Molecular analysis of the development of the vertebrate visual system.
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Animals↗
Biomedical subjects
P Gruss
Publications and source records attributed to P Gruss.
[The expression of Pax7 in C2C12 cell line and its inducible inactivation].
Pax7 is a member of mouse Pax gene family. It was expressed from day 8 to 17 p. c. during murine embryogenesis, mainly restricted to the central nerve systems. Additional Pax7 expression could be followed during myogenesis from the dermamyotome of the somites to the skeletal muscle tissues. For the first time, we demonstrated that Pax7 is expressed in mouse myoblast cell line, C2 C12. The expression of Pax7 was blocked when C2 C12 cells were induced to differentiate into myotube. Moreover, cells of mesodermal origin, in particular of the mouse embryo fibroblast cell line C3 H10 T1/2 which were especially permissive to the action of myogenic HLH proteins, did not express Pax7. These results suggest that Pax7 may be a suppressive factor for the myoblast differentiation of C2 C12 cells in vitro.
Animals↗
Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of Hox genes.
Cdx1 is expressed along the embryonic axis from day 7.5 postcoitum until day 12, by which time the anterior limit of expression has regressed from the hindbrain level to the forelimb bud region. To assign a functional role for Cdx1 in murine embryonic development, we have inactivated the gene via homologous recombination. Viable fertile homozygous mutant mice were obtained that show anterior homeotic transformations of vertebrae. These abnormalities were concomitant with posterior shifts of Hox gene expression domains in the somitic mesoderm. The presence of putative Cdx1-binding sites in Hox gene control regions as well as in vitro transactivation of Hoxa-7 indicates a direct regulation.
Animals↗
Loss of p53 function through PAX-mediated transcriptional repression.
Direct interactions between the genes that regulate development and those which regulate the cell cycle would provide a mechanism by which numerous biological events could be better understood. We have identified a direct role for PAX5 in the control of p53 transcription. In primary human diffuse astrocytomas, PAX5 expression inversely correlated with p53 expression. The human p53 gene harbours a PAX binding site within its untranslated first exon that is conserved throughout evolution. PAX5 and its paralogues PAX2 and PAX8 are capable of inhibiting both the p53 promoter and transactivation of a p53-responsive reporter in cell culture. Mutation of the identified binding site eliminates PAX protein binding in vitro and renders the promoter inactive in cells. These data suggest that PAX proteins might regulate p53 expression during development and propose a novel alternative mechanism for tumour initiation or progression, by which loss of p53 function occurs at the transcriptional level.
3T3 Cells↗
Identification of DNA recognition sequences for the Pax3 paired domain.
The Pax gene family, encoding transcription factors, has been classified into four subfamilies according to their genomic organization, the sequences of the paired domains (PD) and the expression pattern. Pax1 and Pax9 constitute one subfamily, Pax2, Pax5 and Pax8 another, Pax3 and Pax7 another one and Pax4 and Pax6 the fourth subfamily. The PD exhibits DNA-binding activity, and is the most conserved functional motif in all Pax proteins. A high-resolution analysis of a PD structure has been performed [Xu et al., Cell 80 (1995) 639-650] and the DNA-binding characteristics of members of two Pax subfamilies (Pax2, Pax5 and Pax6) have been determined. Here, we have utilized a PCR-based selection approach to identify the DNA-binding sequences of the Pax3/PD, a member of a subfamily which has not yet been characterized. Comparison of the Pax3/PD-binding sequences with those of other PD proteins revealed both similarities and differences in the DNA-recognition sequence. This suggests that different Pax proteins can regulate the expression of the same target gene, but they can also regulate the expression of completely unrelated genes by binding to their DNA regulatory regions.
Animals↗
Cryptorchidism and homeotic transformations of spinal nerves and vertebrae in Hoxa-10 mutant mice.
Homozygous mice mutated by homologous recombination for the AbdB-related Hoxa-10 gene are viable but display homeotic transformations of vertebrae and lumbar spinal nerves. Mutant males exhibit unilateral or bilateral criptorchidism due to developmental abnormalities of the gubernaculum, resulting in abnormal spermatogenesis and sterility. These results reveal an important role of Hoxa-10 in patterning posterior body regions and suggest that Hox genes are involved in specifying regional identity of both segmented and nonovertly segmented structures of the developing body.
Animals↗
Activin A inhibits Pax-6 expression and perturbs cell differentiation in the developing spinal cord in vitro.
We have developed an in vitro model of the isolated chicken neural plate. Here we demonstrate that even in the absence of notochord, the neural plate rapidly develops a typical dorsoventral patterning. This observation suggests that the ventral cell types are specified or at least predetermined prior to notochord formation and that permissive conditions are sufficient for differentiation of ventral structures. Treatment of the neural plate with activin A extinguishes Pax-6 gene expression, whereas the dorsal markers Pax-3 and Pax-7 are still expressed. The absence of Pax-6 transcripts can be correlated with an impeded differentiation of the motor neurons, whereas the floor plate seems to be enlarged. We propose that the region-specific expression of Pax-6 in the spinal cord is under the control of activin-like molecules.
Activins↗
Selection of ten base-pair sequences which enhance the response of the Gal1 minimal promoter to murine Hoxa-7 in yeast.
The mouse homeodomain protein Hoxa-7, expressed under the control of an inducible promoter, was able to inducibly activate reporter genes containing multimerized Hoxa-7 binding sites in Saccharomyces cerevisiae. This tight regulation was exploited in an attempt to screen for Hoxa-7 responsive elements. A reporter library consisting of a randomised 10 bp element inserted into the minimal gal1 promoter was constructed. In a surprisingly small screen, 24 reporters were isolated which had all of the transactivation characteristics expected for a Hoxa-7 binding site insertion. However, further characterisation revealed that the selected elements lacked homeodomain (HD) binding core motifs and were not bound by a purified Hoxa-7/beta-galactosidase fusion protein capable of binding known sites. The minimal promoter context contains 16 HD core motifs in 410 bp. Careful re-examination of basal levels revealed a low residual response of the gal1 minimal promoter to Hoxa-7. The 11 characterised 10 bp inserts amplified Hoxa-7 responsiveness in a manner correlated to increases in basal reporter activity. Thus, a quantitative range of Hox-responsiveness was produced by slight sequence alterations that did not change HD binding sites of their relative spacing in the promoter. These data suggest how, without altering resident HD base contact zones, mouse promoters could be optimised by natural selection to give appropriate quantitative outputs in each anatomical region defined by an assortment of Hox proteins. The selected elements were pyrimidine rich on the sense strand, containing (T)nC motifs, strikingly similar to sequences which enhance Hoxa-7 binding and activation from outside the HD contact zone. A search of defined sequence databases demonstrated that these elements were over-represented in promoters. Two elements altered the mobility shift patterns produced by cell extracts on minimal promoter fragments.
Animals↗
undulated phenotypes suggest a role of Pax-1 for the development of vertebral and extravertebral structures.
undulated extensive mice carry a deletion of at least 28.2 kb, removing the terminal Pax-1 exon including the poly(A) signal. This mutation leads to a drastically reduced amount of Pax-1 transcript. The similarity in phenotype between unex and the point mutation un suggests both to be functionally equivalent. A third mutant, Undulated short tail, harbors a deletion of at least 48.3 kb, eliminating the Pax-1 locus as well as 38 kb of flanking sequences. Phenotypically, this mutant differs from un and unex. The comparative phenotype analysis of all undulated alleles revealed morphological alterations in the vertebral column, the sternum, the scapula, the facial skeleton, and the thymus that correspond to Pax-1 expression domains. The vertebral column phenotype is characterized by the reduction of vertebral components. These reductions can be traced back to the delayed cell accumulation in the embryonic sclerotome while the principal organization of this tissue is maintained. The morphological alterations outside the vertebral column are likewise confined to size reductions. Therefore, Pax-1 may exert similar functions in all of its expression domains.
Amino Acid Sequence↗
Segment-specific expression of the neuronatin gene during early hindbrain development.
The developing hindbrain is segmented in a series of repetitive bulges called neuromeres or rhombomeres. In the mouse, first molecular evidence for segmentation of the hindbrain came from rhombomeres 3- and 5-specific expression of the Krox-20 gene. The hindbrain segments are linked with the expression of different Hox genes which have a role in patterning the hindbrain and branchial region of the vertebrate head. Here we identified by subtractive hybridization a gene, mouse neuronatin, that is downregulated in P19 embryo carcinoma cells that have undergone a partial differentiation process. Neuronatin encodes putative transmembrane proteins of 54, 55, and 81 amino acids that might serve as protein ligands, cofactors, or small cell adhesion molecules. The neuronatin gene is transiently expressed in rhombomeres 3 and 5 during early hindbrain development and in the floor of the foregut pocket. In addition, expression is observed in the early Rathke's pouch, in the derived adenohypophysis, and in the developing inner ear. During later embryogenesis the neuronatin gene is strongly expressed in the major part of the central and peripheral nervous system. These results suggest that neuronatin participates in the maintenance of segment identity in the hindbrain and pituitary development and maturation or maintenance of the overall structure of the nervous system.
Alternative Splicing↗
A transgenic neuroanatomical marker identifies cranial neural crest deficiencies associated with the Pax3 mutant Splotch.
The murine Pax3 gene encodes a transcription factor containing a paired domain as well as a paired-type homeodomain. Its expression during embryonic development is temporally and spatially restricted, including mainly the dorsal part of the neural tube, the mesencephalon, the neural crest derivatives, and the dermomyotome. Development in the absence of Pax3 can be studied in Splotch mutant mice, which bear mutations within the Pax3 gene. Various alleles have been phenotypically and molecularly characterized. Abnormalities have been observed in the brain, the neural tube, the trunk neural crest derivatives and in muscles of these mutants. The importance of PAX3 during human embryonal development is readily seen in Waardenburg patients, who present a dominant inherited syndrome consisting mainly of craniofacial abnormalities, pigmentation deficiencies, and deafness, consecutive to PAX3 mutations. In order to analyze the nervous system of Splotch embryos in more detail, we employed the transgenic mouse line L17. These transgenic mice harbor a beta-galactosidase marker gene under the control of Hoxa-7 promoter elements. Probably in combination with cis-elements adjacent to the integration site of the L17 transgene, the Hoxa-7 elements drive the expression of the marker gene in major parts of the peripheral nervous system, as well as in more restricted parts of the central nervous system. These structures can be visualized during embryonic development, allowing detailed neuroanatomical studies in midgestation embryos. We describe the beta-galactosidase expression in wild-type L17 mice and demonstrate the applicability of L17 mice to the study of the nervous system. We then apply this experimental system to the analysis of Splotch embryos. Our findings underline the importance of Pax3 in the development of neural crest-derived structures, especially of cranial ganglia and nerves. We suggest the use of L17 mice as a valuable tool to perform similar analysis for other embryonal mutant phenotypes.
Animals↗
Pax3: a paired domain gene as a regulator in PNS myelination.
Pax3 RNA is expressed in neural crest when Schwann cell (SC) precursors migrate to the PNS. Pax3 RNA and SC markers were monitored in sciatic nerves of mice during development and nerve repair. An inverse correlation was observed between expression of Pax3 RNA and myelin basic protein (MBP). Inverse correlation was also observed in SC primary cultures. Treating cultures with forskolin, an adenylate cyclase agonist, repressed Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and elevated MBP. Subsequent microinjection with Pax3 expression vector elevated Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and repressed MBP. Thus, Pax3 is likely involved in the differentiation pathway to myelinating SCs. Pax3 repressed a 1.3 kb MBP promoter fragment in cotransfection assays, suggesting that it represses MBP transcription.
Animals↗
Expression of the novel murine homeobox gene Sax-1 in the developing nervous system.
We have isolated the novel murine Sax-1 gene, a member of the NK-1 class of homeobox genes, and report its expression pattern in the developing central nervous system (CNS) in comparison to two other homeobox genes, Evx-1 and Pax-6. Sax-1 was found to be transiently expressed in the developing posterior CNS. First seen in the ectoderm lateral to the primitive streak, the signal later encompassed the neural plate. Posteriorly, the expression domain overlapped with the Evx-1 expression in the streak, while anteriorly it was delimited by the Pax-6 signal in the neural tube. This early phase starting at day 9.5 pc, Sax-1 was expressed in distinct areas of spinal cord, hindbrain and forebrain. Particularly strong signals were detected in rhombomere 1 and in the pretectum. In these areas, subsets of neurons may be marked and specified. In addition to the normal pattern of Sax-1 during development, the expression in different mouse mutants was analysed. In Brachyury curtailed homozygotes, the expression of Sax-1 was found to be reduced during neurulation and even lost at day 9.0 pc. Ventral shift and finally loss of the signal in the ventral spinal cord was observed in Danforth's short tail homozygotes.
Amino Acid Sequence↗
PAX genes: what's new in developmental biology and cancer?
PAX genes encode nuclear transcription factors which are rapidly becoming regarded as major controllers of developmental processes in both vertebrates and invertebrates. Mutations in murine Pax genes underlie three natural mouse alleles and two corresponding human syndromes. Murine Pax genes have been shown to be proto-oncogenes. Furthermore, human PAX genes have recently been demonstrated to play an influential part in some common human cancers. The diversity in effects of PAX genes reflects that of their structure. All encode a DNA-binding domain termed the paired domain and in addition some also encode a second binding domain--the paired type homeobox.
Animals↗
Six3, a murine homologue of the sine oculis gene, demarcates the most anterior border of the developing neural plate and is expressed during eye development.
The Drosophila sine oculis homeobox-containing gene is known to play an essential role in controlling the initial events of pattern formation in the eye disc and is also required for the development of other parts of the fly visual system including the optic lobes. In this paper, we report the isolation of a sequence-related gene referred to as Six3. Based on its amino acid sequence, this gene can be included in the new Six/sine oculis subclass of homeobox genes. Early on, Six3 expression is restricted to the anterior neural plate including areas that later will give rise to ectodermal and neural derivatives. Later, once the longitudinal axis of the brain bends, Six3 mRNA is also found in structures derived from the anterior neural plate: ectoderm of nasal cavity, olfactory placode and Rathke's pouch, and also the ventral forebrain including the region of the optic recess, hypothalamus and optic vesicles. Based on this expression pattern, we conclude that Six3 is one of the most anterior homeobox gene reported to date. The high sequence similarity of Six3 with the Drosophila sine oculis, and its expression during eye development, suggests that this gene is the likely murine homologue. This finding supports the idea that mammals and insects share control genes such as eyeless/Pax6 (Halder, G., Callaerts, P. and Gehring, W. J. (1995) Science 267, 1788-1792), and also possibly other members of the regulatory cascade required for eye morphogenesis. In Small eye (Pax6) mouse mutants Six3 expression is not affected. Finally, based on the chromosomal localization and the expression pattern of the mouse Six3 gene, the human Six3 cognate could be a good candidate to be at least one of the genes affected in patients with holoprosencephaly type 2 due to an interstitial deletion of 2p21-p22. This region shares a homology with the distal region of mouse chromosome 17 where Six3 has been mapped.
Amino Acid Sequence↗
Pax-2 controls multiple steps of urogenital development.
Urogenital system development in mammals requires the coordinated differentiation of two distinct tissues, the ductal epithelium and the nephrogenic mesenchyme, both derived from the intermediate mesoderm of the early embryo. The former give rise to the genital tracts, ureters and kidney collecting duct system, whereas mesenchymal components undergo epithelial transformation to form nephrons in both the mesonephric (embryonic) and metanephric (definitive) kidney. Pax-2 is a transcriptional regulator of the paired-box family and is widely expressed during the development of both ductal and mesenchymal components of the urogenital system. We report here that Pax-2 homozygous mutant newborn mice lack kidneys, ureters and genital tracts. We attribute these defects to dysgenesis of both ductal and mesenchymal components of the developing urogenital system. The Wolffian and Müllerian ducts, precursors of male and female genital tracts, respectively, develop only partially and degenerate during embryogenesis. The ureters, inducers of the metanephros are absent and therefore kidney development does not take place. Mesenchyme of the nephrogenic cord fails to undergo epithelial transformation and is not able to form tubules in the mesonephros. In addition, we show that the expression of specific markers for each of these components is de-regulated in Pax-2 mutants. These data show that Pax-2 is required for multiple steps during the differentiation of intermediate mesoderm. In addition, Pax-2 mouse mutants provide an animal model for human hereditary kidney diseases.
Animals↗
Homeobox genes and connective tissue patterning.
In vertebrates, limb tendons are derived from cells that migrate from the lateral plate mesoderm during early development. While some of the developmental steps leading to the formation of these tissues are known, little is known about the molecular mechanisms controlling them. We have identified two murine homeobox-containing genes, Six 1 and Six 2, which are expressed in a complementary fashion during the development of limb tendons. Transcripts for both genes are found in different sets of phalangeal tendons. Six 1 and Six 2 also are expressed in skeletal and smooth muscle, respectively. These genes may participate in the patterning of the distal tendons of the limb phalanges by setting positional values along the limb axes.
Amino Acid Sequence↗