SIX1 acts synergistically with TBX18 in mediating ureteral smooth muscle formation

Dysfunction of the ureter often leads to urine flow impairment from the kidney to the bladder, causing dilation of the ureter and/or renal pelvis. Six1 is a crucial regulator of renal development: mutations in human SIX1 cause branchio-oto-renal (BOR) syndrome and Six1–/– mice exhibit renal agenesis, although the ureter is present. It remains unclear whether Six1 plays a role in regulating ureter morphogenesis. We demonstrate here that Six1 is differentially expressed during ureter morphogenesis. It was expressed in undifferentiated smooth muscle (SM) progenitors, but was downregulated in differentiating SM cells (SMCs) and had disappeared by E18.5. In Six1–/– mice, the ureteral mesenchymal precursors failed to condense and differentiate into normal SMCs and showed increased cell death, indicating that Six1 is required for the maintenance and normal differentiation of SM progenitors. A delay in SMC differentiation was observed in Six1–/– ureters. A lack of Six1 in the ureter led to hydroureter and hydronephrosis without anatomical obstruction when kidney formation was rescued in Six1–/– embryos by specifically expressing Six1 in the metanephric mesenchyme, but not the ureter, under control of the Eya1 promoter. We show that Six1 and Tbx18 genetically interact to synergistically regulate SMC development and ureter function and that their gene products form a complex in cultured cells and in the developing ureter. Two missense mutations in SIX1 from BOR patients reduced or abolished SIX1-TBX18 complex formation. These findings uncover an essential role for Six1 in establishing a functionally normal ureter and provide new insights into the molecular basis of urinary tract malformations in BOR patients.

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title: Structural changes of smooth muscle in congenital ureteropelvic junction obstruction.
abstract: Ureteropelvic junction (UPJ) obstruction is the most common cause of congenital hydronephrosis. Previous studies have reported that the excess amount of collagen restricting mobility and resiliency of the UPJ is the result of an impaired collagen production by anomalous smooth muscle cells (SMCs). Our purpose was to evaluate the role of SMC differentiation in the pathogenesis of UPJ obstruction.



Surgical specimens of UPJ from 21 patients (8 girls/13 boys) who were subjected to dismembered pyeloplasty were examined immunohistochemically using monoclonal antibodies against smooth muscle (SM) myosin heavy chain isoforms including SM1, SM2, and SMemb. The age ranged from 1 month to 13 years. Ureteropelvic walls taken from 14 forensic autopsy cases, with no urological abnormalities, served as age-matched control group.



The immunohistochemical expression of SM1 and SM2 in UPJ obstruction was significantly increased when compared with controls (P < .05). In contrast, there was no statistical difference of expression of SMemb.



Our findings supported the hypothesis that the primary anomaly in UPJ obstruction may be attributed to a malfunction of SMCs in the ureter.

title: Embryogenesis of the congenital anomalies of the kidney and the urinary tract.
abstract: Ectopia of the initial ureter is the first ontogenic mis-step that leads to many congenital anomalies of the kidney and urinary tract (CAKUT). The ectopia results in hypoplastic kidney, ectopia of the ureteral orifice, urinary outflow obstruction and/or reflux. Recent studies on several mutant mouse models verified that ectopic ureteral budding indeed occurs prior to the formation of CAKUT. Often, the genes involved in navigating the site of ureteral budding also regulate later ontogenic processes of the kidney and other urinary tract systems. These additional functions of the genes underlie the wide spectrum of CAKUT, as the genes are expressed at multiple sites at multiple ontogenic stages, and regulate the morphogenesis of the many portions of the excretory system through their distinctive cellular functions.

title: Branching morphogenesis and kidney disease
abstract: Branching morphogenesis in the kidney is a tightly regulated, complex process and its disruption potentially can lead to a broad spectrum of diseases, ranging from rare hereditary syndromes to common conditions such as hypertension and chronic kidney failure. This review synthesizes data on branching during kidney development derived from in vitro and in vivo rodent studies and to apply them to human diseases. It discusses how the broad organization of molecular interactions during kidney development might provide a mechanistic framework for understanding disorders related to aberrant branching.

title: The cellular basis of kidney development.
abstract: Mammalian kidney development has helped elucidate the general concepts of mesenchymal-epithelial interactions, inductive signaling, epithelial cell polarization, and branching morphogenesis. Through the use of genetically engineered mouse models, the manipulation of Xenopus and chick embryos, and the identification of human renal disease genes, the molecular bases for many of the early events in the developing kidney are becoming increasingly clear. Early patterning of the kidney region depends on interactions between Pax/Eya/Six genes, with essential roles for lim1 and Odd1. Ureteric bud outgrowth and branching morphogenesis are controlled by the Ret/Gdnf pathway, which is subject to positive and negative regulation by a variety of factors. A clear role for Wnt proteins in induction of the kidney mesenchyme is now well established and complements the classic literature nicely. Patterning along the proximal distal axis as the nephron develops is now being investigated and must involve aspects of Notch signaling. The development of a glomerulus requires interactions between epithelial cells and infiltrating endothelial cells to generate a unique basement membrane. The integrity of the glomerular filter depends in large part on the proteins of the nephrin complex, localized to the slit diaphragm. Despite the kidney's architectural complexity, with the advent of genomics and expression arrays, it is becoming one of the best-characterized organ systems in developmental biology.

title: Sequential cell and tissue interactions governing organogenesis of the kidney
abstract: SummaryThe complex development of the metanephric kidney illustrates the decisive role of sequential morphogenetic interactions of varying type in organogenesis. In this review the following steps were singled out from this continuous, strictly controlled organogenesis: determination of the mesoderm during the blastula stage, induction of pronephric nephrons during gastrulation, guided migration of the pronephric duct, mesenchyme-controlled branching of the ureter, induced aggregation of the mesenchymal cells of the metanephric blastema, homotypically controlled polarization of the cells in the renal vesicle and remodelling of the vesicle into the S-shaped body, guided migration of endothelial cells into the nephric blastema and the glomerular crevice, and the matrix interaction(s) completing the formation of the glomerular basement membrane with dual origin.

title: Tailbud-derived mesenchyme promotes urinary tract segmentation via BMP4 signaling
abstract: Urinary tract morphogenesis requires the sub-division of the ureteric bud (UB) into the intra-renal collecting system and ureter, two tissues with unique structural and functional properties. In this report we investigate the cellular and molecular mechanisms that mediate their differentiation. Fate mapping experiments in the developing chick indicate that the UB is surrounded by two distinct mesenchymal populations: nephrogenic mesenchyme derived from the intermediate mesoderm and tailbud-derived mesoderm, which is selectively associated with the domain of the UB that differentiates into the ureter. Functional experiments utilizing murine metanephric kidney explants show that BMP4, a paracrine factor secreted by tailbud-derived mesenchyme, is required for ureter morphogenesis. Conversely, ectopic BMP4 signaling is sufficient to induce ureter morphogenesis in domains of the UB normally fated to differentiate into the intra-renal collecting system. Collectively, these results indicate that the border between the kidney and ureter forms where mesenchymal tissues originating in two different areas of the early embryo meet. These data raise the possibility that the susceptibility of this junction to congenital defects in humans, such as ureteral-pelvic obstructions, may be related to the complex morphogenetic movements that are required to integrate cells from these different lineages into a single functional structure.

title: Renal branching morphogenesis: concepts, questions, and recent advances.
abstract: The ureteric bud (UB) is an outgrowth of the Wolffian duct, which undergoes a complex process of growth, branching, and remodeling, to eventually give rise to the entire urinary collecting system during kidney development. Understanding the mechanisms that control this process is a fascinating problem in basic developmental biology, and also has considerable medical significance. Over the past decade, there has been significant progress in our understanding of renal branching morphogenesis and its regulation, and this review focuses on several areas in which there have been recent advances. The first section deals with the normal process of UB branching morphogenesis, and methods that have been developed to better observe and describe it. The next section discusses a number of experimental methodologies, both established and novel, that make kidney development in the mouse a powerful and attractive experimental system. The third section discusses some of the cellular processes that are likely to underlie UB branching morphogenesis, as well as recent data on cell lineages within the growing UB. The fourth section summarizes our understanding of the roles of two groups of growth factors that appear to be particularly important for the regulation of UB outgrowth and branching: GDNF and FGFs, which stimulate this process via tyrosine kinase receptors, and members of the TGFbeta family, including BMP4 and Activin A, which generally inhibit UB formation and branching.

title: Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney.
abstract: Signaling by the ureteric bud epithelium is essential for survival, proliferation and differentiation of the metanephric mesenchyme during kidney development. Most studies that have addressed ureteric signaling have focused on the proximal, branching, ureteric epithelium. We demonstrate that sonic hedgehog is expressed in the ureteric epithelium of the distal, non-branching medullary collecting ducts and continues into the epithelium of the ureter -- the urinary outflow tract that connects the kidney with the bladder. Upregulation of patched 1, the sonic hedgehog receptor and a downstream target gene of the signaling pathway in the mesenchyme surrounding the distal collecting ducts and the ureter suggests that sonic hedgehog acts as a paracrine signal. In vivo and in vitro analyses demonstrate that sonic hedgehog promotes mesenchymal cell proliferation, regulates the timing of differentiation of smooth muscle progenitor cells, and sets the pattern of mesenchymal differentiation through its dose-dependent inhibition of smooth muscle formation. In addition, we also show that bone morphogenetic protein 4 is a downstream target gene of sonic hedgehog signaling in kidney stroma and ureteral mesenchyme, but does not mediate the effects of sonic hedgehog in the control of mesenchymal proliferation.

title: Bone morphogenetic protein signaling in the developing kidney: present and future.
abstract: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily. A critical role for BMP signaling in the development of the metanephric kidney is supported by a growing number of studies using in vitro assays and in vivo animal models. Here we review current knowledge of BMPs, BMP receptors and regulators of the BMP signaling pathway in the developing kidney. We highlight major gaps in our knowledge of the roles of BMP signaling in the development of the normal and abnormal kidney and identify areas and techniques likely to improve our understanding.

title: Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system.
abstract: The vertebrate urogenital system forms due to inductive interactions between the Wolffian duct, its derivative the ureteric bud, and their adjacent mesenchymes. These establish epithelial primordia within the mesonephric (embryonic) and metanephric (adult) kidneys and the Müllerian duct, the anlage of much of the female reproductive tract. We show that Wnt9b is expressed in the inductive epithelia and is essential for the development of mesonephric and metanephric tubules and caudal extension of the Müllerian duct. Wnt9b is required for the earliest inductive response in metanephric mesenchyme. Further, Wnt9b-expressing cells can functionally substitute for the ureteric bud in these interactions. Wnt9b acts upstream of another Wnt, Wnt4, in this process, and our data implicate canonical Wnt signaling as one of the major pathways in the organization of the mammalian urogenital system. Together these findings suggest that Wnt9b is a common organizing signal regulating diverse components of the mammalian urogenital system.

title: Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis
abstract: Antagonists act to restrict and negatively modulate the activity of secreted signals during progression of embryogenesis. In mouse embryos lacking the extra-cellular BMP antagonist gremlin 1 (Grem1), metanephric development is disrupted at the stage of initiating ureteric bud outgrowth. Treatment of mutant kidney rudiments in culture with recombinant gremlin 1 protein induces additional epithelial buds and restores outgrowth and branching. All epithelial buds express Wnt11, and Gdnf is significantly upregulated in the surrounding mesenchyme, indicating that epithelial-mesenchymal (e-m) feedback signalling is restored. In the wild type, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct and ureteric bud and Grem1 is upregulated in the mesenchyme around the nascent ureteric bud prior to initiation of its outgrowth. In agreement, BMP activity is reduced locally as revealed by lower levels of nuclear pSMAD protein in the mesenchyme. By contrast, in Grem1-deficient kidney rudiments, pSMAD proteins are detected in many cell nuclei in the metanephric mesenchyme, indicative of excessive BMP signal transduction. Indeed, genetic lowering of BMP4 levels in Grem1-deficient mouse embryos completely restores ureteric bud outgrowth and branching morphogenesis. The reduction of BMP4 levels in Grem1 mutant embryos enables normal progression of renal development and restores adult kidney morphology and functions. This study establishes that initiation of metanephric kidney development requires the reduction of BMP4 activity by the antagonist gremlin 1 in the mesenchyme, which in turn enables ureteric bud outgrowth and establishment of autoregulatory GDNF/WNT11 feedback signalling.

title: Bone morphogenetic protein 4 regulates the budding site and elongation of the mouse ureter.
abstract: In the normal mouse embryo, Bmp4 is expressed in mesenchymal cells surrounding the Wolffian duct (WD) and ureter stalk, whereas bone morphogenetic protein (BMP) type I receptor genes are transcribed either ubiquitously (Alk3) or exclusively in the WD and ureter epithelium (Alk6). Bmp4 heterozygous null mutant mice display, with high penetrance, abnormalities that mimic human congenital anomalies of the kidney and urinary tract (CAKUT), including hypo/dysplastic kidneys, hydroureter, ectopic ureterovesical (UV) junction, and double collecting system. Analysis of mutant embryos suggests that the kidney hypo/dysplasia results from reduced branching of the ureter, whereas the ectopic UV junction and double collecting system are due to ectopic ureteral budding from the WD and accessory budding from the main ureter, respectively. In the cultured metanephros deprived of sulfated glycosaminoglycans (S-GAGs), BMP4-loaded beads partially rescue growth and elongation of the ureter. By contrast, when S-GAGs synthesis is not inhibited, BMP4 beads inhibit ureter branching and expression of Wnt 11, a target of glial cell-derived neurotrophic factor signaling. Thus, Bmp4 has 2 functions in the early morphogenesis of the kidney and urinary tract. One is to inhibit ectopic budding from the WD or the ureter stalk by antagonizing inductive signals from the metanephric mesenchyme to the illegitimate sites on the WD. The other is to promote the elongation of the branching ureter within the metanephros, thereby promoting kidney morphogenesis.

title: BMP‐4 affects the differentiation of metanephric mesenchyme and reveals an early anterior‐posterior axis of the embryonic kidney
abstract: Bone morphogenetic protein‐4 (BMP‐4), a member of the transforming growth factor‐β (TGF‐β) family, regulates several developmental processes during animal development. We have now studied the effects of BMP‐4 in the metanephric kidney differentiation by using organ culture technique. Human recombinant BMP‐4 diminishes the number of ureteric branches and changes the branching pattern. Our data suggest that BMP‐4 affects the ureteric branching indirectly via interfering with the differentiation of the nephrogenic mesenchyme. The clear positional preference of the defects to posterior mesenchyme might reflect an early anterior‐posterior patterning of the metanephric mesenchyme. The smooth muscle α‐actin expressing cell population around the ureteric stalk, highly expressing Bmp‐4 mRNA, is also expanded in kidneys treated with BMP‐4. Thus, BMP‐4 may be a physiological regulator of the development of the periureteric smooth muscle layer and ureteric elongation. Dev Dyn;217:146–158.

title: Antagonism of BMP4 signaling disrupts smooth muscle investment of the ureter and ureteropelvic junction.
abstract: Congenital ureteropelvic junction obstruction has been associated with aberrant ureteral smooth muscle organization. Recent evidence has shown that BMP4 may be involved in ureteral morphogenesis. We determined whether the disruption of BMP4 signaling results in abnormal smooth muscle investment of the ureter and ureteropelvic junction.



We used a Cre mediated Bmp4 knockout system to conditionally excise the Bmp4 gene in developing mouse embryos. Kidney rudiments were isolated from embryos at varying gestational ages from WT and conditional knockout mice. Metanephric kidney explants were cultured in the presence or absence of the BMP antagonist Noggin. Agarose beads pre-incubated with Gremlin, another BMP antagonist, were used for localized disruption of BMP signaling. Frozen sections and whole metanephric explants were then analyzed by immunofluorescence.



Bmp4 gene excision resulted in a dose dependent loss of ureteral smooth muscle. Antagonism of BMP signaling inhibited ureteral smooth muscle investment in a dose dependent manner and was paralleled by a dose dependent decrease in the immediate downstream targets of BMP signaling, phosphorylated Smad1, 5 and 8. Localized antagonism of BMP resulted in the focal disruption of ureteral smooth muscle investment.



We report that decreased BMP signaling, whether by the loss of BMP4 in vivo or direct antagonism in vitro, results in a gradual reduction of the normal, well organized coat of smooth muscle surrounding the ureter. Our results also suggest that this occurs via a direct Smad dependent pathway. This raises the possibility that abnormalities in BMP4 signaling may have a role in the development of congenital ureteropelvic junction obstruction.

title: Tbx18 regulates the development of the ureteral mesenchyme.
abstract: Congenital malformations of the urinary tract are a major cause of renal failure in children and young adults. They are often caused by physical obstruction or by functional impairment of the peristaltic machinery of the ureter. The underlying molecular and cellular defects are, however, poorly understood. Here we present the phenotypic characterization of a new mouse model for congenital ureter malformation that revealed the molecular pathway important for the formation of the functional mesenchymal coating of the ureter. The gene encoding the T-box transcription factor Tbx18 was expressed in undifferentiated mesenchymal cells surrounding the distal ureter stalk. In Tbx18-/- mice, prospective ureteral mesenchymal cells largely dislocalized to the surface of the kidneys. The remaining ureteral mesenchymal cells showed reduced proliferation and failed to differentiate into smooth muscles, but instead became fibrous and ligamentous tissue. Absence of ureteral smooth muscles resulted in a short hydroureter and hydronephrosis at birth. Our analysis also showed that the ureteral mesenchyme derives from a distinct cell population that is separated early in kidney development from that of other mesenchymal cells of the renal system.

title: Transcription factor SIX5 is mutated in patients with branchio-oto-renal syndrome.
abstract: Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by the association of branchial arch defects, hearing loss, and renal anomalies. Mutations in EYA1 are known to cause BOR. More recently, mutations in SIX1, which interacts with EYA1, were identified as an additional cause of BOR. A second member of the SIX family of proteins, unc-39 (SIX5), has also been reported to directly interact with eya-1 in Caenorhabditis elegans. We hypothesized that this interaction would be conserved in humans and that interactors of EYA1 represent good candidate genes for BOR. We therefore screened a cohort of 95 patients with BOR for mutations in SIX5. Four different heterozygous missense mutations were identified in five individuals. Functional analyses of these mutations demonstrated that two mutations affect EYA1-SIX5 binding and the ability of SIX5 or the EYA1-SIX5 complex to activate gene transcription. We thereby identified heterozygous mutations in SIX5 as a novel cause of BOR.

title: SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes.
abstract: Urinary tract malformations constitute the most frequent cause of chronic renal failure in the first two decades of life. Branchio-otic (BO) syndrome is an autosomal dominant developmental disorder characterized by hearing loss. In branchio-oto-renal (BOR) syndrome, malformations of the kidney or urinary tract are associated. Haploinsufficiency for the human gene EYA1, a homologue of the Drosophila gene eyes absent (eya), causes BOR and BO syndromes. We recently mapped a locus for BOR/BO syndrome (BOS3) to human chromosome 14q23.1. Within the 33-megabase critical genetic interval, we located the SIX1, SIX4, and SIX6 genes, which act within a genetic network of EYA and PAX genes to regulate organogenesis. These genes, therefore, represented excellent candidate genes for BOS3. By direct sequencing of exons, we identified three different SIX1 mutations in four BOR/BO kindreds, thus identifying SIX1 as a gene causing BOR and BO syndromes. To elucidate how these mutations cause disease, we analyzed the functional role of these SIX1 mutations with respect to protein-protein and protein-DNA interactions. We demonstrate that all three mutations are crucial for Eya1-Six1 interaction, and the two mutations within the homeodomain region are essential for specific Six1-DNA binding. Identification of SIX1 mutations as causing BOR/BO offers insights into the molecular basis of otic and renal developmental diseases in humans.

title: Ear and kidney syndromes: molecular versus clinical approach.
abstract: The association between ear and kidney anomalies is not usually due to an insult to the embryo. In recent years, many essential development control genes that coordinate the assembly and function of kidney and ear have been discovered through the generation of animal mutants and have increased our understanding of the mechanisms of human oto-renal diseases. Here, we describe ear and kidney clinical syndromes and their molecular expression.

title: Eya protein phosphatase activity regulates Six1–Dach–Eya transcriptional effects in mammalian organogenesis
abstract: The precise mechanistic relationship between gene activation and repression events is a central question in mammalian organogenesis, as exemplified by the evolutionarily conserved sine oculis (Six), eyes absent (Eya) and dachshund (Dach) network of genetically interacting proteins. Here, we report that Six1 is required for the development of murine kidney, muscle and inner ear, and that it exhibits synergistic genetic interactions with Eya factors. We demonstrate that the Eya family has a protein phosphatase function, and that its enzymatic activity is required for regulating genes encoding growth control and signalling molecules, modulating precursor cell proliferation. The phosphatase function of Eya switches the function of Six1–Dach from repression to activation, causing transcriptional activation through recruitment of co-activators. The gene-specific recruitment of a co-activator with intrinsic phosphatase activity provides a molecular mechanism for activation of specific gene targets, including those regulating precursor cell proliferation and survival in mammalian organogenesis.

title: Six1 is required for the early organogenesis of mammalian kidney
abstract: The murine Six gene family, homologous to Drosophila sine oculis (so) which encodes a homeodomain transcription factor, is composed of six members (Six1-6). Among the six members, only the Six2 gene has been previously shown to be expressed early in kidney development, but its function is unknown. We have recently found that the Six1 gene is also expressed in the kidney. In the developing kidney, Six1 is expressed in the uninduced metanephric mesenchyme at E10.5 and in the induced mesenchyme around the ureteric bud at E11.5. At E17.5 to P0, Six1 expression became restricted to a subpopulation of collecting tubule epithelial cells. To study its in vivo function, we have recently generated Six1 mutant mice. Loss of Six1 leads to a failure of ureteric bud invasion into the mesenchyme and subsequent apoptosis of the mesenchyme. These results indicate that Six1 plays an essential role in early kidney development. In Six1-/- kidney development, we have found that Pax2, Six2 and Sall1 expression was markedly reduced in the metanephric mesenchyme at E10.5, indicating that Six1 is required for the expression of these genes in the metanephric mesenchyme. In contrast, Eya1 expression was unaffected in Six1-/- metanephric mesenchyme at E10.5, indicating that Eya1 may function upstream of Six1. Moreover, our results show that both Eya1 and Six1 expression in the metanephric mesenchyme is preserved in Pax2-/- embryos at E10.5, further indicating that Pax2 functions downstream of Eya1 and Six1 in the metanephric mesenchyme. Thus, the epistatic relationship between Pax, Eya and Six genes in the metanephric mesenchyme during early kidney development is distinct from a genetic pathway elucidated in the Drosophila eye imaginal disc. Finally, our results show that Eya1 and Six1 genetically interact during mammalian kidney development, because most compound heterozygous embryos show hypoplastic kidneys. These analyses establish a role for Six1 in the initial inductive step for metanephric development.

title: Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system.
abstract: Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.

title: A myocardial lineage derives from Tbx18 epicardial cells
abstract: Understanding the origins and roles of cardiac progenitor cells is important for elucidating the pathogenesis of congenital and acquired heart diseases. Moreover, manipulation of cardiac myocyte progenitors has potential for cell-based repair strategies for various myocardial disorders. Here we report the identification in mouse of a previously unknown cardiac myocyte lineage that derives from the proepicardial organ. These progenitor cells, which express the T-box transcription factor Tbx18, migrate onto the outer cardiac surface to form the epicardium, and then make a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Tbx18-expressing cardiac progenitors also give rise to cardiac fibroblasts and coronary smooth muscle cells. The pluripotency of Tbx18 proepicardial cells provides a theoretical framework for applying these progenitors to effect cardiac repair and regeneration.

title: Altered myogenesis in Six1-deficient mice
abstract: Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, we constructed Six1-deficient mice by replacing its first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles in particular certain hypaxial muscles. Six1–/– embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1–/– embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2.

title: Eya1 is required for the morphogenesis of mammalian thymus, parathyroid and thyroid.
abstract: Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system. We have now examined the role of Eya1 during the morphogenesis of organs derived from the pharyngeal region, including thymus, parathyroid and thyroid. The thymus and parathyroid are derived from 3rd pharyngeal pouches and their development is initiated via inductive interactions between neural crest-derived arch mesenchyme, pouch endoderm, and possibly the surface ectoderm of 3rd pharyngeal clefts. Eya1 is expressed in all three cell types during thymus and parathyroid development from E9.5 and the organ primordia for both of these structures failed to form in Eya1(-/-) embryos. These results indicate that Eya1 is required for the initiation of thymus and parathyroid gland formation. Eya1 is also expressed in the 4th pharyngeal region and ultimobranchial bodies. Eya1(-/-) mice show thyroid hypoplasia, with severe reduction in the number of parafollicular cells and the size of the thyroid lobes and lack of fusion between the ultimobranchial bodies and the thyroid lobe. These data indicate that Eya1 also regulates mature thyroid gland formation. Furthermore, we show that Six1 expression is markedly reduced in the arch mesenchyme, pouch endoderm and surface ectoderm in the pharyngeal region of Eya1(-/-) embryos, indicating that Six1 expression in those structures is Eya1 dependent. In addition, we show that in Eya1(-/-) embryos, the expression of Gcm2 in the 3rd pouch endoderm is undetectable at E10.5, however, the expression of Hox and Pax genes in the pouch endoderm is preserved at E9.5-10.5. Finally, we found that the surface ectoderm of the 3rd and 4th pharyngeal region show increased cell death at E10.5 in Eya1(-/-) embryos. Our results indicate that Eya1 controls critical early inductive events involved in the morphogenesis of thymus, parathyroid and thyroid.

title: Expression of green fluorescent protein in the ureteric bud of transgenic mice: a new tool for the analysis of ureteric bud morphogenesis.
abstract: The growth and branching of the ureteric bud is a complex process that is ultimately responsible for the organization of the collecting duct system as well as the number of nephrons in the metanephric kidney. While the genes involved in the regulation of this process have begun to be elucidated, our understanding of the cellular and molecular basis of ureteric bud morphogenesis remains rudimentary. Furthermore, the timing and sequence of branching and elongation that gives rise to the collecting system of the kidney can only be inferred from retrospective staining or microdissection of fixed preparations. To aid in the investigation of these issues, we developed strains of transgenic mice in which a green fluorescent protein (GFP) is expressed in the ureteric bud under the control of the Hoxb7 promoter. In these mice, GFP is expressed in every branch of the ureteric bud throughout renal development, and in its derivative epithelia in the adult kidney. As GFP fluorescence can be easily visualized in living tissue, this allows the dynamic pattern of ureteric bud growth and branching to be followed over several days when the kidneys are cultured in vitro. Using confocal microscopy, branching of the ureteric bud in all three dimensions can be analyzed. These mice represent an extremely powerful tool to characterize the normal patterns of ureteric bud morphogenesis and to investigate the response of the bud to growth factors, matrix elements, and other agents that regulate its growth and branching.

title: Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia
abstract: Haploinsufficiency for human EYA1, a homologue of the Drosophila melanogaster gene eyes absent (eya), results in the dominantly inherited disorders branchio-oto-renal (BOR) syndrome and branchio-oto (BO) syndrome, which are characterized by craniofacial abnormalities and hearing loss with (BOR) or without (BO) kidney defects. To understand the developmental pathogenesis of organs affected in these syndromes, we inactivated the gene Eya1 in mice. Eya1 heterozygotes show renal abnormalities and a conductive hearing loss similar to BOR syndrome, whereas Eya1 homozygotes lack ears and kidneys due to defective inductive tissue interactions and apoptotic regression of the organ primordia. Inner ear development in Eya1 homozygotes arrests at the otic vesicle stage and all components of the inner ear and specific cranial sensory ganglia fail to form. In the kidney, Eya1 homozygosity results in an absence of ureteric bud outgrowth and a subsequent failure of metanephric induction. Gdnf expression, which is required to direct ureteric bud outgrowth via activation of the c-ret Rtk (refs 5, 6, 7, 8), is not detected in Eya1–/– metanephric mesenchyme. In Eya1–/– ear and kidney development, Six but not Pax expression is Eya1 dependent, similar to a genetic pathway elucidated in the Drosophila eye imaginal disc. Our results indicate that Eya1 controls critical early inductive signalling events involved in ear and kidney formation and integrate Eya1 into the genetic regulatory cascade controlling kidney formation upstream of Gdnf. In addition, our results suggest that an evolutionarily conserved Pax-Eya-Six regulatory hierarchy is used in mammalian ear and kidney development.

title: Molecular effects of Eya1 domain mutations causing organ defects in BOR syndrome.
abstract: Eya1 is a critical gene for mammalian organogenesis. Mutations in human EYA1 cause branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by varying combinations of branchial, otic and renal anomalies, whereas deletion of mouse Eya1 results in the absence of multiple organ formation. Eya1 and other Eya gene products share a highly conserved 271 amino acid Eya domain that is required for protein-protein interaction. Recently, several point mutations that result in single amino acid substitutions in the conserved Eya domain region of EYA1 have been identified in BOR patients; however, the molecular and developmental basis of organ defects that occurred in BOR syndrome is unclear. To understand how these point mutations cause disease, we have analyzed the functional importance of these Eya domain missense mutations with respect to protein complex formation and cellular localization. We have demonstrated that these point mutations do not alter protein localization. However, four mutations are crucial for protein-protein interactions in both yeast and mammalian cells. Our results provide insights into the molecular mechanisms of organ defects detected in human syndromes.

title: Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4.
abstract: Mouse olfactory epithelium (OE) originates from ectodermally derived placode, the olfactory placode that arises at the anterior end of the neural plate. Tissue grafting and recombination experiments suggest that the placode is derived from a common preplacodal domain around the neural plate and its development is directed by signals arising from the underlying mesoderm and adjacent neuroectoderm. In mice, loss of Six1 affects OE morphogenesis but not placode formation. We show here that embryos lacking both Six1 and Six4 failed to form the olfactory placode but the preplacodal region appeared to be specified as judged by the expression of Eya2, which marks the common preplacodal domain, suggesting a synergistic requirement of Six1 and Six4 in patterning the preplacodal ectoderm to a morphologic placode. Our results show that Six1 and Six4 are coexpressed in the preplacodal ectoderm from E8.0. In the olfactory pit, Six4 expression was observed in the peripheral precursors that overlap with Mash1-expressing cells, the early committed neuronal lineage. In contrast, Six1 is highly distributed in the peripheral regions where stem cells reside at E10.5 and it overlaps with Sox2 expression. Both genes are expressed in the basal and apical neuronal progenitors in the OE. Analyses of Six1;Six4 double mutant embryos demonstrated that the slightly thickened epithelium observed in the mutant was not induced for neuronal development. In contrast, in Six1(-/-) embryos, all neuronal lineage markers were initially expressed but the pattern of their expression was altered. Although very few, the pioneer neurons were initially present in the Six1 mutant OE. However, neurogenesis ceased by E12.5 due to markedly increased cell apoptosis and reduced proliferation, thus defining the cellular defects occurring in Six1(-/-) OE that have not been previously observed. Our findings demonstrate that Six1/4 function at the top of early events controlling olfactory placode formation and neuronal development. Our analyses show that the threshold of Six1/4 may be crucial for the expression of olfactory specific genes and that Six1 and Six4 may act synergistically to mediate olfactory placode specification and patterning through Fgf and Bmp signaling pathways.

title: The role of Six1 in mammalian auditory system development
abstract: The homeobox Six genes, homologues to Drosophila sine oculis (so) gene, are expressed in multiple organs during mammalian development. However, their roles during auditory system development have not been studied. We report that Six1 is required for mouse auditory system development. During inner ear development, Six1 expression was first detected in the ventral region of the otic pit and later is restricted to the middle and ventral otic vesicle within which, respectively, the vestibular and auditory epithelia form. By contrast, Six1 expression is excluded from the dorsal otic vesicle within which the semicircular canals form. Six1 is also expressed in the vestibuloacoustic ganglion. At E15.5, Six1 is expressed in all sensory epithelia of the inner ear. Using recently generated Six1 mutant mice, we found that all Six1+/- mice showed some degree of hearing loss because of a failure of sound transmission in the middle ear. By contrast, Six1-/- mice displayed malformations of the auditory system involving the outer, middle and inner ears. The inner ear development in Six1-/- embryos arrested at the otic vesicle stage and all components of the inner ear failed to form due to increased cell death and reduced cell proliferation in the otic epithelium. Because we previously reported that Six1 expression in the otic vesicle is Eya1 dependent, we first clarified that Eya1 expression was unaffected in Six1-/- otic vesicle, further demonstrating that the Drosophila Eya-Six regulatory cassette is evolutionarily conserved during mammalian inner ear development. We also analyzed several other otic markers and found that the expression of Pax2 and Pax8 was unaffected in Six1-/- otic vesicle. By contrast, Six1 is required for the activation of Fgf3 expression and the maintenance of Fgf10 and Bmp4 expression in the otic vesicle. Furthermore, loss of Six1 function alters the expression pattern of Nkx5.1 and Gata3, indicating that Six1 is required for regional specification of the otic vesicle. Finally, our data suggest that the interaction between Eya1 and Six1 is crucial for the morphogenesis of the cochlea and the posterior ampulla during inner ear development. These analyses establish a role for Six1 in early growth and patterning of the otic vesicle.

title: Smad transcription factors.
abstract: Smad transcription factors lie at the core of one of the most versatile cytokine signaling pathways in metazoan biology-the transforming growth factor-beta (TGFbeta) pathway. Recent progress has shed light into the processes of Smad activation and deactivation, nucleocytoplasmic dynamics, and assembly of transcriptional complexes. A rich repertoire of regulatory devices exerts control over each step of the Smad pathway. This knowledge is enabling work on more complex questions about the organization, integration, and modulation of Smad-dependent transcriptional programs. We are beginning to uncover self-enabled gene response cascades, graded Smad response mechanisms, and Smad-dependent synexpression groups. Our growing understanding of TGFbeta signaling through the Smad pathway provides general principles for how animal cells translate complex inputs into concrete behavior.

title: Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1.
abstract: Previous studies have suggested a role of the homeodomain Six family proteins in patterning the developing vertebrate head that involves appropriate segmentation of three tissue layers, the endoderm, the paraxial mesoderm and the neural crest cells; however, the developmental programs and mechanisms by which the Six genes act in the pharyngeal endoderm remain largely unknown. Here, we examined their roles in pharyngeal pouch development. Six1-/- mice lack thymus and parathyroid and analysis of Six1-/- third pouch endoderm demonstrated that the patterning of the third pouch into thymus/parathyroid primordia is initiated. However, the endodermal cells of the thymus/parathyroid rudiments fail to maintain the expression of the parathyroid-specific gene Gcm2 and the thymus-specific gene Foxn1 and subsequently undergo abnormal apoptosis, leading to a complete disappearance of organ primordia by E12.5. This thus defines the thymus/parathyroid defects present in the Six1 mutant. Analyses of the thymus/parathyroid development in Six1-/-;Six4-/- double mutant show that both Six1 and Six4 act synergistically to control morphogenetic movements of early thymus/parathyroid tissues, and the threshold of Six1/Six4 appears to be crucial for the regulation of the organ primordia-specific gene expression. Previous studies in flies and mice suggested that Eya and Six genes may function downstream of Pax genes. Our data clearly show that Eya1 and Six1 expression in the pouches does not require Pax1/Pax9 function, suggesting that they may function independently from Pax1/Pax9. In contrast, Pax1 expression in all pharyngeal pouches requires both Eya1 and Six1 function. Moreover, we show that the expression of Tbx1, Fgf8 and Wnt5b in the pouch endoderm was normal in Six1-/- embryos and slightly reduced in Six1-/-;Six4-/- double mutant, but was largely reduced in Eya1-/- embryos. These results indicate that Eya1 appears to be upstream of very early events in the initiation of thymus/parathyroid organogenesis, while Six genes appear to act in an early differentiation step during thymus/parathyroid morphogenesis. Together, these analyses establish an essential role for Eya1 and Six genes in patterning the third pouch into organ-specific primordia.

title: Uroplakins as markers of urothelial differentiation.
abstract: In studying the physiology and diseases of a given organ, the identification of the major differentiation products of that particular organ is a crucially important step. Detailed knowledge on the structure and function of these molecules, which are frequently accumulated in large quantities during cellular differentiation, help elucidate the molecular basis of important physiological processes, such as muscle contraction. Mutations of many of the tissue-specific products have been found to be the underlying causes for many organ-restricted diseases. The availability of promoters of some of these tissue-specific genes enables one to drive various transgenes including mutated differentiation products, specific oncogenes, dominant negative mutants of various receptors, to express uniquely in the target organ. The resultant pathology and abnormal physiology can yield great insights on the functions of the differentiation products per se and various test molecules, as well as the mechanisms of many human diseases. It is in this context that the recent identification of uroplakins as the major differentiation products of mammalian urothelium is important, as this has made it possible to employ many approaches, that have proven to be fruitful in other organ systems, for elucidating the biochemical mechanisms of urothelial growth and differentiation. In this paper, we will discuss what has been learned recently about the uroplakin molecules and their possible role in urothelial differentiation.

title: Eya 1 acts as a critical regulator for specifying the metanephric mesenchyme.
abstract: Although it is well established that the Gdnf-Ret signal transduction pathway initiates metanephric induction, no single regulator has yet been identified to specify the metanephric mesenchyme or blastema within the intermediate mesoderm, the earliest step of metanephric kidney development and the molecular mechanisms controlling Gdnf expression are essentially unknown. Previous studies have shown that a loss of Eya 1 function leads to renal agenesis that is a likely result of failure of metanephric induction. The studies presented here demonstrate that Eya 1 specifies the metanephric blastema within the intermediate mesoderm at the caudal end of the nephrogenic cord. In contrast to its specific roles in metanephric development, Eya 1 appears dispensable for the formation of nephric duct and mesonephric tubules. Using a combination of null and hypomorphic Eya 1 mutants, we now demonstrated that approximately 20% of normal Eya 1 protein level is sufficient for establishing the metanephric blastema and inducing the ureteric bud formation but not for its normal branching. Using Eya 1, Gdnf, Six 1 and Pax 2 mutant mice, we show that Eya 1 probably functions at the top of the genetic hierarchy controlling kidney organogenesis and it acts in combination with Six 1 and Pax 2 to regulate Gdnf expression during UB outgrowth and branching. These findings uncover an essential function for Eya 1 as a critical determination factor in acquiring metanephric fate within the intermediate mesoderm and as a key regulator of Gdnf expression during ureteric induction and branching morphogenesis.

title: A human homologue of the Drosophila eyes absent gene underlies Branchio-Oto-Renal (BOR) syndrome and identifies a novel gene family
abstract: A candidate gene for Branchio-Oto-Renal (BOR) syndrome was identified at chromosome 8q13.3 by positional cloning and shown to underlie the disease. This gene is a human homologue of the Drosophila eyes absent gene (eya), and was therefore called EYA1. A highly conserved 271-amino acid C-terminal region was also found in the products of two other human genes (EYA2 and EYA3), demonstrating the existence of a novel gene family. The expression pattern of the murine EYA1 orthologue, Eya1, suggests a role in the development of all components of the inner ear, from the emergence of the otic placode. In the developing kidney, the expression pattern is indicative of a role for Eya1 in the metanephric cells surrounding the ‘just-divided’ ureteric branches.

title: Branchio-oto-renal syndrome: reduced penetrance and variable expressivity in four generations of a large kindred.
abstract: The family on which this report is based is of interest because it contains individuals with the branchio-oto-renal (BOR) syndrome who have renal hypoplasia or malformations of the kidney or collecting system including duplication; only branchial and ear anomalies; and apparent nonpenetrance of the syndrome. This report provides evidence to support the hypothesis that in some families variable expressivity includes duplication of the urinary collecting system in individuals with other manifestations of the BOR syndrome as well as individuals with branchial and ear anomalies who have apparently normal kidneys.