Pax2 mutation

Kidney organogenesis in vertebrates can be viewed as occurring in four general stages or developmental events. The early patterning of the mesoderm during gastrulation to generate nephrogenic cell populations in the intermediate mesoderm is followed by the formation of a nephric primordium during somitogenesis and the caudal growth of the pronephric or Wolffian duct, which will form the future collecting system of the kidney. Induction of nephron development from nephrogenic mesenchyme and the patterning of nephron primordia into glomerular and tubule domains is closely followed by vascularization of the glomerulus and the onset of blood filtration. Pax genes are important developmental regulators and function at multiple stages of vertebrate kidney organogenesis. In this report, the zebrafish pax2.1 mutant no isthmus was used to investigate the role for pax2.1 in development of the pronephros. During metanephric development, pax2 is expressed strongly in the branching ureteric bud and in comma- and S-shaped mesenchymal condensations. A requirement for pax2.1 in multiple aspects of pronephric development is demonstrated, including tubule and duct epithelial differentiation and cloaca morphogenesis. Morphological analysis demonstrates that noi minus larvae specifically lack pronephric tubules, while glomerular cell differentiation is unaffected. In addition, pax2.1 expression in the lateral cells of the pronephric primordium is required to restrict the domains of Wilms' tumor suppressor (wt1) and vascular endothelial growth factor (VEGF) gene expression to medial podocyte progenitors. Ectopic podocyte-specific marker expression in pronephric duct cells correlates with loss of expression of the pronephric tubule and duct-specific markers mAb 3G8 and a Na+/K+ ATPase a1 subunit. The results suggest that the failure in pronephric tubule differentiation in noi arises from a patterning defect during differentiation of the pronephric primordium and that mutually inhibitory regulatory interactions play an important role in defining the boundary between glomerular and tubule progenitors in the forming nephron (Majumdar,2000).

A new mouse frameshift mutation (Pax2[1Neu]) is described with a 1-bp insertion in the Pax2 gene. This mutation is identical to a previously described mutation in a human family with renal-coloboma syndrome. Heterozygous mutant mice exhibit defects in the kidney, the optic nerve, and the retinal layer of the eye; in homozygous mutant embryos, development is severely affected in the optic nerve, metanephric kidney, and ventral regions of the inner ear. A deletion of the cerebellum and the posterior mesencephalon is observed in homozygous mutant embryos, demonstrating that in contrast to mutations in Pax5, which is also expressed early in the mid-hindbrain region, loss of Pax2 gene function alone results in the early loss of the mid-hindbrain region. The mid-hindbrain phenotype is similar to Wnt1 and En1 mutant phenotypes, suggesting the conservation of gene regulatory networks between vertebrates and Drosophila (Favor, 1996).

The inner ear is a complex sensory organ responsible for balance and sound detection in vertebrates. It originates from a transient embryonic structure, the otic vesicle, which contains all of the information to develop autonomously into the mature inner ear. The development of the otic vesicle is reviewed here, bringing together classical embryological experiments and recent genetic and molecular data. The specification of the prospective ectoderm and its commitment to the otic fate are very early events and can be related to the expression of genes with restricted expression domains. A combinatorial gene expression model for placode specification and diversification, based on classical embryological evidence and gene expression patterns, is discussed. The formation of the otic vesicle is dependent on inducing signals from endoderm, mesoderm and neuroectoderm. Ear induction consists of a sequence of discrete instructions from those tissues that confer the final identity on the otic field, rather than a single all-or-none process. The head ectoderm develops three pairs of sensory placodes from anterior to posterior -- nose, lens and ear -- along with those placodes that generate the neurons of some cranial sensory ganglia. Homeobox genes of the Sine oculis (six) and Distal-less related Dlx families are expressed but these factors are expressed in more than one sensory placode. Specific combinations of genes rather than single gene expression thus appears to be characteristic for each placode. The important role of the neural tube in otic development is highlighted by the abnormalities observed in mouse mutants for the Hoxa1, kreisler and fgf3 genes and those reported in retinoic acid-deficient quail. Still, the nature of the relation between the neural tube and otic development remains unclear. Gene targeting experiments in the mouse have provided evidence for genes potentially involved in regional and cell-fate specification in the inner ear. The disruption of the mouse Brn3.1 gene identifies the first mutation affecting sensory hair-cell specification; mutants for the Pax2 and Nkx5.1 genes show that these two genes are required for the the development of specific regions of the otic vesicle. Several growth-factors contribute to the patterned cell proliferation of the otic vesicle. Among these, IGF-I and FGF-2 are expressed in the otic vesicle and may act in an autocrine manner. Little is known about early mechanisms involved in guiding ear innervation. However, targeted disruption of genes coding for neurotrophins and Trk receptors have shown that once synaptic contacts are established, they depend on specific trophic interactions that involve these two gene families. The accessibility of new cellular and molecular approaches are opening new perspectives in vertebrate developmental biology (Torres, 1998).

The Kidney and retinal defects (Krd) mouse carries a 7-cM transgene-induced deletion on chromosome 19 that includes the Pax2 locus. Adult mice heterozygous for the Krd deletion (Krd/+) are haploid for Pax2 and have a variable, semidominant phenotype characterized by structural defects of the kidney, retina, and optic disc. Renal and ocular anomalies present in heterozygous Pax2 mutants in both mice and humans support the hypothesis that haploinsufficiency of Pax2 underlies the Krd phenotype. To understand the embryonic basis of ocular defects observed in adult Krd/+ mice, immunohistochemistry, digital three-dimensional reconstructions, and quantitative morphometry were used to examine Pax2 protein distribution and ocular development in normal and Krd/+ mice from E10.5 to post-natal day 2. In +/+ embryos, Pax2 immunopositive (Pax2+) cells demarcate the embryonic fissure as it forms in the ventral optic cup and optic stalk. After closure of the embryonic fissure, Pax2 immunostaining disappears from the ventral retina, but persists in a cuff of cells encircling the developing optic disc, the site where ganglion cell axons exit the retina. In Krd/+ embryos, Pax2+ cells in the posterior optic cup and the optic stalk undergo abnormal morphogenetic movements and the embryonic fissure fails to form normally. This results in an abnormal organization of the Pax2+ cells and ganglion cell axons at the nascent optic disc. The abnormal morphogenetic movements of the Pax2+ cells in the embryonic retina and optic stalk and the initial misrouting of the ganglion cell axons give rise to retinal and optic disc defects observed in the adult Krd/+ mice. These results indicate a requirement for full diploid expression of Pax2 for normal morphogenesis of a portion of the embryonic fissure and the optic groove (Otteson, 1998).

Based on morphology and position, it is believed that the Pax2+ cells at the optic disc play a role in guiding ganglion cell axons out of the retina. The Pax2+ cells that encircle the optic disc are a subset of the channel-forming cells that create extracellular spaces or channels among their endfeet at the inner surface of the pia that are believed to play a role in guiding ganglion cell axons out of the retina into the optic nerve. Netrin-1 (see Drosophila Netrin), a secreted protein that is required for axon guidance of commissural neurons within the developing brain, is expressed by the Pax2+ cells at the optic disc and optic nerve; Netrin-1 mutants show significant axon pathfinding defects at the optic disc. In the Krd/+ embryos, the disorganization of the Pax2-expressing cells at the optic disc would be predicted to result in both an abnormal organization of the network of extracellular channels and a dispersion of the associated chemoattractant guidance cues to ectopic locations. Either or both of these events could give rise to the intraretinal defects in axon pathfinding observed in Krd/+ embryos (Otteson, 1998)

The development of two major subdivisions of the vertebrate nervous system, the midbrain and the cerebellum, is controlled by signals emanating from a constriction in the neural primordium called the midbrain/hindbrain organizer. The closely related transcription factors Pax-2 and Pax-5 exhibit an overlapping expression pattern very early in the developing midbrain/hindbrain junction. Experiments carried out in fish with neutralizing antibodies against Pax-b, the orthologue of Pax-2 in mouse, places this gene family in an regulatory cascade necessary for the development of the midbrain and the cerebellum. The targeted mutation of Pax-5 has been reported to have only slight effects in the development of structures derived from the isthmic constriction, whereas the Pax-2 null mutant mice show a background-dependent phenotype with varying penetrance. To test a possible redundant function between Pax-2 and Pax-5, the brain phenotypes of mice expressing different dosages of both genes were analyzed. A conserved biological function of both proteins is demonstrated by the midbrain/hindbrain regionalization. One allele of Pax-2, but not Pax-5, is necessary and sufficient for midbrain and cerebellum development in C57BL/6 mice (Schwarz, 1997).

The nested expression patterns of the paired-box containing transcription factors Pax2/5 and Pax6 demarcate the midbrain and forebrain primordium at the neural plate stage. In Pax2/5 deficient mice, the mesencephalon/metencephalon primordium is completely missing, resulting in a fusion of the forebrain to the hindbrain. Morphologically, in the alar plate the deletion is characterized by the substitution of the tectum (dorsal midbrain) and cerebellum (dorsal metencephalon) by the caudal diencephalon and in the basal plate by the replacement of the midbrain tegmentum by the ventral metencephalon (pons). Molecularly, the loss of the tectum is demonstrated by an expanded expression of Pax6, (the molecular determinant of posterior commissure), and a rostral shift of the territory of expression of Gbx2 and Otp (markers for the pons), toward the caudal diencephalon. These results suggest that an intact territory of expression of Pax2/5 in the neural plate, nested between the rostral and caudal territories of expression of Pax6, is necessary for defining the midbrain vesicle (Schwarz, 1999).

The molecular basis of the Pax2 and Pax6 function in the establishment of visual system territories has been studied. Loss-of-function mutants have revealed crucial roles for Pax2 in the generation of the optic stalk and for Pax6 in the development of the optic cup. Ectopic expression of Pax6 in the optic stalk under control of Pax2 promoter elements results in a shift of the optic cup/optic stalk boundary, indicated by the presence of retinal pigmented cells on the optic stalk. By studying mouse embryos at early developmental stages an expansion of Pax2 expression domain was detected in the Pax6-/- mutant and an expansion of the Pax6 expression domain in the Pax2-/- embryo. These results suggest that the position of the optic cup/optic stalk boundary depends on Pax2 and Pax6 expression, hinting at a possible molecular interaction. Using gel shift experiments, the presence was confirmed of Pax2- and Pax6- binding sites on the retina enhancer of the Pax6 gene and on the Pax2 upstream control region, respectively. Co-transfection experiments have revealed a reciprocal inhibition of Pax2 promoter/enhancer activity by Pax6 protein and vice versa. Based on these findings, a model for Pax gene regulation that establishes the proper spatial regionalization of the mammalian visual system is proposed (Schwartz, 2000).

The following molecular model for visual system regionalization is proposed. The signaling molecule Sonic hedgehog, which establishes the midline of the brain and subdivides the eye domain, is presumably the initial activator of Pax2. Strong evidence comes from analysis in mice lacking sonic hedgehog function. With regard to eye patterning, it has been demonstrated that sonic hedgehog is required for Pax2 expression and optic stalk formation. These results are consistent with previous evidence from zebrafish studies suggesting that sonic hedgehog activity from the ventral midline normally stimulates expression of Pax2 in the adjacent optic stalk precursors and represses expression of Pax6. This in turn restricts Pax6 expression to distal portions of the optic vesicle. Unfortunately, no candidate gene activating Pax6 in the prosencephalic portion of the neural plate has been described. A possible candidate directly or indirectly acting on Pax6 activation, based on the spatial-temporal expression pattern, could be the homeobox protein Otx2 or the cell-signaling molecule Notch1 (Schwartz, 2000 and references therein).

After their initial activation, the Pax2 and Pax6 expression becomes independent of the activating factors, owing to their autocatalytic enhancement. Pax6 protein can bind its own enhancer and has the potential to stimulate transcription. The Pax2 protein, in turn, has the capacity to bind to the Pax6 enhancer and represses transcription. The Pax6 protein has the same function on the Pax2 enhancer. Although the Pax2 promoter region contains one site for Pax2 protein binding, no Pax2 autocatalytic enhancement was observed in the cell culture assays. Sonic hedgehog could be hypothesized to be the Pax2 activator, since the sonic hedgehog gradient always reaches the Pax2-expressing territory; alternatively, the promoter region used in the transfection experiment does not contain the element(s) that are essential for Pax2 self-activation. Consequently, in a region where the activity of these genes overlaps, reciprocal inhibition establishes a boundary, finally leading to a steady state where no cells express both proteins at the same time. This in consequence leads to the formation of the optic stalk and optic cup boundary. However, it is not known whether the Pax2 and Pax6 boundary and therefore the division of a Pax2- and a Pax6- positive domain (which follow very different developmental fates) is the first separation generating positional information for the eye field (Schwartz, 2000 and references therein).

In Drosophila, similar to the vertebrate system where Pax2 expression is restricted to the optic stalk and Pax6 expression to the optic cup and the lens, sparkling and eyeless are expressed in the homolog structures. sparkling expression is found in the precursors of cone and primary pigment cells, whereas eyeless expression is restricted to regions anterior to the morphogenetic furrow in the undifferentiated part of the eye disc epithelium. In addition, the phenotype of the eyeless or the sparkling mutation is comparable to the Pax6-/- and Pax2-/- phenotypes in the mouse. In conclusion, Pax2 and Pax6 and their Drosophila homologs sparkling and eyeless, play important and strikingly conserved roles in the morphogenesis and regional specification of the vertebrate or insect eyes.The visual system is most probably not the only example of reciprocal inhibition between Pax genes in the regionalization of the body plan. These genes are expressed in adjacent territories in other embryonic structures. The regionalization of the midbrain/forebrain boundary is crucially dependant upon Pax6 expression in the prosencephalon, and on Pax2 and Pax5 expression in the mesencephalon. In the spinal cord, Pax1/9 and Pax3/7 could be good candidates for such a regionalization mechanism in more caudal parts of the neuroectoderm. Therefore, the molecular mechanism described here for the eye may be of more general importance (Schwartz, 2000).

Renal-coloboma syndrome is a recently described autosomal dominant syndrome of abnormal optic nerve and renal development. Two families have been reported with both renal-coloboma syndrome and mutations of the PAX2 gene. The PAX2 gene, which encodes a DNA-binding protein, is expressed in the developing ear, CNS, eye, and urogenital tract. Ocular and/or renal abnormalities have been consistently noted in the five reports to date of patients with renal-coloboma syndrome, but PAX2 expression patterns suggest that auditory and CNS abnormalities may be additional features of renal-coloboma syndrome. To determine whether additional clinical features are associated with PAX2 mutations, PCR-SSCP has been used to identify PAX2 gene mutations in patients. Four patients are repored with mutations in exon 2: one has severe ocular and renal disease, microcephaly, and retardation; another has ocular and renal disease with high-frequency hearing loss. Unexpectedly, extreme variability in clinical presentation is observed between a mother, her son, and an unrelated patient, all of whom had the same PAX2 mutation as previously described in two siblings with renal-coloboma syndrome. These results suggest that a sequence of seven Gs in PAX2 exon 2 may be particularly prone to mutation (Schimmenti, 1997).

Human dysplastic kidneys are developmental aberrations, responsible for many cases of chronic renal failure in very houng children. Such kidneys contain poorly differentiated metanephric cells in addition to metaplastic elements. Apoptosis is prominent in undifferentiated cells around dysplastic tubules, perhaps explaining the tendency of some of these organs to regress. In contrast, apoptosis is rare in dysplastic epithelia, which are thought to be ureteric bud malformations. On occasion, these tubules form cysts which distend the abdominal cavity (the multicystic dysplastic kidney); dysplastic kidneys rarely become malignant. Dysplastic tubules maintain a high rate of proliferation postnatally; PAX2, a potentially oncogenic transcription factor, is expressed in these epithelia. In contrast, both cell proliferation and PAX2 are downregulated during normal maturation of human collecting ducts. BCL2, a protein that prevents apoptosis in renal mesenchymal to epithelial conversion, is expressed ectopically in dysplastic kidney epithelia. It is proposed that dysplastic cyst formation may be understood in terms of aberrant temporal and spatial expression of master genes, which are tightly regulated in the normal program of human nephrogenesis (Winyard, 1996).

During gestation, the gene Pax2 is expressed in the mid-hindbrain area, the developing eye and the inner ear. Pax2 null mutant mice were generated that demonstrate the need for Pax2 in the establishment of axonal pathways along the optic stalks and ventral diencephalon. In null mutant brains, the optic tracts remain totally ipsilateral due to agenesis of the optic chiasma. Pax2 mutants show extension of the pigmented retina into the optic stalks and failure of the optic fissure to close, resulting in coloboma. In the inner ear, Pax2 mutants show agenesis of the cochlea and the spiral ganglion, i.e., the parts of the organ responsible for auditory function and in whose primordium Pax2 is expressed. These results identify Pax2 as a major regulator of patterning during organogenesis of the eye and inner ear and indicate its function in morphogenetic events required for closure of the optic fissure and neural tube (Torres, 1996).

The murine cpk mouse develops a rapid-onset polycystic kidney disease (PKD) with many similarities to human PKD. During kidney development, the transcription factor Pax2 is required for the specification and differentiation of the renal epithelium. In humans, Pax2 is also expressed in juvenile cystic kidneys where it correlates with cell proliferation. In this report, Pax2 expression is demonstrated in the cystic epithelium of the mouse cpk kidneys. To assess the role of Pax2 during the development of polycystic kidney disease, the progression of renal cysts was examined in cpk mutants carrying one or two alleles of Pax2. Reduced Pax2 gene dosage results in a significant inhibition of renal cyst growth while maintaining more normal renal structures. The inhibition of cyst growth is not due to reduced proliferation of the cystic epithelium, but rather to increased cell death in the Pax2 heterozygotes. Increased apoptosis with reduced Pax2 gene dosage was also observed in normal developing kidneys. Thus, increased cell death is an integral part of the Pax2 heterozygous phenotype and may be the underlying cause of Pax gene haploinsufficiency. That the cystic epithelium requires Pax2 for continued expansion underscores the embryonic nature of the renal cystic cells and may provide new insights toward growth suppression strategies (Ostrom, 2000).

Pax2 and Pax5 arose by gene duplication at the onset of vertebrate evolution and have since diverged in their developmental expression patterns. They are expressed in different organs of the mouse embryo except for their coexpression at the midbrain-hindbrain boundary (MHB), which functions as an organizing center to control midbrain and cerebellum development. During MHB development, Pax2 expression is initiated prior to Pax5 transcription, and Pax2-/- embryos fail to generate the posterior midbrain and cerebellum, whereas Pax5-/- mice exhibit only minor patterning defects in the same brain regions. To investigate whether these contrasting phenotypes are caused by differences in the temporal expression or biochemical activity of these two transcription factors, a knock-in (ki) mouse, which expresses a Pax5 minigene under the control of the Pax2 locus, has been generated. Midbrain and cerebellum development was entirely rescued in Pax25ki/5ki embryos. Pax5 could furthermore completely substitute for the Pax2 function during morphogenesis of the inner ear and genital tracts, despite the fact that the Pax5 transcript of the Pax25ki allele is expressed only at a fivefold lower level than the wild-type Pax2 mRNA. As a consequence, the Pax25ki allele is able to rescue most but not all Pax2 mutant defects in the developing eye and kidney, both of which are known to be highly sensitive to Pax2 protein dosage. Together these data demonstrate that the transcription factors Pax2 and Pax5 have maintained equivalent biochemical functions since their divergence early in vertebrate evolution (Bouchard, 2000).

The outgrowth of the ureteric bud from the posterior nephric duct epithelium and the subsequent invasion of the bud into the metanephric mesenchyme initiate the process of metanephric, or adult kidney, development. The receptor tyrosine kinase RET and glial cell-derived neurotrophic factor (GDNF) form a signaling complex that is essential for ureteric bud growth and branching morphogenesis of the ureteric bud epithelium. Pax2 expression in the metanephric mesenchyme is independent of induction by the ureteric bud. Pax2 mutants are deficient in ureteric bud outgrowth and do not express GDNF in the uninduced metanephric mesenchyme. Furthermore, Pax2 mutant mesenchyme is unresponsive to induction by wild-type heterologous inducers. In normal embryos, GDNF is sufficient to induce ectopic ureter buds in the posterior nephric duct, a process inhibited by bone morphogenetic protein 4. However, GDNF replacement in organ culture is not sufficient to stimulate ureteric bud outgrowth from Pax2 mutant nephric ducts, indicating additional defects in the nephric duct epithelium of Pax2 mutants. Pax2 can activate expression of GDNF in cell lines derived from embryonic metanephroi. Furthermore, Pax2 protein can bind to upstream regulatory elements within the GDNF promoter region and can transactivate expression of reporter genes. Thus, activation of GDNF by Pax2 coordinates the position and outgrowth of the ureteric bud such that kidney development can begin (Brophy, 2001).

The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. This study introduces zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However, during larval stages, both conserved and divergent features of development are found, in comparison to mammals. A major difference is that in fish, evidence is found for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. pax2.1 and pax8, members of the zebrafish pax2/5/8 paralog group, are expressed in the thyroid primordium, whereas in mice, only Pax8 has a function during thyroid development. Analysis of the zebrafish pax2.1 mutant no isthmus (noi–/–) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi–/–, but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi–/–. Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. It is suggested that the noi–/– mutant serves as a new zebrafish model for hypothyroidism (Wendl, 2002).

The transcription factors Pax2 and Pax6 are co-expressed in the entire optic vesicle (OV) prior and concomitant with the establishment of distinct neuroretinal, retinal, pigmented-epithelial and optic-stalk progenitor domains, suggesting redundant functions during retinal determination. Pax2; Pax6 compound mutants display a dose-dependent reduction in the expression of the melanocyte determinant Mitf, accompanied by transdifferentiation of retinal pigmented epithelium (RPE) into neuroretina (NR) in Pax2-/-; Pax6+/- embryos, which strongly resembles the phenotype of Mitf-null mutants. In Pax2-/-; Pax6-/- OVs Mitf fails to be expressed and NR markers occupy the area that usually represents the Mitf+ RPE domain. Furthermore, both Pax2 and Pax6 bind to and activate a MITF RPE-promoter element in vitro, whereas prolonged expression of Pax6 in the Pax2-positive optic stalk leads to ectopic Mitf expression and RPE differentiation in vivo. Together, these results demonstrate that the redundant activities of Pax2 and Pax6 direct the determination of RPE, potentially by directly controlling the expression of RPE determinants (Bäumer, 2003).

The midbrain-hindbrain domain (MH) of the vertebrate embryonic neural tube develops in response to the isthmic organizer (IsO), located at the midbrain-hindbrain boundary (MHB). MH derivatives are largely missing in mutants affected in IsO activity; however, the potentialities and fate of MH precursors in these conditions have not been directly determined. To follow the dynamics of MH maintenance in vivo, artificial chromosome transgenesis was used in zebrafish to construct lines where egfp transcription is driven by the complete set of regulatory elements of her5, the first known gene expressed in the MH area. In these lines, egfp transcription faithfully recapitulates her5 expression from its induction phase onwards. Using the stability of GFP protein as lineage tracer, her5, first demonstrated at gastrulation, is a selective marker of MH precursor fate. By comparing GFP protein and her5 transcription, the spatiotemporal dynamics of her5 expression that conditions neurogenesis progression towards the MHB over time was further revealed. The molecular identity of GFP-positive cells was traced in the acerebellar (ace) and no-isthmus (noi) mutant backgrounds to analyze directly fgf8 and pax2.1 mutant gene activities for their ultimate effect on cell fate. Most MH precursors are maintained in both mutants but express abnormal identities, in a manner that strikingly differs between the ace and noi contexts. These observations directly support a role for Fgf8 in protecting anterior tectal and metencephalic precursors from acquiring anterior identities, while Pax2.1 controls the choice of MH identity as a whole. Together, these results suggest a model where an ordered MH pro-domain is identified at gastrulation, and where cell identity choices within this domain are subsequently differentially controlled by Fgf8 and Pax2.1 functions (Tallafuß, 2003).

Regulation of Pax2 Expression

Organization of the inner ear into auditory and vestibular components is dependent on localized patterns of gene expression within the otic vesicle. Surrounding tissues are known to influence compartmentalization of the otic vesicle, yet the participating signals remain unclear. This study identifies Sonic hedgehog (Shh) secreted by the notochord and/or floor plate as a primary regulator of auditory cell fates within the mouse inner ear. Whereas otic induction proceeds normally in Shh-/- embryos, morphogenesis of the inner ear is greatly perturbed by midgestation. Ventral otic derivatives including the cochlear duct and cochleovestibular ganglia fail to develop in the absence of Shh. The origin of the inner ear defects in Shh-/- embryos can be traced back to alterations in the expression of a number of genes involved in cell fate specification including Pax2, Otx1, Otx2, Tbx1, and Ngn1. Several of these genes are targets of Shh signaling given their ectopic activation in transgenic mice that misexpress Shh in the inner ear. Taken together, these data support a model whereby auditory cell fates in the otic vesicle are established by the direct action of Shh (Riccomagno, 2002).

The failure in cochlear duct outgrowth in Shh-/- embryos is most likely mediated by the lack of Pax2, Otx1, and Otx2, genes previously ascribed with required roles in this process. Furthermore, the observations that Shh is both necessary and sufficient for the expression of Pax2 along the medial wall of the otic vesicle implicates Pax2 as a downstream effector of Shh signaling in the otocyst. The regulation of Pax2 by Shh in inner ear development resembles the relationship between Pax2 and Shh in the formation of another placode-derived sensory organ, the eye. In generating the proximal-distal axis of the optic cup, Shh signaling from the ventral forebrain promotes Pax2-expressing proximal fates (optic fissure, optic stalk) at the expense of Pax6-expressing distal fates (prospective retina, pigmented epithelium, and lens. To maintain the border between proximal and distal lineages, Pax2 and Pax6 antagonize each other by mutual transcriptional repression. The commonality in response by Pax genes to Hh signaling can be broadened to include Pax1 in the ventral somite and Pax6 in the ventral neural tube. In both of these cases, Pax family members with opposing functions are expressed adjacent to sites of Pax1 and Pax6 activity. This is not a general rule, since Pax genes are not expressed complementary to Pax2 in the inner ear, although other transcription factors may be fulfilling an antagonistic role in this tissue. The observations thus add to the growing list of functions for Pax transcription factors in mediating cellular responses to Shh signaling (Riccomagno, 2002).

The vertebrate inner ear arises from an ectodermal thickening, the otic placode, that forms adjacent to the presumptive hindbrain. Previous studies have suggested that competent ectodermal cells respond to Fgf signals from adjacent tissues and express two highly related paired box transcription factors Pax2a and Pax8 in the developing placode. Compromising the functions of both Pax2a and Pax8 together blocks zebrafish ear development, leaving only a few residual otic cells. This suggests that Pax2a and Pax8 are the main effectors downstream of Fgf signals. The results further provide evidence that pax8 expression and pax2a expression are regulated by two independent factors, Foxi1 and Dlx3b, respectively. Combined loss of both factors eliminates all indications of otic specification. It is suggested that the Foxi1-Pax8 pathway provides an early 'jumpstart' of otic specification that is maintained by the Dlx3b-Pax2a pathway (Hans, 2004).

It is proposed that induction of otic fate by Fgf signals takes place only when cells are competent to respond, and that this competence is provided by Foxi1 and Dlx3b. A direct role for Foxi1 and Dlx3b in competence needs to be demonstrated, for example by ectopic expression and transplantation experiments. Foxi1 and Dlx3b function by regulating pax8 and pax2a expression, respectively, in an Fgf-dependent fashion. In Dlx3b-deficient embryos, expression of pax8 is indistinguishable from that in wild-type embryos, presumably owing to normal Foxi1 and Fgf signaling. However, otic pax2a expression is initiated only very late and weakly. By contrast, otic pax8 expression fails and pax2a expression is present although delayed in foxi1 mutants. Inhibition of both factors, Foxi1 and Dlx3b, completely blocks otic specification even in the presence of functional Fgf signaling. By activating Pax8, Foxi1 thus provides competence to otic precursor cells to respond to early Fgf signaling; Dlx3b and Pax2a subsequently maintain this competence (Hans, 2004).

Pax2, kidney differentiation and Wilms' tumor

The pattern-forming event of kidney tubulogenesis is initiated by the inductive transition of mesenchymal cells to epithelial phenotype, a transition that is critically dependent on the regulated expression of the developmental control gene, Pax-2. Because of a defined role in in vitro renal tubulogenesis, an evaluation was made of the effects of epidermal growth factor (EGF), transforming growth factor (TGF)-beta 1 and retinoic acid on Pax-2 gene expression in proximal tubule cells (PTC). Rabbit cultured PTC grown to confluent quiescent conditions were analyzed for the effect of various factors on Pax-2 gene expression. PTC express high levels of Pax-2. A 24-hour exposure to EGF, a potent mitogen of PTC, increases this level of expression. In contrast, Pax-2 gene expression is suppressed by treating PTC with retinoic acid, a well-described differentiating factor, and with TGF-beta 1, a recognized antiproliferative agent for these cells, which suggests that Pax-2 has a role in renal cell proliferation. The mechanism of the effect of TGF-beta 1 on Pax-2 mRNA levels was further detailed. TGF-beta 1 does not affect Pax-2 transcription rates; however, in a dose-dependent manner, it diminishes the stability of Pax-2 mRNA. TGF-beta 1 reduces Pax-2 mRNA stability from a control half-life of 120 min to a half-life of less than 60 min. This study demonstrates that various soluble inductive factors affect Pax-2 gene expression in renal tubule cells. TGF-beta 1 downregulates Pax-2 gene expression through a posttranscriptional process, an acknowledged mechanism for modulating important growth regulatory gene products (Liu, 1997).

PAX2 is a member of the paired box family of genes with an important role in kidney, genital tract and eye development. Another gene essential for kidney and genital tract development is the Wilms tumour gene, WT1. PAX2 and WT1 encode transcription factors expressed during fetal kidney development in patterns that overlap both spatially and temporally. The overlap of PAX2 and WT1 expression in fetal kidney prompted a determination of whether PAX2 regulates the WT1 gene. To investigate this possibility, the WT1 promoter and a series of WT1 promoter deletion fragments were cloned into a luciferase reporter vector, and used in co-transfection experiments with PAX2 expression constructs. PAX2 transactivates the WT1 promoter up to 35-fold in CHO-K1 cells, and from four- to sevenfold in 293 cells. Two regions of the WT1 promoter are required in the same promoter construct for efficient transactivation by PAX2 in CHO-K1 cells. Purified recombinant PAX2 protein is found to bind to two sites in the WT1 promoter, at -205/-230 and +377/+402. Removal of WT1 promoter sequences containing the -205/-230, or +377/+402 binding sites abolishes transactivation of the WT1 promoter by PAX2 in CHO-K1 cells, and has a differential effect on transactivation of the WT1 promoter in 293 cells, depending on the PAX2 isoform used. A fragment containing the -205/-230 site alone can be transactivated by PAX2. These findings suggest that PAX2 is a tissue-specific modulator of WT1 expression, and is involved in cell growth control via WT1 (McConnell, 1997).

The Wilms' tumor suppressor gene, wt1, encodes a zinc finger protein that functions as a transcriptional regulator. Expression of the wt1 gene is developmentally regulated and restricted to a small set of tissues that include the fetal urogenital system, mesothelium, and spleen. In the developing kidney, induction of neprohogenesis by the ureter is accompanied by an increase in the expression levels of the Pax-2 gene. This is followed by an increase in wt1 expression, as mesenchymal cells condense and differentiate. PAX2 isoforms are capable of transactivating the wt1 promoter. Deletion mutagenesis of the wt1 promoter identifies an element responsible for mediating PAX2 responsiveness, located between nucleotides -33 and -71 relative to the first wt1 transcription start site. Consistent with its identity as a PAX responsive element, multimerization of this motif upstream of a heterologous minimal promoter enhances reporter activity when co-transfected with a Pax-2 expression vector. PAX2 can also stimulate expression of the endogenous wt1 gene. These results suggest that a role for PAX2 during mesenchyme-to-epithelium transition in renal development is to induce wt1 expression (Dehbi, 1996a).

The patterns of expression of the human PAX2 gene in Wilms' tumors and human fetal kidney were examined by Northern blot and in situ hybridization. In situ hybridization analysis reveals that PAX2 is expressed in nephrogenic structures in fetal kidney and also in Wilms' tumors. This pattern of expression suggests that PAX2 may have a role in differentiation of tissues in the kidney. In fetal kidney, PAX2 expression rapidly attenuates following the initial differentiation, but no evidence of attenuation was found in Wilms' tumors. The timing of PAX2 expression is restricted to fetal development, although high levels of expression were also observed in nephrogenic rests of residual normal juvenile kidney tissue adjacent to a Wilms' tumor. Nephrogenic rests are the presumptive precursors of Wilms' tumors but are not necessarily neoplastic. The failure of PAX2 expression to attenuate in Wilms' tumor and nephrogenic rests may be associated with events leading to the onset of Wilms' tumors. By somatic cell hybrid mapping, the PAX2 gene was localized to chromosome 10q22.1-q24.3, although this region has not previously been implicated in Wilms' tumor (Eccles, 1992).

Pax2 is a transcription factor with important functions during kidney development. Ectopic expression of Pax2 in podocytes has been reported in various glomerular diseases, but the functional relevance remains unknown. An inducible mouse model was developed that allows activation of Pax2 specifically in podocytes. Persistent expression of Pax2 does not interfere with the initial differentiation of podocytes, but mice ectopically expressing PAX2 develop end-stage renal failure soon after birth. Similarly, activation of PAX2 in healthy adult animals results in renal disease within 3 weeks after podocyte-specific induction of a deleter Cre. PAX2 activation causes repression of the podocyte key regulator molecule Wt1 and consequently a dramatic reduction of nephrin expression. Recruitment of the groucho-related protein TLE4 may be involved in converting Pax2 into a transcriptional repressor of Wt1. Finally, treatment of mice with an angiotensin-converting enzyme (ACE) inhibitor normalizes renal function and induces upregulation of the important structural molecule nephrin via a Wt1-independent pathway. These data demonstrate the functional significance of PAX2 reexpression in mature podocytes for the development of glomerular diseases and suggest that reactivation of PAX genes in terminally differentiated cells leads to a more dedifferentiated phenotype (Wagner, 2006).

Pax2 miscellaneous targets

The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues. Through alternative splicing, the murine Pax-2 gene encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences, as well as the three immunopurified Pax-2 binding sites, demonstrate that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2. The genomic clones can now be used as sequence tags to identify potential target loci (Phelps, 1996).

The paired domain transcription factor Pax2 is required for the formation of the isthmic organizer (IsO) at the midbrain-hindbrain boundary, where it initiates expression of the IsO signal Fgf8. To gain further insight into the role of Pax2 in mid-hindbrain patterning, novel Pax2-regulated genes were sought by cDNA microarray analysis of FACS-sorted GFP+ mid-hindbrain cells from wild-type and Pax2–/– embryos carrying a Pax2GFP BAC transgene. Five genes have been identified that depend on Pax2 function for their expression in the mid-hindbrain boundary region. These genes code for the transcription factors En2 and Brn1 (Pou3f3), the intracellular signaling modifiers Sef and Tapp1, and the non-coding RNA Ncrms. The Brn1 gene was further identified as a direct target of Pax2; two functional Pax2-binding sites in the promoter and in an upstream regulatory element of Brn1 are essential for lacZ transgene expression at the mid-hindbrain boundary. Moreover, ectopic expression of a dominant-negative Brn1 protein in chick embryos implicates Brn1 in Fgf8 gene regulation. Together, these data defined novel functions of Pax2 in the establishment of distinct transcriptional programs and in the control of intracellular signaling during mid-hindbrain development (Bouchard, 2005).

Holoprosencephaly (HPE) is the most common congenital malformation of the forebrain in human. Several genes with essential roles during forebrain development have been identified because they cause HPE when mutated. Among these are genes that encode the secreted growth factor Sonic hedgehog (Shh) and the transcription factors Six3 and Zic2. In the mouse, Six3 and Shh activate each other's transcription, but a role for Zic2 in this interaction has not been tested. This study demonstrates that in zebrafish, as in mouse, Hh signaling activates transcription of six3b in the developing forebrain. zic2a is also activated by Hh signaling, and represses six3b non-cell-autonomously, i.e. outside of its own expression domain, probably through limiting Hh signaling. Zic2a repression of six3b is essential for the correct formation of the prethalamus. The diencephalon-derived optic stalk (OS) and neural retina are also patterned in response to Hh signaling. This study shows that zebrafish Zic2a limits transcription of the Hh targets pax2a and fgf8a in the OS and retina. The effects of Zic2a depletion in the forebrain and in the OS and retina are rescued by blocking Hh signaling or by increasing levels of the Hh antagonist Hhip, suggesting that in both tissues Zic2a acts to attenuate the effects of Hh signaling. These data uncover a novel, essential role for Zic2a as a modulator of Hh-activated gene expression in the developing forebrain and advance understanding of a key gene regulatory network that, when disrupted, causes HPE (Sanek, 2009).

Evolutionary homologs continued: part 4/5 | part 5/5 | back to part 1/5 | part 2/5

sparkling: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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