Vertebrate Pax2 cloning, expression and regulation

Little is known about the factors that control the specification of the mid-hindbrain domain (MHD) within the vertebrate embryonic neural plate. Because the head-trunk junction of the Drosophila embryo and the MHD have patterning similarities, vertebrate genes have been sought related to the Drosophila head gap gene buttonhead (btd), which in the fly specifies the head-trunk junction. The identification of a zebrafish gene which, like btd, encodes a zinc-finger transcriptional activator of the Sp-1 family (hence its name, bts1 for btd/Sp-related-1) is reported; bts1 shows a restricted expression in the head. During zebrafish gastrulation, bts1 is transcribed in the posterior epiblast, including the presumptive MHD, and precedes in this area the expression of other MHD markers such as her5, pax2.1 and wnt1. Ectopic expression of bts1 combined to knock-down experiments demonstrates that Bts1 is both necessary and sufficient for the induction of pax2.1 within the anterior neural plate, but is not involved in regulating her5, wnt1 or fgf8 expression. These results confirm that early MHD development involves several genetic cascades that independently lead to the induction of MHD markers, and identify Bts1 as a crucial upstream component of the pathway selectively leading to pax2.1 induction. In addition, they imply that flies and vertebrates, to control the development of a boundary embryonic region, have probably co-opted a similar strategy: the restriction to this territory of the expression of a Btd/Sp-like factor (Tallafuss, 2001).

The mammalian Pax2, Pax5 and Pax8 genes code for highly related transcription factors, which play important roles in embryonic development and organogenesis. The characterization of all members of the zebrafish Pax2/5/8 family is reported. These genes have arisen by duplications before or at the onset of vertebrate evolution. Due to an additional genome amplification in the fish lineage, the zebrafish contains two Pax2 genes, the previously known Pax[b] gene (here renamed as Pax2.1) and a novel Pax2.2 gene. The zebrafish Pax2.1 gene most closely resembles the mammalian Pax2 gene in its expression pattern, as it is transcribed first in the midbrain-hindbrain boundary region, then in the optic stalk, otic system, pronephros and nephric ducts, and last in specific interneurons of the hindbrain and spinal cord. Pax2.2 differs from Pax2.1 by the absence of expression in the nephric system and by a delayed onset of transcription in other Pax2.1 expression domains. Pax8 is also expressed in the same domains as Pax2.1, but its transcription is already initiated during gastrulation in the primordia of the otic placode and pronephric anlage, thus identifying Pax8 as the earliest developmental marker of these structures. The zebrafish Pax5 gene, in contrast to its mouse ortholog, is transcribed in the otic system in addition to its prominent expression at the midbrain-hindbrain boundary. The no isthmus (noi) mutation is known to inactivate the Pax2.1 gene, thereby affecting the development of the midbrain-hindbrain boundary region, pronephric system, optic stalk and otic region. Although the different members of the Pax2/5/8 family may potentially compensate for the loss of Pax2.1 function, it is demonstrated here that only the expression of the Pax2.2 gene remains unaffected in noi mutant embryos. The expression of Pax5 and Pax8 is either not initiated at the midbrain-hindbrain boundary or is later not maintained in other expression domains. Consequently, the noi mutation of zebrafish is equivalent to combined inactivation of the mouse Pax2 and Pax5 genes with regard to the loss of midbrain-hindbrain boundary development (Pfeiffer, 1998).

The patterns of the Gbx2, Pax2, Wnt1, and Fgf8 gene expression were analyzed in the chick with respect to the caudal limit of the Otx2 anterior domain, taken as a landmark of the midbrain/hindbrain (MH) boundary. The Gbx2 anterior boundary is always concomitant with the Otx2 posterior boundary. The ring of Wnt1 expression is included within the Otx2 domain and Fgf8 transcripts included within the Gbx2 neuroepithelium. Pax2 expression is centered on the MH boundary with a double decreasing gradient. A new nomenclature is proposed to differentiate the vesicles and constrictions observed in the avian MH domain at stage HH10 and HH20, based on the localization of the Gbx2/Otx2 common boundary (Hidalgo-Snachez, 1999).

Transcriptional factors and signaling molecules are responsible for regionalization of the central nervous system. In the early stage of neural development, Pax6 is expressed in the prosencephalon, while En1 and Pax2 are expressed in the mesencephalon. Pax6 was misexpressed in the mesencephalon to elucidate the mechanism of the di-mesencephalic boundary formation. Histological analysis, expression patterns of diencephalic marker genes, and fiber trajectory of the posterior commissure indicate that Pax6 misexpression causes a caudal shift of the di-mesencephalic boundary. Pax6 represses En1, Pax2 and other tectum (mesencephalon)-related genes such as En2, Pax5, Pax7, but induces Tcf4, a diencephalon marker gene. To know how Pax6 represses En1 and Pax2, a dominant-active or negative form of Pax6 was ectopically expressed. The dominant-active form of Pax6 shows a similar but more severe phenotype than Pax6, while the dominant-negative form shows an opposite phenotype, suggesting that Pax6 acts as a transcriptional activator. Thus Pax6 may repress tectum-related genes by activating an intervening repressor. The results of misexpression experiments, together with normal expression patterns of Pax6, En1 and Pax2, suggest that repressive interaction between Pax6 and En1/Pax2 defines the di-mesencephalic boundary (Matsunaga, 2000).

Generation of cell diversity in the vertebrate central nervous system starts during gastrulation stages in the ectodermal germ layer and involves specialized cell groups, such as the organizer located at the midbrain-hindbrain boundary (MHB). Mutations in the zebrafish no isthmus (noi) gene alter development of the MHB, and affect the pax2.1 gene (formerly pax(zf-b)). Analysis of the structure of pax2.1 reveals at least 12 normal splice variants. The noi alleles can be arranged, by molecular and phenotypic criteria, into a series of five alleles of differing strength, ranging from a null allele to weak alleles. In keeping with a role in development of the MHB organizer, gene expression is already affected in the MHB primordium of the gastrula neural ectoderm in noi mutants. engrailed gene eng3 activation depends completely on noi function, and eng2 activation is strongly dependent on noi function. In contrast, onset of wnt1, fgf8 and her5 expression occurs normally in the null mutants, but is eliminated later on. These observations suggest that three signaling pathways, involving pax2.1, wnt1 and fgf8, are activated independently in the early anterior-posterior patterning of this area. In addition, analysis of the allelic series unexpectedly suggests that noi activity is also required during dorsal-ventral patterning of the MHB in somitogenesis stages, and possibly in a later eng expression phase. It is proposed that noi/pax2.1 participates in sequential signaling processes as a key integrator of midbrain-hindbrain boundary development (Lun, 1998).

The isolation of zebrafish Fgf8 and its expression during gastrulation, somitogenesis, fin bud and early brain development is described. By demonstrating genetic linkage and by analysing the structure of the Fgf8 gene, it is shown that acerebellar is a zebrafish Fgf8 mutation that may inactivate Fgf8 function. Homozygous acerebellar embryos lack a cerebellum and the midbrain-hindbrain boundary organizer. Fgf8 function is required to maintain, but not initiate, expression of Pax2.1 and other marker genes in this area. Fgf8 and Pax2.1 are activated in adjacent domains that only later become overlapping; activation of Fgf8 occurs normally in no isthmus embryos that are mutant for Pax2.1. These findings suggest that multiple signaling pathways are independently activated in the midbrain-hindbrain boundary primordium during gastrulation, and that Fgf8 functions later during somitogenesis to polarize the midbrain. Fgf8 is also expressed in a dorsoventral gradient during gastrulation and ectopically expressed Fgf8 can dorsalize embryos. Nevertheless, acerebellar mutants show only mild dorsoventral patterning defects. Also, in spite of the prominent role suggested for Fgf8 in limb development, the pectoral fins are largely unaffected in the mutants. Fgf8 is therefore required in development of several important signaling centers in the zebrafish embryo, but may be redundant or dispensable for others (Reifers, 1998).

Kidney organogenesis is initiated with the formation of the pronephric kidney and requires Pax-2 gene function. Pax-2 cDNAs from the frog Xenopus laevis have been isolated and characterized. Expression of Xenopus Pax-2 (XPax-2) genes is confined to the nervous system, sensory organs, the visceral arches, and the developing excretory system. The earliest expression site is detected at stage 13 (early neurula) in the anterior third of the neural plate. XPax-2 expression is associated with two distinct wedge-shaped patches of cells flanking the midline, separated by a small region devoid of expression. In the course of neural tube closure, the two patches of XPax-2 expression converge toward the dorsal midline. These cells will ultimately form the posterior portion of the mid-brain at the midbrain-hindbrain boundary. DNA sequencing of XPax-2 cDNAs isolated from head and pronephric kidney libraries reveals seven novel alternatively spliced Pax-2 isoforms. They all retain DNA-binding domains, but can differ significantly in their C termini, with some isoforms containing a novel Pax-2 exon. The spectrum of XPax-2 splice events was investigated in pronephric kidneys, animal cap cultures and in whole embryos. Splicing of XPax-2 transcripts is found to be extensive and temporally regulated during Xenopus embryogenesis. Since all investigated tissues express essentially the full spectrum of XPax-2 splice variants, it is concluded that splicing of XPax-2 transcripts does not occur in a tissue-specific manner (Heller, 1997).

This report describes Pax2, a member of the paired box gene family that is expressed during embryogenesis. Two overlapping cDNA clones were isolated and sequenced. At least two forms of the Pax2 protein can be deduced from the cDNA sequence. In addition to the highly conserved paired domain, an octapeptide sequence is located downstream. Expression of Pax2 is primarily restricted to the developing embryo in the excretory and central nervous systems. The transient nature of Pax2 expression during kidney organogenesis correlates with polarization and induction of epithelial structures and may indicate an important morphogenetic role for this gene (Dressler, 1990).

PAX2 is one of nine PAX genes that have been described in vertebrates. Each PAX gene contains a conserved paired box domain that was first identified in Drosophila. PAX2 encodes a transcription factor that has a critical role in the development of the urogenital tract, the eyes, and the CNS. To facilitate further analysis of PAX2 mutations in human disease, the complete structure of the human PAX2 gene has been determined. Five genomic lambda clones containing human PAX2 gene sequences were isolated. Sequencing and restriction mapping of these clones show that human PAX2 is composed of 12 exons spanning approximately 70 kb. Two alternatively spliced exons and a dinuclotide repeat polymorphism are also found in PAX2 (Sanyanusin, 1996).

Pax6 (Drosophila homolog: Eyeless) and Pax2 are members of the Pax family of transcription factors: both are expressed in the developing visual system of zebrafish embryos. Pax6 protein is present in all cells that form the neural retina and pigment epithelium, whereas Pax2 is located primarily in cells that will give rise to the optic stalk. In this study, the roles of midline signaling in the regulation of Pax2 and Pax6 distributions and in the subsequent morphogenesis of the eye are examined. Midline signaling is severely perturbed in cyclops mutant embryos; this results in an absence of ventral midline CNS tissue and fusion of the eyes. Mutant embryos ectopically express Pax6 in a bridge of tissue around the anterior pole of the neural keel in the position normally occupied by cells that form the optic stalks. In contrast, Pax2 protein is almost completely absent from this region in mutant embryos. Concommitant with the changes in Pax protein distribution, cells in the position of the optic stalks differentiate as retina. These results suggest that a signal emanating from the midline, which is absent in cyclops mutant embryos, may be required to promote Pax2 and inhibit Pax6 expression in cells destined to form the optic stalks. Sonic hedgehog (Shh also known as Vhh-1 and Hhg-1) is a midline signaling molecule that is absent from the neuroepithelium of cyclops mutant embryos at early developmental stages. To test the possibility that Shh might be able to regulate the spatial expression of Pax6 and Pax2 in the optic primordia, Shh was overexpressed in the developing CNS. The number of cells containing Pax2 is increased following shh overexpression; embryos develop hypertrophied optic stalk-like structures. Complimentary to the changes in Pax2 distribution, there are fewer Pax6-containing cells and therefore pigment epithelium and neural retina are reduced in size. These results suggest that Shh or a closely related signalling molecule emanating from midline tissue in the ventral forebrain either directly or indirectly induces the expression of Pax2 and inhibits the expression of Pax6 and thus may regulate the partitioning of the optic primordia into optic stalks and retinal tissue (Macdonald, 1996).

The secreted signaling molecule encoded by the wnt1 (See Drosophila Wingless) gene and the paired box-containing pax2 gene are both thought to play integral roles in patterning the zebrafish rostral nervous system. Using a double-label analysis, the expression patterns of wnt1 RNA and pax2 protein during zebrafish embryogenesis were examined to determine whether they are expressed in identical or overlapping patterns in individual embryos. During gastrulation, wnt1 RNA is detected in a pattern similar but not identical to the pax2 protein. Later, wnt1 and pax2 co-localize to the midbrain-hindbrain boundary. Exogenous retinoic acid, a teratogen that is known to affect the formation of the midbrain-hindbrain boundary, has a profound affect on both wnt1 and pax2 expression at gastrulation. When pax2 is overexpressed in zebrafish embryos, the wnt1 pattern of expression expands ventrally in the prospective rostral neuroepithelium. Despite the widespread and random distribution of exogenous pax2 RNA, it alone is unable to induce wnt1 expression in other ectopic sites. These results are consistent with the coordinate expression of wnt1 and pax2 activity in a pathway responsible for establishing the midbrain-hindbrain boundary and support the earlier interpretation that pax2 may regulate wnt1 expression, although only in a subset of embryonic cells. These data suggest that a predisposition for the regionalization of the central nervous system exists at gastrulation (Kelly, 1995).

Members of the PAX family of transcription factors are candidates for controlling cell identity in the spinal cord. Cells have been morphologically analyzed that express one of these transcription factors, PAX2, demonstrating that multiple interneuron cell types express PAX2. Two ventral populations of PAX2-expressing interneurons in the spinal cord are marked by coexpression of the transcription factors EN1 and EVX1. Interestingly, the expression domains of PAX2, EN1 and EVX1 in postmitotic neurons correlate closely with those of Pax6 and Pax7 in the ventricular zone, implicating these patterning genes in the regulation of PAX2, EN1 and EVX1. PAX2 is first expressed in newly postmitotic cells in the process of migrating laterally from the ventricular zone into the mantle zone. One patterning genes, Pax6, is required for the correct specification of ventral PAX2+ interneurons that coexpress EN1. These results demonstrate that the early activity of patterning genes in the ventricular zone determines interneuron identity in the spinal cord (Burrill, 1997).

Nkx5-1 and Nkx5-2 (two genes without direct known Drosophila homologs) are two highly related homeobox genes that are expressed during development of the mouse inner ear. The detailed expression of both genes within the developing ear is presented and a comparison is made to the expression of other potential control genes in this organ. Both genes are active between E13.5 and birth in non-sensory epithelium of the semicircular canals, utricle and saccule. Nkx5-1 and Nkx5-2 are also expressed in the cochlea, where the expression is restricted to the stria vascularis. The endolymphatic duct is devoid of any Nkx5 transcripts. Pax2 is expressed in epithelial cells of the ventral part of the membranous labyrinth where it overlaps with the Nkx5 expression domain. sek (a receptor tyrosine kinase of the Eph family) shows a complementary pattern to Nkx5 in the vestibular epithelium. In the cochlea sek is expressed throughout the mesenchyme and epithelium but not in the stria vascularis. Pax2 and sek are limited to the ventral part of the vestibulum, whereas Nkx5 genes are active throughout. These data suggest that Nkx5 genes, Pax2 and sek play different roles in the patterning of inner ear structures (Rinkwitz-Brandt, 1996).

The expression of Pax2, a murine gene containing a paired-box, was examined in the developing central nervous system by in situ hybridization. Pax2 expression is detected along the boundaries of primary division in the neural tube. Initially, Pax2 is expressed in the ventricular zone in two compartments of cells on either side of the sulcus limitans and along the entire rhombencephalon and spinal cord. At later times, Pax2 is restricted to progeny cells that have migrated to specific regions of the intermediate zone. In the eye, Pax2 expression is restricted to the ventral half of the optic cup and stalk and later to the optic disc and nerve. In the ear, expression is restricted to regions of the otic vesicle that form neuronal components. The transient and restricted nature of Pax2 expression suggests that this murine segmentation gene homolog may also establish compartmental boundaries and contribute to the specification of neuronal identity, as do certain Drosophila segmentation genes (Nornes, 1990).

The first morphologically discernible structures of the inner ear development in the chick embryo are the otic placodes, which arise as two ectodermal thickenings, lateral to prospective rhombomeres 5 and 6 at the 3- to 5-somite stage. The placodes subsequently invaginate to form the otic pit at about the 12- to 14-somite stage and eventually form the closed otic vesicles at the 24- to 30-somite stage. The early stages of otic placode development depend on signals from neighboring tissues, including the hindbrain. The identity of these signals and of the responding placodal genes, however, is not known. A chick homeobox gene cNkx5-1 has been identifed, which is expressed in the otic placode beginning at stage 10 and which exhibits a dynamic expression pattern during formation and further differentiation of the otic vesicle. In a series of heterotopic transplantation experiments, cNkx5-1 has been shown to be activated in ectopic positions. However, significant differences in otic development and cNkx5-1 gene activity have been observed when placodes are transplanted into the more rostral positions within the head mesenchyme or into the wing buds of older hosts. These results indicate that only the rostral tissues are able to induce and/or maintain ear development. Ectopically induced cNkx5-1 expression always reproduces the endogenous pattern within the lateral wall of the otocyst that is destined to form vestibular structures. In contrast, cPax2, which is expressed in the medial wall of the early otic vesicle, and later in the formation of the cochlea, never resumes its correct expression pattern after transplantation. These experiments illustrate that only some aspects of gene expression and presumably pattern formation during inner ear development can be established and maintained ectopically. In particular, the dorsal vestibular structures seem to be programmed earlier and differently from the ventral cochlear part (Herbrand, 1998).

Although the development of the vertebrate eye is well described, the number of transcription factors, known to be key to this process, is still limited. The localized expression of the orphan nuclear receptor Tlx (a homolog of Drosophila Tailless) in the optic cup and discrete parts of the central nervous system suggests the possible role of Tlx in the formation or function of these structures. Analyses of Tlx targeted mice reveal that, in addition to the central nervous system cortical defects, lack of Tlx function results in progressive retinal and optic nerve degeneration with associated blindness. An extensive screen of Tlx-positive and Tlx-negative P19 neural precursors has identified Pax2 as a candidate target gene. This identification is significant, because Pax2 is known to be involved in retinal development in both the human and the mouse eye. Pax2 is a direct target and the Tlx binding site in its promoter is conserved between mouse and human. These studies show that Tlx is a key component of retinal development and vision and an upstream regulator of the Pax2 signaling cascade (Yu, 2000).

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

Pax2 is the earliest known gene to be expressed throughout the mid-hindbrain region in late gastrula embryos of the mouse and is essential for the formation of an organizing center at the midbrain-hindbrain boundary (MHB), which controls midbrain and cerebellum development. Transgenic analysis has been used to identify three MHB-specific enhancers in the upstream region of the mouse Pax2 gene. A 120 bp enhancer (at -3.7 kb), in cooperation with the endogenous promoter, is sufficient to induce transgene expression in the anterior neural plate of late gastrula embryos. The activity of this early enhancer is severely reduced by mutation of three homeodomain-binding sites, two of which are part of a recognition sequence for POU homeodomain proteins. Oct3/4 (Pou5f1), the mouse ortholog of zebrafish Pou2, efficiently binds to this sequence, suggesting its involvement in the regulation of the early Pax2 enhancer. Starting at the four-somite stage, Pax2 is expressed at the MHB under the control of two enhancers located at -4.1 kb and -2.8 kb. The distal late enhancer contains a 102 bp sequence that is not only highly conserved between the mouse and pufferfish Pax2 genes, but also contributes to the enhancer activity of both genes in transgenic mice. The proximal 410 bp enhancer, which overlaps with a kidney-specific regulatory element, contains a functional Pax2/5/8-binding site and thus maintains Pax2 expression at the MHB under auto- and cross-regulatory control by Pax2/5/8 proteins. Importantly, the early and proximal late enhancers are not only sufficient but also necessary for expression at the MHB in the genomic context of the Pax2 locus, since their specific deletion interfers with correct temporal expression of a large Pax2 BAC transgene. Hence, separate enhancers under the control of distinct transcription factors activate and maintain Pax2 expression at the MHB (Pfeffer, 2002).

The pax2.1 gene encodes a paired-box transcription factor that is one of the earliest genes to be specifically activated in development of the midbrain and midbrain-hindbrain boundary (MHB), and is required for the development and organizer activity of this territory. To understand how this spatially restricted transcriptional activity of pax2.1 is achieved, the pax2.1-promoter has been isolated and characterized using a lacZ and a GFP reporter gene in transient injection assays and transgenic lines. Stable transgenic expression of this reporter gene shows that a 5.3-kb fragment of the 5' region contains most, but not all, elements required for driving pax2.1 expression. The expressing tissues include the MHB, hindbrain, spinal cord, ear and pronephros. Transgene activation in the pronephros and developing ear suggests that these pax2.1-expressing tissues are composed of independently regulated subdomains. In addition, ectopic but spatially restricted activation of the reporter genes in rhombomeres 3 and 5 and in the forebrain, none of which normally express endogenous pax2.1, demonstrates the importance of negative regulation of pax2.1. Comparison of transgene expression in wild-type and homozygous pax2.1 mutant no isthmus (noi) embryos reveals that the transgene contains control element(s) for a novel, positive transcriptional feedback loop in MHB development. Transcription of endogenous pax2.1 at the MHB is known to be initially Pax2.1 independent, during activation in late gastrulation. In contrast, transgene expression requires the endogenous Pax2.1 function. Transplantations, mRNA injections and morpholino knock-down experiments show that this feedback regulation of pax2.1 transcription occurs cell-autonomously, and that it requires the engrailed-type genes eng2 and eng3 as known targets for Pax2.1 regulation. eng2 and eng3 are necessary to maintain, but not initiate, expression of pax2.1 at the MHB. It is suggested that this novel feedback loop may allow continuation of pax2.1 expression, and hence development of the MHB organizer, to become independent of the patterning machinery of the gastrula embryo (Picke, 2002).

A consideration of the transcriptional control elements excluded from the described promoter/enhancer fragment of pax2.1 illustrates the importance of pathways that ultimately lead to tissue-specific transcriptional repression of pax2.1, although the function of pax2.1 repression in these tissues is currently unclear. Examples for this are the ectopic, spatially-restricted expression domains of placZ5.3 in the forebrain and in rhombomeres 3 (r3) and 5 (r5) from stages of late gastrulation onwards (Picke, 2002).

The zinc-finger transcription factor Krox20 (Egr2 -- Zebrafish Information Network) is transcribed from the end of gastrulation in r3 and r5 of zebrafish embryos and could therefore mediate regulation of pax2.1 in the hindbrain. Krox20 directly controls expression of Hox genes, Eph receptors and follistatin in r3 and r5, and indeed the pax2.1 upstream sequence contains six potential Krox20 binding sites. Alternatively, pax2.1 could be activated independently in r3 and r5 by different factors, or regulated via a diffusible factor in a non cell-autonomous manner from adjacent rhombomeres (Picke, 2002).

The ectopic activation of placZ5.3 in the fore- and hind-brain, in addition to the pax2.1-like domain at the MHB, is especially evident at the 20-somite stage where the overall expression pattern has a 'multiple-stripe' appearance, akin to a segmental pattern. The Drosophila Pax-2 ortholog shaven (DPax2), is at embryonic stages expressed in a segmental pattern in the developing external sensory organs of the CNS and PNS. There is as yet no direct evidence for a similar metameric expression pattern of Pax2 orthologs in other organisms, although it has been previously noted that pax2.1-expressing interneurons in the hindbrain and spinal cord form repetitive clusters along the AP axis of the embryo. However, the Drosophila engrailed ortholog AmphiEn of the basal chordate Amphioxus is expressed in 'metameric' stripes along the AP axis of the segmentally organized mesoderm, suggesting a relationship between segmentation in protostomes and deuterostomes. Although it is unclear whether metameric subdivisions exist in the midbrain and isthmic regions, metamerism is a well established concept for the vertebrate hindbrain. Therefore, an interesting possibility is presented: that the partially metameric pattern produced by the pax2.1 promoter/enhancer fragment reflects an ancestral state of pax2 gene regulation in a 'metameric' pattern, which in modern vertebrates, possibly through evolution of additional silencer elements, became restricted to one stripe at the MHB (Picke, 2002).

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein kinases is stimulated in response to a wide array of cellular stresses and proinflammatory cytokines. Mice lacking individual members of the Jnk family (Jnk1, Jnk2, and Jnk3) are viable and survive without overt structural abnormalities. Mice with a compound deficiency in Jnk expression can survive to birth, but fail to close the optic fissure (retinal coloboma). JNK initiates a cytokine cascade of bone morphogenetic protein-4 (BMP4) and sonic hedgehog (Shh) that induces the expression of the paired-like homeobox transcription factor Pax2 and closure of the optic fissure. BMP4 is under the control of JNK. In vitro studies using retinal explant cultures indicate that the function of BMP4, in part, is to induce the expression of Shh in a cytokine cascade that leads to the expression of the paired-like homeobox transcription factor Pax2. Interestingly, the role of JNK to regulate BMP4 expression during optic fissure closure is conserved in Drosophila during dorsal closure, a related morphogenetic process that requires JNK-regulated expression of the BMP4 ortholog Decapentaplegic (Weston, 2003).

BMP4 has been implicated in the regulation of Shh expression in the mouse. To test whether BMP4 regulates the expression of Shh and Pax2 in the eye, retinal explant cultures were examined. Consistent with in vivo results, strong expression of both Shh and Pax2 was detected in control retinas, but not in mutant retinas. When the mutant retinas were cultured in the presence of BMP4 for 48 h, induced expression of both Shh and Pax2 was detected, indicating that BMP4 is sufficient to cause expression of Shh and Pax2, and that the BMP4-Shh-Pax2 pathway is intact in the JNK-deficient mutant embryonic eyes. In contrast, no BMP4-stimulated expression of Shh or Pax2 in the mutant retinas was detected in the absence or presence of an antagonistic antibody to Shh. Similarly, when control retinas were cultured in the presence of BMP4 plus the antagonistic antibody to Shh, there was a dramatic decrease in both Shh and Pax2 expression. These data imply that BMP4 induces the expression of Shh and Pax2 in mutant retinas, and that Shh is upstream of Pax2 expression. This signaling cascade is initiated by JNK and is absent in JNK-deficient retinas (Weston, 2003).

It is striking that the effects of Pax2 deficiency are similar to those caused by JNK deficiency. For example, both of these mutations cause failure of optic fissure closure (coloboma) and renal epithelial cell necrosis. Furthermore, both mutations alter the expression of Shh at the basis of the diencephalon in E9.5 embryos. These similar phenotypes are most likely accounted for by the observation that Pax2 expression is markedly reduced in the eyes and kidney epithelium of JNK-deficient mice. A further contributing factor may be that JNK can phosphorylate Pax2. However, because the level of Pax2 mRNA and protein in JNK-deficient eyes is extremely low, the role of altered Pax2 phosphorylation is unclear (Weston, 2003).

The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4 methyltransferase complex

The MLL family of histone methyltransferases maintains active chromatin domains by methylating histone H3 on lysine 4 (H3K4). How MLL complexes recognize specific chromatin domains in a temporal and tissue-specific manner remains unclear. This study shows that the DNA-binding protein PAX2 promotes assembly of an H3K4 methyltransferase complex through the ubiquitously expressed nuclear factor PTIP (pax transcription activation domain interacting protein). PTIP copurifies with ALR, MLL3, and other components of a histone methyltransferase complex. PTIP promotes assembly of the ALR complex and H3K4 methylation at a PAX2-binding DNA element. Without PTIP, Pax2 binds to this element but does not assemble the ALR complex. Embryonic lethal ptip-null mutants and conditional mutants both show reduced levels of methylated H3K4. Thus, PTIP bridges DNA-binding developmental regulators to histone methyltransferase-dependent epigenetic regulation (Patel, 2007).

Pax2 mutation

Evolutionary homologs continued: part 3/5 | part 4/5 | part 5/5 | back to part 1/5 |

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

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