1 Department of Molecular, Cellular and Developmental Biology and
2 Department of Radiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
* Present address: Skirball Institute, New York University, New York, NY, USA
Author for correspondence (e-mail: robert.lazzarini{at}mssm.edu)
Accepted 9 April 2002
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SUMMARY |
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Key words: glial cells missing, Secondary neurulation, Cell fate specification, Neural tube defects, Tail bud, Mouse
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INTRODUCTION |
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We have assessed the potential role of Gcm1 in murine CNS development using transgenic mice that express Gcm1 under the control of the mouse Hoxa7 enhancer. We show that ectopic expression of Gcm1 during early embryogenesis leads to two severe neural tube defects that have counterparts in human disease: failure of the neural tube to close (spina bifida or more precisely, myelocele) and multiple neural tubes (diastematomyelia). The dysraphisms develop during the period of transgene expression and within the zone of expression. After transgene expression ceases, the dysraphisms are progressively resolved and the open neural tube closes. Neonatal animals, while showing signs of scarring and tissue resorption, have a closed vertebral column containing the multiple neural tubes (as in human diastematomyelia). The animals live a normal life span, are fertile and do not exhibit any obvious weakness or motor disabilities.
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MATERIALS AND METHODS |
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X-gal staining and histology
Embryos between days 8.5 and 16.5 (days of vaginal plug was designated as day 0.5) were dissected in cold phosphate buffer (PBS) and fixed in 2% paraformaldehyde in 0.1 M Pipes (piperazine-N,N'-bis-2-ethanesulfonic acid; Sigma, St Louis, MO) pH 6.9 containing 2 mM MgCl2 and 1.25 mM EGTA for 30 minutes at 4°C. Embryos were then washed in 0.1 M PBS containing 0.02% NP40 for 20 minutes and stained in the dark at 37°C in 1 mg/ml X-gal, 5 mM K3Fe(CN)6, 5 mM K4 Fe(CN)6, 2 mM MgCl2, 0.02% NP40 and 0.01% sodium deoxycholate in 0.1 M PBS pH 7.4. After the staining, embryos were washed in PBS and fixed in 4% paraformaldehyde overnight, cleared in 70% ethanol and photographed as whole mounts. They were then dehydrated and embedded in paraffin. Ten µm sections were cut, dewaxed and counterstained with 0.1% Eosin or with Hematoxylin and Eosin for histological examination of the embryos.
Northern blot
For northern blot analysis, total RNA were extracted from E9.5 transgenic and wild-type embryos using guanidine thiocyanate-cesium chloride method (Sambrook et al., 1989). Poly(A) selected RNA were purified on oligo(dT) resine using mRNA isolation kit (Ambion). RNA were then separated on 1% agarose gel in 6.3% formaldehyde containing buffers, transferred to nylon membrane (Nytran) and hybridized with a 810-bp Gcm1-specific cDNA probe-labeled by random priming (NEB blot). After hybridization with the Gcm1 probe, the blot was stripped and probed with a GAPDH-specific probe used as standard control of RNA loading.
In situ hybridization
In situ hybridization was carried out on 10 µm paraffin sections of embryos as described by Wilkinson (Wilkinson et al., 1987). The following probes were used to generate sense and antisense riboprobes: Shh probe corresponding to a 642-bp EcoRI fragment (a gift from Dr A. McMahon, Harvard University), Pax3 probe [530-bp PstI/HindIII fragment from the 3'-end of the gene (Goulding et al., 1991
)], Gcm1 probe [800-bp fragment, bases 710-1510 in the sequence in Altshuller et al. (Altshuller et al., 1996
)], Tbx6 probe was generated from an EST clone (IMAGE consortium clone ID 1446422), Fgfr1 probe, corresponding to a 400-bp fragment covering the Ig.II and 5' half of the Ig.III domains, was generated by PCR using Fgfr1 cDNA as a template (a gift from Dr M. Goldfarb, Mount Sinai School of Medicine, New York), Notch1 probe corresponds to a 422-bp fragment (a gift from Dr J. Kitajewski, Columbia University, New York, NY). Sense and anti-sense 35S-UTP-labelled riboprobes were generated with T3 or T7 RNA polymerases using a standard in vitro transcription protocol.
Immunohistochemistry
Embryos were dissected in cold PBS and fixed in 4% (w/v) paraformaldehyde overnight at 4°C and embedded in paraffin according to standard protocols. Ten µm sections were deparafinized in xylene and hydrated through a graded ethanol series, then treated with 1% H2O2/10% methanol in 0.1 M PBS for 20 minutes, washed extensively in PBS and blocked for 1 hour in 10% normal goat serum, 1% gelatin, 5% BSA, 0.05% sodium azide in 0.1 M PBS. They were incubated overnight with the primary antibody diluted in 10% normal goat serum/0.1 M PBS in a humid chamber. Sections were then washed in 0.1 M PBS/0.1% Triton X-100 (Sigma) 3 time for 20 minutes and then incubated with the appropriate peroxidase-conjugated secondary antibody for 1 hour at room temperature. Sections were then washed in 0.1 M PBS/0.1% Triton X-100 and transferred in 0.1 M Tris-HCl pH 7.6. Peroxidase histochemistry was performed in 0.1 M Tris-HCl pH 7.6 containing 0.03% diaminobenzidine (DAKO, Carpinteria, CA), 0.1% NiCl2 and 0.003% H2O2 and stopped in water. Sections were dehydrated, cleared in xylene and mounted in cytoseal (Stephens Scientific). The following primary antibody were used: a mouse monoclonal anti-Islet-1 (1/20 dilution, mouse IgG2b, 39.4D5; DSHB, University of Iowa), a mouse monoclonal anti-tubulin ß3 (mouse IgG2b, clone SDL.3D10, 1/100 dilution; Sigma), a mouse monoclonal anti-MAP2 (mouse IgG1, 1/100 dilution; Sigma), and a mouse monoclonal anti-neurofilament 160 (mouse IgG1, 1/200 dilution; Sigma).
Magnetic resonance imaging
Each E16.5 embryo was positioned within a 1.5 cm diameter polyethylene tube filled with Fomblin (perfluoropolyether; Ausimont, Thorofare, New Jersey) used as a wetting and embedding agent to prevent dehydration and to reduce artifacts at tissue margins. Where present, air bubbles were aspirated from the interstices of the embryo with a very fine needle and syringe. The sample was wedged in place between two styrofoam plugs to reduce tissue vibration and the tube was sealed to prevent evaporation and re-entry of air bubbles.
The magnetic resonance images in the axial, coronal and sagittal planes were obtained on a 9.4 T superconducting magnet with a vertical 89 mm bore using a 25 mm birdcage coil. An automated water cooling system maintained the temperature within the bore at less than 30°C (Bruker Avance System with microimaging; Bruker Analytik, Rheinstetten, Germany). Pilot studies of diverse T1, T2 and intermediate-weighted (Int-w) sequences led us to select the Int-w sequence for spinal cord analysis, specifically: TR=2000 mseconds, TE=45 mseconds, slice thickness 0.5 mm, field of view 15x15 mm, data matrix 512x512, and number of excitations=50. This corresponds to an in-plane resolution of 29x29 µm and a slice thickness of 500 µm. Each sample was run overnight for a total acquisition time of 14 hours, 17 minutes.
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RESULTS |
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Five independent Gcm1 founder transgenic mice were generated with these constructs (see Fig. 1A). In all animals, ectopic expression of Gcm1 leads to congenital CNS abnormalities. Most analyses described here were carried out on two permanent transgenic lines that were established. To illustrate the domain of transgene expression, embryos (E9.5 to E16.5) bearing the Hoxa7-lacZ transgene were stained with X-gal as whole mounts. As shown in Fig. 1D,E, the expression of the Hoxa7-Gcm1 transgene is detectable at E9.5 in the most caudal region of the embryos with an anterior limit of expression at the level of somite 18-20 (mid-thoracic level). ß-galactosidase activity was observed in the neural tube and somites as well as in mesenchymal cells of the tail bud (Fig. 1D,E). A rostral-caudal gradient of lacZ activity is also noted at this stage. At E10.5, lacZ activity shifts caudally and remains robust in the tail bud (Fig. 1E). At later stages, ß-galactosidase staining is only detectable in the tail bud and in the tail until E12.5. Furthermore, expression of Gcm1 in E9.5 transgenic embryos was confirmed by the detection on northern blots of a single transcript of 1.8 kb, which corresponds to the predicted size of the transgene mRNA (Fig. 1B).
Gcm1 expression induces severe neural tube malformations
Gross morphologic examination of transgenic embryos revealed two types of severe neural tube defects. All transgenic mice derived from the 5 founders showed either one or both of the two pathologic phenotypes: failure of neural tube closure (spina bifida) and the presence of ectopic neural tubes (diastematomyelia). A summary of the examination of 62 transgenic embryos, shown in Table 1, reveals that 100% of the embryos had multiple neural tubes and 26% had open neural tube. No signs of embryonic lethality were observed. Most remarkably the neonatal animals exhibit a progressive resolution of their spina bifida and by 2 months of age show no outward signs of the embryonic neural defect.
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DISCUSSION |
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The transgene that we employed initiates expression of Gcm1 at a time and place that are of critical importance to neurulation: E9.5 and the tail bud, the time and place of the posterior neuropore closure and secondary neurulation (Nievelstein et al., 1993; Schoenwolf, 1984
). At that time the caudal tip of the neural tube lies adjacent to the tail bud (Griffith et al., 1992
). The embryonic tail bud comprises a seemingly homogeneous mass of cells and represents the remains of Hensens node and the primitive streak. This cell mass has remarkable developmental potential and gives rise, during subsequent development, to a variety of structures including the secondary neural tube, the caudal notochord, hind gut, part of the vertebral column and musculature; all without the previous formation of the three germ layers (Griffith et al., 1992
; Tam and Trainor, 1994
). This seemingly direct generation of ecto-, endo- and mesodermal cell types is in stark contrast to the generation of similar cell types in the rostral regions, and the details of this process are incompletely understood.
After closure of the posterior neuropore (E9.5-10) the primary neural tube is extended caudally through a process of secondary neurulation (Naidich et al., 1996; Nievelstein et al., 1993
; Schoenwolf, 1984
). At that time the closed primary neural tubes extends to the level of somite 32-34, the future level of the third sacral vertebra and thus primary neurulation forms the parts of the wild-type spinal cord that have dorsal and ventral roots. The secondary neurulation produces the caudal spinal cord, including the portions that are the primordia of the filum terminale, the ventriculus terminalis and part of the conus medullaris. In mice, the observed earliest event in secondary neurulation is the continuous accretion of aggregates of the tail bud mesenchymal cells in the form of medullary rosettes at the caudal end of the primary neural tube (Muller and ORahilly, 1987
; Muller and ORahilly, 1988
; Nievelstein et al., 1993
; Schoenwolf, 1984
). These cells take on a columnar epithelial appearance and form a neurocoele by cavitation, the lumen being always in contact with the lumen of the primary neural tube.
Several lines of evidence suggest that the complex phenotype of the transgenic mice described here (spina bifida and diastematomyelia) is a direct result of two distinct phenomena: the inhibition of posterior neuropore closure and the indirect stimulation of secondary neurulation. That the spina bifida observed in the transgenic mice results from an inhibition of neuropore closure by Gcm1 is supported by our estimates of the anatomical level of the defect. The position of the closure defect in affected transgenic mice, whether visualized in histologic sections or in whole mounts by MR imaging, is same in different animals and corresponds to the position of the posterior neuropore. The value of 26% reported in Table 1 for the proportion of transgenic embryos with recognizable spina bifida may be strongly affected by two competing secondary processes: progressive resolution of the defect and the neuroepithelial hyperplasia, which exaggerates the defect.
The possibility that Gcm1 directly stimulates secondary neurulation is difficult to reconcile with our inability to detect any Gcm1 mRNA in the wild-type tail bud during active secondary neurulation (Fig. 8). Rather, we believe that the transgene-derived Gcm1 indirectly stimulates secondary neurulation that results in the generation of ectopic neural tubes. This hypothesis is supported by two lines of evidence. The time and place of transgene expression indicate that the ectopic neural tubes are not derived from primary neurulated tissue. Further, the appearance of numerous, small "free-floating" neural tube-like structures at the very tip of the tail bud suggests that the transgene-derived Gcm1 stimulates the neuroepithelial differentiation of tail bud mesenchyme, a process already underway at that time and place. There is mounting evidence to suggest that when tail bud mesenchymal cells are prevented from differentiating into paraxial mesoderm they follow a pathway leading to the formation of secondary neural tubes. Much of the evidence comes from studies of the effects of mutations in the transcriptional or signaling factors known to be active in the mesenchyme to paraxial mesoderm transformation, an early stage in the differentiation of somites. Transcriptional cascades initiated by FGFR1 (Ciruna and Rossant, 2001) or Wnt3a (Yamaguchi et al., 1999a
; Yamaguchi et al., 1999b
; Yoshikawa et al., 1997
) have been identified in the somite progenitor cells. Interruption of these cascades by null mutations in the genes encoding them leads not only to a deficit in paraxial mesoderm but also to the formation of ectopic neural tubes. Thus mutations in either Fgfr1 (Ciruna et al., 1997
; Deng et al., 1997
), Wnt3a (Yoshikawa et al., 1997
), Tbx6 (Chapman and Papaioannou, 1998
) or mutations in both Lef1 and Tcf1 (Galceran et al., 1999
) lead to the formation of ectopic neural tubes. The transformation of paraxial mesoderm progenitors to neural progenitors that is apparent in these null mutants has been attributed to diversion of the progenitor cells to a default neural pathway when mesodermal differentiation is inhibited. Our results are compatible with this interpretation if ectopic expression of Gcm1 in tail bud mesenchymal cells specifically interferes with their differentiation to paraxial mesoderm. Gcm-induced interference with both epidermal and mesodermal differentiation has been observed in Drosophila (Akiyama-Oda et al., 1998
; Bernardoni et al., 1998
; Reifegerste et al., 1999
). In our study, we showed that ectopic expression of Gcm1 in multipotential mesenchymal cells induces the down regulation of two factors normally required for mesodermal differentiation, Notch1 and Tbx6, in cells assuming neurepithelial cell type. We believe that the ectopic neural tubes in our transgenic mice result from the incomplete suppression of the differentiation of mesenchyme to paraxial mesoderm and the diversion of precursors to the default neural pathway. However the possibility that Gcm1 may directly induce neural-specifying genes can not be ruled out.
A striking difference between our transgenic mice and those bearing the aforementioned null mutations is that our transgenic animals are viable, fertile and live a normal life span. They show no obvious signs of posterior limb weakness. All mesoderm-derived organs and tissues are present in normal amount. Thus, the cells fated to the ectopic neural tubes seem to be in addition to, not instead of, paraxial mesoderm tissues. Thus, we further speculate that this interference with mesenchymal cell differentiation occasions a limited hyperplasia that brings the paraxial mesoderm generation to the normal range and accounts for the increase in neuroepithelial cells that we see in the transgenic embryos. These results are corroborated by the observation that the tail buds of E9.5 transgenic mice are consistently larger than those of their wild-type littermates. This hyperplasia seems restricted to the neuroepithelium and is evident in the MRI images of Fig. 4.
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ACKNOWLEDGMENTS |
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