Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).

A Distal-less-like gene is induced in the regenerating central nervous system of the urodele Pleurodeles waltl.

We report the cloning of a Distal-less-like gene (PwDlx-3) and its pattern of expression during embryonic development and adult tail regeneration in the urodele Pleurodeles waltl. Using RT-PCR and in situ hybridization experiments we determined that, during regeneration, PwDlx-3 is expressed in the epidermis, the cells associated with muscle masses and in the ventrolateral parts of the ependymal tube. PwDlx-3 localization in the muscle masses and in cells of the ependymal tube, which give rise during regeneration to the ventral roots and the spinal ganglia, suggests that this gene might be expressed in cells which have some neural crest cell potentialities. PwDlx-3 is the first homeobox gene shown to be expressed in the regenerating spinal cord but not in the adult one and could thus be involved in the regeneration of the nervous system.

Vertebrate orthologues of the Drosophila region-specific patterning gene teashirt.

In Drosophila the teashirt gene, coding for a zinc finger protein, is active in specific body parts for patterning. For example, Teashirt is required in the trunk (thorax and abdomen) tagmata of the embryo, parts of the intestine and the proximal parts of appendages. Here we report the isolation of vertebrate cDNAs related to teashirt. As in Drosophila, human and murine proteins possess three widely spaced zinc finger motifs. Additionally, we describe the expression patterns of the two murine genes. Both genes show regionalized patterns of expression, in the trunk, in the developing limbs and the gut.

Expression of polysialylated neural cell adhesion molecule (PSA-N-CAM) in developing, adult and regenerating caudal spinal cord of the urodele amphibians.

The patterns of expression of polysialylated ("embryonic") form of Neural Cell Adhesion Molecule (PSA/E-N-CAM) and of all N-CAM isoforms were investigated by indirect immunofluorescence and immunoblotting during the development of the Central Nervous System (CNS) and during the regeneration of the caudal Spinal Cord (SC) of the amphibian urodeles Pleurodeles waltl (Pw) and Notophthalmus viridescens (Nv). In this study, a monoclonal antibody to group B Meningococcus (anti-Men-B) which recognizes alpha-2,8-linked sialic units of PSA-N-CAM, and polyclonal anti-total N-CAM antibodies were used. Total-N-CAM immunoreactivities were consistently detected throughout the CNS of developing and adult newts. PSA-N-CAM expression predominated in "embryonic" developing CNS and was reduced to certain CNS areas in the adult urodeles. In the case of SC, the expression level of this isoform of N-CAM dramatically decreased to become low and nearly restricted to some ependymoglial cell surfaces. Interestingly, during newt tail regeneration, PSA-N-CAM was intensely reexpressed in regenerating SC, at the surface of ependymoglial cell processes and in axonal compartments. Expression was maximal at 4 to 6 weeks following amputation, and then gradually returned to a normal adult low level in well differentiated SC. These findings strongly supported the view that the expression of PSA-N-CAM was associated with the properties of plasticity shown by the SC ependymoglial tissue in newts, during tail regeneration. On the other hand, the high level of PSA-N-CAM expression in axonal compartments of regenerating as well as developing SC suggested that these isoforms of N-CAM could be implicated in axonal outgrowth within the "tunnels" defined by the radial ependymoglial processes. This transient PSA-N-CAM expression could therefore be considered both as a negative modulator of cell-cell and cell-substrate interactions and as a permissive factor for neuron differentiation.

Patterns of dystrophin expression in developing, adult and regenerating tail skeletal muscle of Amphibian urodeles.

The patterns of expression of dystrophin were investigated by indirect immunofluorescence and by immunoblotting in developing, adult and regenerating tail skeletal muscle of newts Pleurodeles waltl and Notophthalmus viridescens. In this study, a monoclonal antibody H-5A3 directed against the C-terminal region (residues 3357-3660) and a polyclonal antibody raised to the central domain (residues 1173-1738) of the chicken skeletal muscle dystrophin were used. Western blot analysis showed that these antibodies recognized a 400 kDa band of dystrophin (and may be of dystrophin-related protein) in the adult muscle tissues and in newt tail regenerates. During skeletal muscle differentiation or epimorphic regeneration (blastema), anti-dystrophin immunoreactivity gradually accumulated over the periphery of the myofibers. Dystrophin and laminin were first and concomitantly observed at the ends of the newly formed myotubes where they were anchored on connective tissue septa or bone processes by dystrophin-rich myotendinous structures. It is noteworthy that neuromuscular junctions, which most probably also contain dystrophin, are established in urodeles near the ends of the myofibers as shown by histochemical localization of AChE activity or fluorescent bungarotoxin detection of AChRs. In the stump transition zone close to the tail amputation level where tissue regeneration of injured muscle fibers took place, dystrophin staining located on the cytoplasmic surface of myofibers progressively disappeared during the dedifferentiation process which seemed to occur during muscle regeneration as suggested by electron microscopy. Furthermore, double labeling experiments using anti-dystrophin and anti-laminin antibodies showed a good correlation between the remodeling processes of the muscle fiber basal lamina and the loss of dystrophin along the sarcolemma of damaged and presumably dedifferentiating muscle cells.

Expression patterns of dystrophin products, especially of apodystrophin-1/Dp71, in the neural retina of Amphibian urodele Pleurodeles waltl.

The expression patterns of the DMD (Duchenne Muscular Dystrophy) gene products, especially of Dp71 (apodystrophin-1) were investigated by immunofluorescence and immunoblotting in the retina of the Amphibian urodele Pleurodeles waltl. H-5A3 monoclonal antibody (mAb), directed against the C-terminal region of dystrophin/utrophin, and 5F3 mAb, directed against the last 31 amino acids of dystrophin and specific of Dp71, were used. Western blot analyses with H-5A3 mAb revealed distinct dystrophin-family isoforms in adult newt retinal extracts: a doublet 400-420 kDa, Dp260 isoform, a protein at about 120 kDa, and a diffuse zone at 70-80 kDa, which might correspond to Dp71. Reactivity with H-5A3 mAb appeared nearly restricted to the outer plexiform synaptic layer. On the other hand, Dp71-specific 5F3 mAb recognized trhee polypeptide bands at 70-80, 60-65 and 50-55 kDa in adult newt retina corresponding most probably to alternative spliced isoforms of Dp71. In immunohistochemistry by conventional epifluorescence microscopy, 5F3 labeling was mainly observed in the plexiform layers, the outer nuclear layer, and the photoreceptor inner segments, especially at the myoid regions. Analysis by confocal scanning laser microscopy (CSLM) revealed that 5F3 labeling was, in addition, present in the pigmented epithelium and the inner nuclear layer. Furthermore, CSLM showed that 5F3 staining at the myoids was concentrated at discrete domains underneath the plasma membrane. Our findings raised the question concerning the functional significance of Dp71 isoforms, especially at the myoid where Dp71 was detected for the first time, although it occurred here highly expressed. Putative role(s) played in this retinal compartment and other ones by Dp71 and/or other dystrophin isoforms were discussed.

Tenascin expression in developing, adult and regenerating caudal spinal cord in the urodele amphibians.

Tenascin (Tn) protein and transcripts were analyzed in developing, adult and regenerating caudal spinal cord (SC) of Pleurodeles waltl. A polyclonal antibody (PAb) against Xenopus Tn and a newt Tn cDNA probe were used. In Western blots, anti-Tn PAb recognized Tn polypeptides of 200-220 kDa in tail regenerate extracts, but also the homolog of Tn/Cytotactin/J1 in brain and SC of adult newt. Immunofluorescence studies showed some reactivity around ependymoglial cells and strong labeling in the nervous tracts, in the developing as well as in the regenerating SC or adult SC. Immunogold electron microscopy revealed the presence of Tn throughout the ependymoglial cells, particularly near and along the plasma membrane of radial processes surrounding axons, especially growth cones. Tn could be more precisely found within rough endoplasmic reticulum and Golgi structures, or again in the surrounding extracellular space. This suggested that Tn was at least produced by radial glial profiles forming axonal compartments in which axons grew. Using the DNA probe for Tn, expression of Tn mRNA was also examined by Northern blot and RNAase protection analyses and by in situ hybridization, respectively. The levels of transcripts, barely detectable in adult tail, increased in regenerates from 3 days through 4-8 weeks post-amputation. In situ Tn mRNA were mainly localized in the mesenchyme, especially at the epithelial-mesenchymal interface, and in the developing cartilage, at the early regeneration stages, whereas high amounts of transcripts were seen not only at these stages, but also later, in the regenerating SC. Our main results supported the view that, in the caudal SC of newts, Tn, synthesized by radial ependymoglial cells, was similarly expressed during regeneration as well as larval development, and exhibited a sustained high accumulation level in the adult SC. On the basis of the multifunctional properties of Tn, the putative roles played by Tn as a substrate for neuronal pathfinding and boundary shaping were discussed.

Forebrain-specific promoter/enhancer D6 derived from the mouse Dach1 gene controls expression in neural stem cells.

Drosophila dachshund is involved in development of eye and limbs and in the development of mushroom bodies, a brain structure required for learning and memory in flies. Its mouse homologue mDach1 is expressed in various embryonic tissues, including limbs, the eye, the dorsal spinal cord and the forebrain. We have isolated a forebrain-specific 2.5-kb enhancer element termed D6 from the mouse mDach1 gene and created D6-LacZ and D6-green fluorescent protein (GFP) reporter gene mouse lines. In embryonic stages, the D6 enhancer activity is first detected at embryonic day 10.5 in scattered cells of the outbuldging cortical vesicles. By embryonic day 12.5, D6 activity expands throughout the developing neocortex and the hippocampus. In the adult mouse brain, D6 enhancer is active in neurons of the cortical plate, in the CA1 layer of the hippocampus and in cells of the subventricular zone and the ventricular ependymal zone. Adult mice also show D6 activity in the olfactory bulb and in the mamillary nucleus. Cultured D6-positive cells, which were derived from embryonic and postnatal brains, show characteristics of neural stem cells. They form primary and secondary neurospheres that differentiate into neurons and astrocytes as examined by cell-specific markers.Our results show that D6 enhancer exerts highly tissue-specific activity in the neurons of the neocortex and hippocampus and in neural stem cells. Moreover, the fluorescence cell sorting of D6-GFP cells from embryonic and postnatal stages allows specific selection of primary neural progenitors and their analysis.

Possible roles for Wnt genes in growth and axial patterning during regeneration of the tail in urodele amphibians.

Urodele amphibians are nearly the only adult vertebrates able to regenerate their missing or amputated tail. An interesting aspect of this biological model lies in the ability of regenerates to differentiate the spinal cord (SC), the vertebral cartilage, and muscles. The main questions addressed in this study concern the possible roles of Wnt genes in these regenerative processes. We have previously reported the expression pattern of a Pleurodeles Waltl wnt-10a gene (Pwnt-10a) in tail blastema (Caubit et al. [1997] Dev. Dyn. 208:139-148). We report here the cloning and tissue distribution of three additional Wnt genes (Pwnt-5a, Pwnt-5b, and Pwnt-7a) in adult and regenerating tail tissues and in the central nervous system (CNS) of adult newt. In adult and regenerating tails, Pwnt-5a and Pwnt-5b transcripts exhibit a graded distribution along the antero-posterior (A-P) axis, the maximal accumulation of these transcripts being detected in the mesenchyme within the subectodermal apical region of the normal tail and blastema. In contrast to Pwnt-5a and Pwnt-5b, Pwnt-7a is expressed in adult normal tail skin and in the epidermis of the regenerating tail. In the adult CNS, Pwnt-5a, Pwnt-5b, Pwnt-7a, and Pwnt-10a genes are expressed in sharp overlapping but not identical domains along the A-P axis. The sustained expression of Wnt genes in the adult newt and the spatial distribution of transcripts in adult and regenerating tail tissues suggest roles of these genes in continuous growth capacities in the urodeles and may explain the ability for CNS and tail regeneration.

Reactivation and graded axial expression pattern of Wnt-10a gene during early regeneration stages of adult tail in amphibian urodele Pleurodeles waltl.

Adult urodele amphibians such as Pleurodeles waltl are able to regenerate their amputated limbs or tail. The mechanisms implicated in growth control and formation of the blastema are unknown but it has been proposed that regeneration in newts may proceed through reactivation of genes involved in embryonic development. Knowing the role of Wnt genes in the patterning of the primary and secondary axes of the vertebrate embryo, we suspected that some of these genes could be involved in axial pattern during newt tail regeneration. Pwnt-10a gene, cloned from a newt tail regenerate cDNA library, showed an expression pattern compatible with such a role in tail regenerates. Pwnt-10a, which is highly expressed during embryonic development (from gastrula to tailbud-stage) and weakly expressed in the adult tail, is strongly re-expressed during tail regeneration. In the blastemal mesenchyme Pwnt-10a transcripts exhibited a graded distribution along the antero-posterior axis, the mRNA accumulation being maximal in the caudal most part corresponding to the growing zone. These findings strongly support the view that Pwnt-10a may act in cooperation with other factors to control growth and patterning in newt tail regeneration. Until now Wnt-10a was only known to be involved in central nervous system development; our results suggest that this gene may also play a role in other developmental processes.

Conservation of BF-1 expression in amphioxus and zebrafish suggests evolutionary ancestry of anterior cell types that contribute to the vertebrate telencephalon.

The forkhead domain containing transcription factor BF-1 has been shown to play a major role in the correct development of the cerebral hemispheres in the mouse. BF-1 orthologs have been isolated from zebrafish and the cephalocordate amphioxus. In both species, BF-1 is expressed in the anterior neural tube. In zebrafish zBF-1 expression is restricted to anterior portions of the otic vesicle and to the presumptive telencephalon. In amphioxus AmphiBF-1 is transiently seen in the frontal part of the first somite and, at 3 days of development, in a small number of cells in the cerebral vesicle (cv). The anterior expression of BF-1 in chordates and vertebrates and of slp-1/2 in Drosophila suggests that BF-1 is crucial for an evolutionarily conserved specification of anterior neuronal cell types.

Two Nkx-3-related genes are expressed in the adult and regenerating central nervous system of the urodele Pleurodeles waltl.

We report the isolation and characterization of two NK-3-related genes (PwNkx-3.2 and PwNkx-3.3) and their expression patterns during embryonic development, in the adult CNS, and during tail regeneration in the urodele Pleurodeles waltl. PwNkx-3.2 is the ortholog of the mouse and Xenopus genes, Bapx 1 and Xbap, but PwNkx-3.3 has no known homologue in any other vertebrate. We demonstrate that PwNkx-3.2 and PwNkx-3.3 exhibit graded axial expression patterns in adult spinal cord. During tail regeneration, the two genes are expressed in the wound epidermis, the regenerating muscle masses, the regenerating neural tube, the spinal ganglia, and the cartilage rod. The spatial distribution of transcripts in the CNS suggests that these genes could participate in maintaining the position information along the anteroposterior axis and may explain the ability of the adult CNS to regenerate. During tail regeneration, both genes could be implicated in the reformation of the axial skeleton.

The spatial restrictions of 5'HoxC genes expression are maintained in adult newt spinal cord.

Urodele amphibians are the only adult vertebrates possessing the capacity to regenerate their limbs and tail after amputation. Epimorphic regeneration is characterized by the accumulation of undifferentiated and dividing mesenchymal cells originating from the tissues of the stump, which form a blastema. It has been proposed that the ability to regenerate precisely the amputated structures depends on a 'positional memory' of the cells at the level of amputation plane and that a continuum of positional value would be present in adult urodeles along the appendages able to regenerate. Hox genes are good candidates for playing a role in providing the capacity for regeneration and for carrying positional information. Here, we report the cloning of four AbdB-like genes (Hoxa9, Hoxc10, Hoxc12 and Hoxc13) in the newt Pleurodeles waltl (Pw). To analyse their expression pattern along the antero-posterior (AP) axis of adult urodele central nervous system (CNS), we used the reverse transcription-polymerase chain reaction (RT-PCR) and showed that the 5'HoxC genes expression pattern conforms to the usual spatial colinearity rule. In addition, the expression level in tail regenerates of PwHoxc13, PwHoxc12, and PwHoxc10 was respectively 20, 7 and 2 fold higher than in adult tail. These last results suggest that 5'HoxC genes could specify positional memory in adult spinal cord (SC) and could be involved in axial patterning of the tail during regeneration.

Clonal cell cultures from adult spinal cord of the amphibian urodele Pleurodeles waltl to study the identity and potentialities of cells during tail regeneration.

The urodele amphibians are nearly the only adult vertebrates able to regenerate their missing or amputated tail. The most striking feature of this model lies in the ability of the spinal cord (SC) to differentiate, within the regenerating tail, a new ependymal tube from which the SC and the peripheral nervous system originate. A fundamental question is whether, in response to tail excision, the ependymoglia of the old SC stump behaves as an embryonic neuroepithelium. To evaluate this possibility, cell lines from primary cell cultures of adult SC were established for the first time in newts, and two cell clones, immunochemically characterized as ependymoglial cell populations, could be obtained. To analyze the potentialities of these clonal cells, after transplantation in tail regenerates, cell-marking experiments, using either in vitro transfection with lacZ gene or the lineage tracer lysinated rhodamine dextran (LRD), were performed. One to 2 weeks postimplantation, most of labeled derivatives were identified as melanocytes. Interestingly, labeled cells were also seen integrated in the ependymoglia of the regenerating SC. Two to 6 weeks after implantation in young regenerates, we also observed LRD-labeled elongated cells close to nerves or myofibers which were unambiguously identified as Schwann cells by galactocerebroside staining. Taken together, these findings showed that clonal cells derived from adult newt SC cultures could largely find, in regenerate mesenchyme, suitable environmental conditions to differentiate into melanocytes or Schwann cells. Because these two cells types arise from neural crest cells during embryo-genesis, this supports the interesting view that multipotent cells are still present in the SC of adult urodeles.

Mouse Dac, a novel nuclear factor with homology to Drosophila dachshund shows a dynamic expression in the neural crest, the eye, the neocortex, and the limb bud.

Dac is a novel nuclear factor in mouse and humans that shares homology with Drosophila dachshund (dac). Alignment with available sequences defines a conserved box of 117 amino acids that shares weak homology with the proto-oncogene Ski and Sno. Dac expression is found in various neuroectodermal and mesenchymal tissues. At early developmental stages Dac is expressed in lateral mesoderm and in neural crest cells. In the neural plate/tube Dac expression is initially seen in the prosencephalon and gets gradually restricted to the presumptive neocortex and the distal portion of the outgrowing optic vesicle. Furthermore, Dac transcripts are detected in the mesenchyme underlying the Apical Ectodermal Ridge (AER) of the extending limb bud, the dorsal root ganglia and chain ganglia, and the mesenchyme of the growing genitalia. Dac expression in the Gli 3 mutant extra toes (Xt/Xt) shows little difference compared to the expression in wild-type limb buds. In contrast, a significant expansion of Dac expression are observed in the anterior mesenchyme of the limb buds of hemimelic extra toes (Hx/+) mice. FISH analysis reveals that human DAC maps to chromosome 13q22.3-23 and further fine-mapping defined a position of the DAC gene at 54cM or 13q21.1, a locus that associates with mental retardation and skeletal abnormalities.

Formation of the peripheral nervous system during tail regeneration in urodele amphibians: ultrastructural and immunohistochemical studies of the origin of the cells.

In the regenerating newt tail, epimorphic regeneration--which recapitulates morphologically normal embryonic development--proceeds along a rostrocaudal differentiation gradient. Innervation of the new myomeres results from the spinal roots of segments rostral to the amputation plane and from ventral roots emerging from the lateroventral region of the regenerating spinal cord, in which motor neurons are differentiating. Electron microscopy and an indirect immunofluorescence study with anti-glial fibrillary acid protein (GFAP) confirm that the ventrolateral part of the regenerated ependymal tube gives rise to cells of the ventral root sheath and the spinal ganglia. Anti-GFAP and anti-neurofilament antibodies showed that ependymoglial cells and Schwann cells may play a role in neuronal pathfinding by helping guide and stabilize pioneering axons as they extend toward the myomeres. The carbohydrate epitope NC-1 is expressed in the spinal cord, in sheath cells of the spinal ganglia and in the non-myelin-forming Schwann cells of the peripheral nervous system. L1, a Ca++ independent neural cell adhesion molecule, was detected in the axonal compartments of the regenerating spinal cord, on immature and/or non-myelin-forming Schwann cells within the peripheral nervous system (PNS), and on nerve fibers within the regenerate. These immunohistochemical observations collectively support the hypothesis that Schwann cells already present in the blastema could be involved in organizing neural pathways.