification during development and regeneration. These observations have important implications for tissue engineering and repair and, ultimately, may lead to treatments that prevent loss of the urothelial barrier, a major cause of voiding dysfunction and bladder pain syndrome.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.06.043 Byline: Joshua W. Mugford, Jing Yu, Akio Kobayashi, Andrew P. McMahon Keywords: Cap mesenchyme; Metanephros; Kidney; Progenitor; Transcription factor; Nephrogenesis Abstract: The functional unit of the kidney is the nephron. During its organogenesis, the mammalian metanephric kidney generates thousands of nephrons over a protracted period of fetal life. All nephrons are derived from a population of self-renewing multi-potent progenitor cells, termed the cap mesenchyme. However, our understanding of the molecular and cellular mechanisms underlying nephron development is at an early stage. In order to identify factors involved in nephrogenesis, we performed a high-resolution, spatial profiling of a number of transcriptional regulators expressed within the cap mesenchyme and early developing nephron. Our results demonstrate novel, stereotypic, spatially defined cellular sub-domains within the cap mesenchyme, which may, in part, reflect induction of nephron precursors. These results suggest a hitherto unappreciated complexity of cell states that accompany the assembly of the metanephric kidney, likely reflecting diverse regulatory actions such as the maintenance and induction of nephron progenitors. Author Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA Article History: Received 1 May 2009; Revised 23 June 2009; Accepted 29 June 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.09.010 Byline: Joshua W. Mugford, Petra Sipila, Jill A. McMahon, Andrew P. McMahon Keywords: Osr1; Metanephros; Kidney; Progenitor Abstract: The mammalian metanephric kidney is derived from the intermediate mesoderm. In this report, we use molecular fate mapping to demonstrate that the majority of cell types within the metanephric kidney arise from an Osr1.sup.+ population of metanephric progenitor cells. These include the ureteric epithelium of the collecting duct network, the cap mesenchyme and its nephron epithelia derivatives, the interstitial mesenchyme, vasculature and smooth muscle. Temporal fate mapping shows a progressive restriction of Osr1.sup.+ cell fates such that at the onset of active nephrogenesis, Osr1 activity is restricted to the Six2.sup.+ cap mesenchyme nephron progenitors. However, low-level labeling of Osr1.sup.+ cells suggests that the specification of interstitial mesenchyme and cap mesenchyme progenitors occurs within the Osr1.sup.+ population prior to the onset of metanephric development. Furthermore, although Osr1.sup.+ progenitors give rise to much of the kidney, Osr1 function is only essential for the development of the nephron progenitor compartment. These studies provide new insights into the cellular origins of metanephric kidney structures and lend support to a model where Osr1 function is limited to establishing the nephron progenitor pool. Author Affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA Article History: Received 15 July 2008; Revised 4 September 2008; Accepted 4 September 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.03.044 Byline: Joshua W. Mugford (a), Petra Sipila (a), Akio Kobayashi (b), Richard R. Behringer (b), Andrew P. McMahon (a) Keywords: Kidney; Mesonephros; Metanephros; Hoxd11; Hox genes Abstract: The mammalian kidney consists of an array of tubules connected to a ductal system that collectively function to control water/salt balance and to remove waste from the organisms' circulatory system. During mammalian embryogenesis, three kidney structures form within the intermediate mesoderm. The two most anterior structures, the pronephros and the mesonephros, are transitory and largely non-functional, while the most posterior, the metanephros, persists as the adult kidney. We have explored the mechanisms underlying regional specific differentiation of the kidney forming mesoderm. Previous studies have shown a requirement for Hox11 paralogs (Hoxa11, Hoxc11 and Hoxd11) in metanephric development. Mice lacking all Hox11 activity fail to form metanephric kidney structures. We demonstrate that the Hox11 paralog expression is restricted in the intermediate mesoderm to the posterior, metanephric level. When Hoxd11 is ectopically activated in the anterior mesonephros, we observe a partial transformation to a metanephric program of development. Anterior Hoxd11.sup.+ cells activate Six2, a transcription factor required for the maintenance of metanephric tubule progenitors. Additionally, Hoxd11.sup.+ mesonephric tubules exhibit an altered morphology and activate several metanephric specific markers normally confined to distal portions of the functional nephron. Collectively, our data support a model where Hox11 paralogs specify a metanephric developmental program in responsive intermediate mesoderm. This program maintains tubule forming progenitors and instructs a metanephric specific pattern of nephron differentiation. Author Affiliation: (a) Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA (b) Program in Developmental Biology, Baylor College of Medicine and Department of Molecular Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Article History: Received 17 January 2008; Revised 31 March 2008; Accepted 31 March 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.02.039 Byline: Charisios D. Tsiairis, Andrew P. McMahon Keywords: Disp1; Ihh gradient; Growth plate; PTHrP; Endochondral ossification Abbreviations: Disp1, Dispatched1; Ihh, Indian hedgehog; N-Shh, non-cholesterol modified Sonic hedgehog; PTHrP, parathyroid hormone related peptide; Shh, sonic hedgehog; Ptch1, Patched1; Smo, Smoothened; PPR, Parathyroid hormone-PTHrP receptor; Disp2, Dispatched2 Abstract: Dispatched1 (Disp1) is required for the release of cholesterol modified hedgehog (Hh) proteins from producing cells. We investigated the role of Disp1 in Indian hedgehog (Ihh) signaling in the developing bone bypassing the lethality of the Disp1.sup.C829F allele at early somite stages through the supply of non-cholesterol modified Sonic hedgehog (N-Shh). The long bones that develop in the absence of wild-type Disp1, while clearly shorter, have a juxtaposition of proliferating and non-proliferating hypertrophic chondrocytes that is markedly more normal in organization than those of ihh null mutants. Direct analysis of Ihh trafficking in the target field demonstrates that Ihh is distributed well beyond Ihh expressing cells though the range of movement and signaling action is more restricted than in wild-type long bones. Consequently, a PTHrP-Ihh feedback loop is established, but over a shorter distance, reflecting the reduced range of Ihh movement. These analyses of the Disp1.sup.C829F mutation demonstrate that Disp1 is not absolutely required for the paracrine signaling role of Ihh in the skeleton. However, Disp1 is critical for the full extent of signaling within the chondrocyte target field and consequently the establishment of a normal skeletal growth plate. Author Affiliation: Department of Molecular and Cellular Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA Article History: Received 8 August 2007; Revised 26 January 2008; Accepted 19 February 2008

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

Research has been conducted on the Hedgehog signaling pathway. Results suggest that Hedgehog signaling is important for organizing ventral cell pattern via the differential cell affinity control.

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 authors review publications on the Hedgehog family of intercellular signaling proteins. The topics of interest include mediated by Hedgehog local interactions in the embryonic ectoderm of Drosophila, long-range signaling in Drosophila imaging discs, Hedgehog reception and control of Hh secretion and distribution.

Research shows that in the mouse embryo the multipass membrane protein Smoothened, involved in Hedgehog signaling, acts epistatic to Ptc1 to facilitate Shh and Ihh signaling. Data further show that Hedgehog signaling within the mouse node determines left/right asymmetry.

Results demonstrate that long range signaling by the sonic hedgehog is mediated by a lipophilic modification of the N-terminus with a cholesterol moiety, which aids in the sonic hedgehog's ability to signal over a few hundred microns. The lipophilic modification also modulates the signal by the sonic hedgehog's receptor, Ptc1.

The role of Nkx genes, transcription factors, in the ventral spinal cord as it relates to neural patterning is discussed in this perspective article. Three central questions can be seen from the general model. One deals with how the Sonic hedgehog (Shh) concentration gradient is generated, another with how the gradient is translated into distinct transcriptional responses, and the third with what the transcriptional regulators that lock in a particular neuronal or glial fate are. Evidence indicates cell type diversity in the dorsal and ventral central nervous system depends on the starting action of qualitatively distinct signals that act in a concentration-dependent way to set up autonomous subdomains of homeodomain protein activity along the D-V axis of the neural tube.

The essential function of Wnt-4 in mammary gland development is discussed. It is downstream of progesterone signaling. If the progesterone receptor is not present in mammary epithelium, ductal sidebranching does not take place. Ectopic expression of the photooncogene Wnt-1 overcomes this lack and its effects.

The Hedgehog signaling pathway is discussed in this review article which tells that new discoveries in the pathway shed light on how shaky understanding of the moleular and cellular mechanisms underlying this vital signaling pathway is. The signaling regulates a great variety of developmental events in the fly embryo and the vertebrate one and is central for several cancers. The model now widely accepted is that Hedgehog signaling leads to ligand-dependent receptor inactivation, the likely receptor encoded by the segment polarity gene, patched. The important role of cholesterol in this signaling is related to Patched, with sequestering and binding and the difference between the two of interest.

Results show that the transcriptional factor T (Brachyury) is involved in the Wnt signaling pathway and it modulates a balance between mesodermal and neural cell during gastrulation of mouse embryo.

Differentiation and proliferation of chondrocytes regulated by Indian hedgehog (Ihh) signaling, which is necessary for bone formation, is discussed. Analysis has been carried out in an Ihh null mutant, and a role for an Ihh/PTHrP feedback mechanism in control of chondrocyte maturation is indicated. Ihh signaling seems to be pivotal in coordinating several cellular processes essential for vertebrate skeleton morphogenesis.

Studies on negative feedback mechanisms are anticipated to advance novel information concerning the pattern formation of multicellular organisms. Two types of mechanisms can be determined based on the way they function in a cell-autonomous or non-cell autonomous way. One of the major issues in understanding the biological roles of negative feedback mechanism is the characterization of their thresholds for inhibitor induction.

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.

A ventral-to-dorsal gradient determines the pituitary cell types producing different hormones. An experiment produces in vivo and in vitro proof of the stages involved in the pituitary cell development. The signals that herald the different stages are listed. These cells play a critical role in enabling the pituitary gland to maintain homeostasis.

The mouse gene Noggin appears to play a role in patterning of the neural tube and somite. Noggin encodes an antagonist of bone morphogenetic protein (BMP). Noggin does not appear to be required for neural induction, but plays an essential role in neural tube growth and development. Differentiation of the somite is also deficient in null mutants.

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.

The Sonic hedgehog (Shh) gene is expressed in the areas that mediate central nervous system (CNS) and limb polarity, indicating its role in the inductive interactions that form the CNS. The Shh and two other closely related genes encode putative secreted proteins and are possibly involved in cell-cell interactions. Ectopic expression of Shh in the mouse CNS triggers the flow of plate-expressed genes.

The generation and characterization of a genetically engineered mouse model in which all animals develop short latency malignant osteosarcoma is described. The model based on osteoblast-restricted deletion of p53 and pRb showed strong similarities between murine osteosarcoma and human osteosarcoma could be used to address the molecular genetics of osteosarcoma and develop novel therapeutic strategies.

The signaling of Sonic Hedgehog (Shh) morphogen is promoted by Gas 1 and Cdo proteins. The Shh signaling is crucial for the development of various parts of the central nervous system.

Studies done to determine the ultimate fate of the Shh-producing cells by using recombinase base mapping in the mouse is reported. Studies indicate that, while specification of the anterior digits depends upon differential concentrations of Shh, the length of time of exposure to Shh is critical in the specification of the differences between the most posterior digits.

Hh signaling in NCC is crucial for normal patterning and growth of the face. Fox genes mediate the action of shh in facial development.

Research reveals that besides the involvement of the chemoattractant Netrin-1 in the development of axons, the morphogen Sonic hedgehog mimics chemoattractant activity, suggesting that a morphogen can also serve as an axonal chemoattractant.

Research has been conducted on apical ectodermal ridge formation. The authors have attempted to demonstrate that Wnt3, expressed throughout the limb ectoderm, is important for normal limb development and critical in establishment apical ectodermal ridge.

Research has been conducted on Hip1, a Hedgehog-binding protein. The authors have investigated the developmental role of this protein via generation of Hip1 mutants, and have discused the interactions between Hedgehog and fibroblast growth factor signaling which controls early lung branching.

Sonic Hedgehog (Shh) and Indian Hedgehog (Ihh) are members of the Hedgehog (Hh) family of signaling molecules known to be involved in embryonic patterning and morphogenesis. The Hh proteins undergo an autocatalytic cleavage to yield an N-terminal and a C-terminal peptide, with the signaling capacities confined to the N peptide. Drosophila Hh-N has been shown to act via both short- and long-range signaling. In vertebrates, however, attempts to directly demonstrate Shh (SHH) or Ihh (IHH) proteins at a distance from producing cells have been largely unsuccessful. Furthermore, the fact that the Hh N peptides occur in a cholesterol-modified, membrane-tethered form is not easily reconciled with long-range signaling. This study used optimized immunohistochemistry combined with tissue separation and biochemical analyses in vivo and in vitro to determine the range of action of SHH and IHH in the mouse embryo. In all embryonic structures studied, we detect signaling peptides in producing cells, but we also find that ligands move over considerable distances depending on the tissue. These data provide direct evidence for the presence of Hedgehog signaling peptides in target compartments, suggesting a direct long-range action without a need for secondary mediators. Visualization of Hedgehog proteins in target tissues was achieved only under conditions that allowed proteoglycan/glycosaminoglycan (PG/GAG) preservation. Furthermore, we show that induced changes of the composition of PG/GAG in the tooth alter SHH signaling. These data suggest a crucial role for PG/GAGs in Hedgehog movement. Key Words: Hedgehog; tooth; dentin; cartilage; neural tube; goblet cells; basement membrane; proteoglycans.

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.

The Pax family of transcription factors plays important roles in vertebrate organogenesis. Pax-2 is a critical factor in the development of the mammalian urogenital system. Pax-2 is expressed in the epithelia of the ureter, the Mullerian duct, and the Wolffian duct and in the nephrogenic mesenchyme. Gene targeting in the mouse as well as natural mutations in mouse and man have demonstrated the requirement of Pax-2 in the development of these structures. Little is known about the molecular mechanisms regulating Pax-2 expression in the developing urogenital system. As a first step to reveal these mechanisms and to search for the elements and factors controlling Pax-2 expression we have characterized regulatory sequences of the Pax-2 gene in an in vivo reporter assay in the mouse. An 8.5-kb genomic region upstream of the Pax-2 transcription start site directed reporter gene activity in the epithelium of the pronephric duct at 8.25 days postcoitum (dpc)and in the Wolffian duct starting from 9.0 dpc. Expression in the Wolffian duct and its derivatives, the ureter, the collecting duct system, the seminal vesicles, the vas deferens, and the epididymis, was maintained at least until 18.5 dpc. Hence, an element(s) in the 8.5-kb upstream region is sufficient to initiate and maintain Pax-2 expression in the Wolffian duct and its derivatives. In order to more precisely map the Wolffian duct regulatory sequences, a deletion analysis of the 8.5-kb upstream region was performed in a transient in vivo reporter assay. A 0.4-kb subfragment was required for marker gene expression in the Wolffian duct. Misexpression of fgf8 under the control of the 8.5-kb upstream region resulted in polycystic kidneys, demonstrating the general usefulness of Pax-2 regulatory sequences in misexpression of foreign genes in the ureter and collecting duct system of the kidney in transgenic approaches in mice. [C] 2001 Academic Press Key Words: kidney; metanephros; urogenital system; promoter analysis; enhancer; Wolffian duct; Pax; Pax-2.

Secreted Wnt proteins regulate many developmental processes in multicellular organisms. We have generated a targeted mutation in the mouse Wnt7b gene. Homozygous Wnt7b mutant mice die at midgestation stages as a result of placental abnormalities. Wnt7b expression in the chorion is required for fusion of the chorion and allantois during placental development. The [alpha]4 integrin protein, required for chorioallantoic fusion, is not expressed by cells in the mutant chorion. Wnt7b also is required for normal organization of cells in the chorionic plate. Thus, Wnt7b signaling is central to the early stages of placental development in mammals. Key Words: Wnt7b; placenta; chorion; [alpha]4 integrin.