Polydactyly Nagoya Research Articles

Gli3 Controls Corpus Callosum Formation by Positioning Midline Guideposts During Telencephalic Patterning

The corpus callosum (CC) represents the major forebrain commissure connecting the 2 cerebral hemispheres. Midline crossing of callosal axons is controlled by several glial and neuronal guideposts specifically located along the callosal path, but it remains unknown how these cells acquire their position. Here, we show that the Gli3 hypomorphic mouse mutant Polydactyly Nagoya (Pdn) displays agenesis of the CC and mislocation of the glial and neuronal guidepost cells. Using transplantation experiments, we demonstrate that agenesis of the CC is primarily caused by midline defects. These defects originate during telencephalic patterning and involve an up-regulation of Slit2 expression and altered Fgf and Wnt/β-catenin signaling. Mutations in sprouty1/2 which mimic the changes in these signaling pathways cause a disorganization of midline guideposts and CC agenesis. Moreover, a partial recovery of midline abnormalities in Pdn/Pdn;Slit2(-/-) embryos mutants confirms the functional importance of correct Slit2 expression levels for callosal development. Hence, Gli3 controlled restriction of Fgf and Wnt/β-catenin signaling and of Slit2 expression is crucial for positioning midline guideposts and callosal development.

Normal Location Of Thumb/Big Toe may be Related to Programmed Cell Death in the Preaxial Area of Embryonic Limb

The forelimbs and hindlimbs of newborn Polydactyly Nagoya (Pdn) mice were examined to analyze the roles of programmed cell death (PCD) in the preaxial region of the limb. Special attention was paid to the relationship between the PCD in the preaxial area and the location and shape of the first digit (thumb/big toe). Although a large, bifurcated or duplicated thumb/big toe appeared in Pdn/+ mice, digit I (thumb/big toe) in Pdn/+ mice, as in +/+ ones, was located more ventro-proximally than the other four digits. On the other hand, abnormal preaxial digits of the fore/hindlimb in Pdn/Pdn mice lay distally and were aligned at the radial/tibial end of a serial curved plane formed by digits II-V; that is, a thumb and big toe of normal shape and location were not detectable in any preaxial digits of Pdn/Pdn mice. In the limb development of Pdn mouse embryos on Day 11-12, PCD did not occur in the preaxial mesoderm of fore/hindlimb only in one-fourths of all embryos obtained by Pdn/+ x Pdn/+ mating. In addition to digital rays II-V, extra preaxial digital rays appeared in the prominent preaxial expansion of fore/hindlimbs in these embryos on early Day 12. These abnormal limb configurations in embryos were closely similar to those in Pdn/Pdn newborn mice. The present findings suggest that PCD in the preaxial region not only prevents the formation of extra digits but also determines the location of the thumb/big toe for the normal limb morphogenesis.

Birth defects caused by mutations in human GLI3 and mouse Gli3 genes

ABSTRACT GLI3 is the gene responsible for Greig cephalopolysyndactyly syndrome (GCPS), Pallister-Hall syndrome (PHS) and Postaxial polydactyly type-A (PAP-A). Genetic polydactyly mice such as Pdn/Pdn (Polydactyly Nagoya), Xt(H)/Xt(H) (Extra toes) and Xt(J)/Xt(J) (Extra toes Jackson) are the mouse homolog of GCPS, and Gli3(tmlUrtt)/Gli3(tmlUrt) is produced as the mouse homolog of PHS. In the present review, relationships between mutation points of GLI3 and Gli3, and resulting phenotypes in humans and mice are described. It has been confirmed that mutation in the upstream or within the zinc finger domain of the GLI3 gene induces GCPS; that in the post-zinc finger region including the protease cleavage site induces PHS; and that in the downstream of the GLI3 gene induces PAP-A. A mimicking phenomenon was observed in the mouse homolog. Therefore, human GLI3 and mouse Gli3 genes have a common structure, and it is suggested here that mutations in the same functional regions produce similar phenotypes in human and mice. The most important issue might be that GCPS and PHS exhibit an autosomal dominant trait, but mouse homologs, such as Pdn/Pdn, Xt(H)/Xt(H), Xt(J)/Xt(J) and Gli3(tmlUrt)/Gli3(tmlUrt), are autosomal recessive traits in the manifestation of similar phenotypes to human diseases. It is discussed here how the reduced amounts of the GLI3 protein, or truncated mutant GLI3 protein, disrupt development of the limbs, head and face.

The mouse mutation Pdn (Polydactyly Nagoya) is caused by the integration of a retrotransposon into the Gli3 gene.

Mutations in the Gli3 gene are associated with a preaxial polydactyly in several mouse mutants such as extra-toes (Xt). The semidominant mouse mutant Pdn (Polydactyly Nagoya) is characterized by a mild polydactyly on the anterior side of the hind limbs. Homozygous Pdn mice show a more severe polydactyly, additional skeletal malformations, and abnormal brain development. Herein, we report the molecular basis of Pdn, being the integration of an Early Transposon (ETn) into the Gli3 gene. As a consequence, several novel Gli3 mRNAs are generated by alternatively spliced transcripts.

Neuro-glial neurotrophic interaction in the S-100β retarded mutant mouse (Polydactyly Nagoya). III. Transplantation study

The hippocampus and caudo-dorsal cortex of the homozygote of polydactyly mutant mouse (Polydactyly Nagoya, Pdn/Pdn) were markedly reduced in S-100β positive astrocytes and serotonergic fibers as compared to the heterozygote (Pdn/+) and wild type (+/+) [39]. The Pdn/Pdn mice die within 2 days after birth, so it is impossible to examine postnatal changes. To demonstrate the developmental change of Pdn/Pdn hippocampal tissue, we transplanted hippocampal pieces of neonatal Pdn/Pdn and +/+ mice into the right and left hippocampus of the same adult +/+ mice, respectively, and immunocytochemically examined them. Two weeks after transplantation, +/+ hippocampal tissue contained a large number of glial fibrillary acidic protein (GFAP) and S-100β positive astrocytes and a number of serotonergic fibers. While Pdn/Pdn hippocampal tissue contained numerous GFAP positive astrocytes, S-100β positive astrocytes and serotonergic fibers were not observed. Two months after transplantation, GFAP and S-100β were expressed in the Pdn/Pdn hippocampal tissue similar to the +/+ tissue. Serotonergic fibers were distributed in the +/+ tissue, while no serotonergic fibers were observed in the Pdn/Pdn transplant tissue. In contrast, no difference was observed in the tyrosine hydroxylase positive fibers between Pdn/Pdn and +/+ grafts. The expression of 5-HT1A receptor-like immunoreactivity was higher in the +/+ tissue than that of Pdn/Pdn tissue. The present results suggest that the expression of S-100β in the astrocytes of early stage of transplantation is a critical event for fiber ingrowth of serotonergic neurons and expression of 5-HT1A receptor.

The mouse mutant Polydactyly Nagoya (Pdn) defines a novel allele of the zinc finger gene Gli3.

The mouse mutant Polydactyly Nagoya (Pdn) defines a novel allele of the zinc finger gene Gli3.

Apoptotic degeneration in the arhinencephalic brain of the mouse mutant Pdn / Pdn

The homozygotes of a mouse strain with genetic polydactyly (Polydactyly Nagoya, Pdn) exhibit arhinencephaly and various brain malformations. In the present experiment, abnormal apoptotic degeneration in the arhinencephalic brain of Pdn / Pdn embryos and newborns was investigated immunohistochemically and by molecular genetic techniques. Polyclonal antibody against single-stranded DNA detected the nuclei of programmed dying cells (apoptotic cells) specifically in the interdigital necrotic zone of the normal mouse limb plate on day 14 of gestation. We used this antibody against single-stranded DNA to investigate the apoptotic degeneration in Pdn / Pdn brain. Abnormal apoptosis was observed in the infralimbic cortical plate, hypothalamus and periventricular thalamus on day 0 after birth in Pdn / Pdn brains. The TRPM-2 gene, which has been considered to mediate apoptosis, was expressed in the developing normal and Pdn / Pdn brains. TRPM-2 gene expressions in the brain stem and cerebellum of arhinencephalic Pdn / Pdn fetuses and newborns were higher than those of +/+ littermates. From these facts, it was suggested that the abnormal apoptosis caused a large amount of cell loss in the arhinencephalic mouse brain, and this cell loss induced the expansion of the ventricle, followed by the hydrocephaly.

Neuro-glial neurotrophic interaction in the S-100β retarded mutant mouse (Polydactyly Nagoya). I. Immunocytochemical and neurochemical studies

The homozygote of a mouse strain with genetic polydactyly (Polydactyly Nagoya; Pdn) shows several brain abnormalities, and significant decrease of S-100β in the brain 43. In order to clarify the effects of the retarded production of S-100β on the development of monoaminergic neuronal systems and supporting glial cells, immunocytochemical studies of tyrosine hydroxylase (TH), serotonin (5-HT), S-100β and glial fibrillary acidic protein (GFAP). In addition, high-performance liquid-chromatography (HPLC) measurements of serotonin and 5-hydroxyindoleacetic acid (5-HIAA) of homozygote ( Pdn/Pdn) mouse were examined, and the results were compared with those of other genotypes; heterozygote ( Pdn/+) and wild type (+/+) mice. In all types of mice, S-100β positive cells and serotonergic fibers were widely distributed throughout the brains and serotonergic cell bodies were located in the brainstem. However, the hippocampus and caudo-dorsal cortex of Pdn/Pdn mouse were markedly reduced in S-100β positive cells and in serotonergic fibers. Furthermore, abnormal distribution of GFAP positive cells and fibers were observed in the neocortex and hippocampus of Pdn/Pdn brain. No differences were seen in the distribution of TH neurons or fibers distribution. In the HPLC study, the content of 5-HT and 5-HIAA of the hippocampus and cortex of Pdn/Pdn mouse was lower than those of Pdn/+ and +/+ mice. The present results suggest that the developmental defect of serotonergic fibers in the Pdn mutant mouse is correlate to the deficiency of S-100β in the astrocyte of this mutant.

Neuro-glial neurotrophic interaction in the S-100β retarded mutant mouse (Polydactyly Nagoya). II. Co-cultures study

The homozygote of a mouse strain with genetic polydactyly (Polydactyly Nagoya, Pdn) shows several brain abnormalities, and significant decrease of S-100β in the brain [17]. An accompanying paper [18] demonstrates that the hippocampus and caudo-dorsal cortex of homozygote ( Pdn/Pdn) mouse were markedly reduced in S-100β positive astrocytes and serotonergic fibers, and the content of 5-HT and 5-HIAA of hippocampus and cortex of Pdn/Pdn mouse was lower than those of heterozygote ( Pdn/+) or wild type (+/+) mice. To further clarify the effects of target tissues from different type brains on the development of serotonergic neurons, raphe neurons from Pdn/Pdn or +/+ newborn mice were co-cultured with hippocampus or cortex of +/+ or Pdn/Pdn newborn mice. The growth of the serotonergic neurons in the mesencephalic raphe tissue dissociated cultures was estimated by measuring the specific uptake of [ 3H]5-HT. The development of both genotypes ( Pdn/Pdn and +/+) of serotonergic neurons was enhanced by co-cultures with target tissues (hippocampus and cortex) of +/+ brain. This effect was not observed in the co-cultures with Pdn/Pdn brain as a source of target tissue. The present results support the idea that the developmental defect of serotonergic fibers in the Pdn mutant mouse is caused by the deficiency of S-100β in the astrocyte of this mutant, and suggest that S-100β is a serotonergic growth factor. This mutant mouse is a useful in vivo model to study neural-glial neurotrophic interactions.

Fetal laser surgery in genetic Polydactyly mice

The first digital ray of the hindlimb plate in Slc:ICR mouse fetus was irradiated with 2 watts argon laser beam for 0.3 sec after releasing from the abdominal cavity and envelop of uterine myometrium on day 13 of gestation, and then the fetuses were allowed to develop in the abdominal cavity contacting with the uterus via the placenta exo utero until term. ICR mouse fetuses which received fetal laser surgery lost their first digits completely, resulting in 4-digit hindfoot on day 18 of gestation. The homozygous Polydactyly Nagoya (Pdn/Pdn) mice exhibit 1-3 extra digits both in the fore- and hindlimbs preaxially. The extra digital rays in the left hindlimbs of Pdn/Pdn fetuses were irradiated with 2 watts argon laser beam for 0.3 sec on day 13 of gestation exo utero. The left hindlimbs of the Pdn/Pdn fetuses which received fetal laser surgery lost their preaxial extra digits on day 18 of gestation, resulting in 5 digits, though their 1st digit was triphalangia. The combination of a laser instrument with the fetoscope and/or ultrasound scanner may promote the fetal surgery of congenital anomalies in humans.

Developmental brain abnormalities accompanied with the retarded production of S-100β protein in genetic polydactyly mice

The homozygotes of a mouse strain with genetic polydactyly (Polydactyly Nagoya. Pdn) exhibit various brain malformations including exencephaly in about 20%. In the present report, the brains of homozygotes Pdn/Pdn which were not exencephalic were examined morphologically and biochemically. Homozygous newborn brains showed hydrocephaly, some gyri on the cerebral hemisphere, absence of the corpus callosum, absence of the commissura anterior, absence of the fornix and commissura fornicis, protuberance of the cortical tissue from the brain surface, and abnormal architecture of the hippocampus. An irregular mass of olfactory nerve was observed on the cribriform plate, and the olfactory bulb was deficient. From these findings, we considered Pdn/Pdn as a kind of arhinencephalic mouse. Nervous tissue-related proteins, S-100α, S-100β, creatine kinase B (CK-B), neuron-specific γ-enolase, guanosine triphosphate binding proteins (G o, G i2 and Gβ) were immunoassayed in the cerebrum of Pdn/Pdn embryos and newborns. Among the protein analysed, only S-100β of Pdn/Pdn showed a significantly lower level than that of +/+ cerebrum during the observation period. The newborn brains were examined immunohistochemically using S-100α, S-100β, CK-B, G oα and NSE antibodies. We could find no differences in the staining patterns among the Pdn/ Pdn, Pdn/+ and +/+ brains.

Prevention of polydactyly manifestation in Polydactyly Nagoya (Pdn) mice by administration of cytosine arabinoside during pregnancy.

Male mice heterozygous for the dominant polydactyly gene Pdn (Polydactyly Nagoya) were crossed with normal or heterozygous females of the same strain. Pregnant females were treated with 5 mg/kg of cytosine arabinoside (Ara-C) on day 12 of gestation. The offspring were removed on day 18 of gestation and examined for external malformations, and the fore- and hindlimbs were examined by means of bone- and cartilage-stained cleared specimens. In +/+ x Pdn/+ matings, Pdn/+ fetuses, bearing preaxial polydactyly of the distal phalangeal type in the hindlimb and deformity of the 1st digit in the forelimb, were obtained in about 50% of the nontreated group. In treated fetuses, however, the incidence of polydactyly and deformity of the 1st digit decreased to 1.4 and 10.1%, respectively. Nontreated Pdn/Pdn fetuses exhibited preaxial polydactyly of the duplicated or triplicated metacarpal/metatarsal type both in the fore- and hindlimbs. In the treated Pdn/Pdn fetuses, the number of preaxial extra digits decreased in both limbs. Some hindlimbs of the treated Pdn/Pdn fetuses exhibited five metatarsals, normally. In the vitally stained specimens at 6 and 24 hours after injection of Ara-C, preaxial marginal necrotic zones (fMI) were observed in almost all of the treated embryos from +/+ x Pdn/+ matings. However, approximately half of the embryos did not exhibit fMI in the nontreated control group at the same stage. Those embryos deficient in fMI were regarded as Pdn/+. These findings indicated that a subteratogenic dose of Ara-C prevented the genetic expression of polydactyly in almost all Pdn/+ and some cases of Pdn/Pdn mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of Genetic Polydactyly in Polydactyly Nagoya (Pdn) Mice in Vitro by Surgical Treatment of Foot Plate during Embryogenesis

Abstract Pdn/Pdn fetuses show preaxial Polydactyly of duplicated or triplicated metacarpal/metatarsal type in the fore‐ and hindlimbs. Pdn/+ fetuses show one extra digit of distal phalangeal type preaxially in the hindlimb and deformity of distal phalanx of the 1st digit in the forelimb. Normal patterns of physiological cell death in the preaxial apical ectodermal ridge (AER) and the deep preaxial mesoderm (fpp) were disrupted in Pdn/Pdn embryos. It was supposed that delayed involution of preaxial AER might have caused the abolishment of fpp, which in turn could have induced polydactyly in Pdn/Pdn.In order to induce cell death in the fpp region artificially, tissue destruction of the fpp region of right fore‐ and hindfoot plates of Pdn/Pdn, Pdn/+ and +/+ embryos was performed by an electric knife on day 11.5 of gestation, and the embryos were cultured in the rotator culture system for 20 hours. The non‐treated left foot plates served as the controls.In Pdn/Pdn embryos, the non‐treated left foot plates showed abnormal protuberance and/or the extra digital rays preaxially. But the treated right foot plates did not exhibit these abnormal characteristics. Instead they revealed 5 digital rays of mesodermal condensation in the histological sections.These results indicated that the restriction of the artificial tissue destruction in the fpp region could prevent the manifestation of preaxial extra digits in Pdn mice.

Digital malformations caused by 5-fluorouracil and cytosine arabinoside in genetic polydactyly mouse (Polydactyly Nagoya, Pdn)

Digital malformations caused by 5-fluorouracil and cytosine arabinoside in genetic polydactyly mouse (Polydactyly Nagoya, Pdn)

Morphogenesis of Genetic Preaxial Polydactyly, Polydactyly Nagoya, Pdn, in Mice

Morphogenesis of Genetic Preaxial Polydactyly, Polydactyly Nagoya, Pdn, in Mice

Morphogenesis of hereditary polydactyly (Polydactyly Nagoya, Pdn) in mice

Morphogenesis of hereditary polydactyly (Polydactyly Nagoya, Pdn) in mice

Physiology of nerve growth factor.

Physiology of nerve growth factor.

Developmental abnormalities of the mouse brain in hereditary polydactyly (Polydactyly Nagoya, Pdn)

Developmental abnormalities of the mouse brain in hereditary polydactyly (Polydactyly Nagoya, Pdn)

Polydactyly Nagoya, Pdn: A new mutant gene in the mouse.

A new hereditary polydactyly (gene symbol Pdn) was found in the course of breeding JCL : ICR mice. The genetic analysis indicated that the polydactyly was an autosomal dominant trait. The homozygotes died within two days after birth. The homozygous fetuses or newborn had 1-3 extra-digits both in te fore- and hindlimbs on the preaxial side. They occasionally showed exencephaly, cleft palate, open eyelid, short tibia and fibula or deformed sternum. The heterozygotes had one extra-digit preaxial side. They occasionally showed exencephaly, heterozygotes had one extra-digit preaxially in the hindlimb and an enlarged first digit on the forelimb which often showed bifurcated distal phalanx. A tab on the postaxial side of the forelimb was found in all homozygotes and in some heterozygotes.

Nerve growth factor.

Nerve growth factor.