Targeted disruption of the Nfix gene.
Generation of an Nfix null allele was achieved by homologous recombination of a replacement-type targeting vector. The targeting construct was designed to disrupt Nfix gene expression by an in-frame insertion of a reporter gene (lacZ) and a loxP-flanked PGKneor-pA cassette into exon 2, thereby replacing all but the first 4 bp of exon 2 and 32 bp of the following intron (Fig. ). Since exon 2 encodes the DNA-binding domain of the NFIX protein, deletion of this region results in a nonfunctional NFI fusion protein. The β-Gal activity of the fusion protein expressed under the endogenous Nfix promoter therefore reflects NFIX expression.
FIG. 1. Generation of an Nfix null allele. (A) Depicted are the first three exons (filled boxes) of the Nfix wild-type locus, the targeting vector, and the targeted alleles before (NfixlacZ-neor) and after the excision of the PGKneor-pA (neor) cassette by Cre-mediated (more ...)
The targeting vector was introduced into CJ7 ES cells, and selected clones, which showed homologous recombination, were identified by Southern blot analysis (Fig. ). Deletion of the selection marker gene was achieved by transient Cre-mediated recombination of the two loxP sites and verified by Southern blot analysis (Fig. ). Two targeted NfixlacZ cell clones were injected into blastocysts to generate germ line chimeras. Male chimeras from one ES cell clone were used for matings with C57BL/6 females. Heterozygous offspring were crossed for homozygosity, and DNA from tail tip biopsy samples was taken to detect the mutated allele by PCR (data not shown) and by Southern blot analysis (Fig. ). To determine whether the NfixlacZ allele was a null allele, RT-PCR was performed. Total mRNA transcripts from brain, liver, and lung from 3-week-old littermates of each genotype were reverse transcribed, and cDNA was analyzed with Nfix exon 1- and exon 4-specific primers (Fig. ). A 622-bp fragment was amplified from wild-type and heterozygous cDNA corresponding to the full-length transcript containing exon 2 and exon 3. No such product could be amplified in tissues from homozygous animals. A smaller 147-bp PCR product was detected in homozygous and heterozygous samples and very weakly in wild-type samples. Sequencing of this smaller fragment showed that it resulted from a direct splice of exon 1 to exon 3 (data not shown). As the exon 1 to exon 3 junction is out of frame, the only possibility is that a 32-amino-acid missense polypeptide is synthesized that terminates in exon 4. It is remarkable that in tissues of Nfix−/− mice the exon 1 to exon 3 splice is more frequent, once the exon 2 to exon 3 splice is eliminated.
Loss of NFIX leads to postnatal lethality.
From a total of 35 heterozygous matings, Nfix
homozygous mutant animals were obtained at a normal Mendelian ratio: 95 Nfix+/+
(31.6%), 130 Nfix+/−
(43.3%), and 75 Nfix−/−
(25%) mice. NFIX-deficient mice developed a dome-shaped head, were unable to fully open their eyes, and had an obvious deformation of the spine (Fig. ). They showed an ataxic gait and, when lifted by their tails, they drew their limbs in toward their bodies (feet-clasping posture) (9
), in contrast to the full extension of limbs observed with normal littermates, indicating neurological abnormalities (Fig. ). With the exception of two animals that reached an age of 3 and 7 months, all other Nfix−/−
animals died between P21 and P28 (Fig. ). When born, Nfix−/−
mice were indistinguishable from their wild-type and heterozygous littermates, but by P5, daily weighing showed that Nfix−/−
mice were unable to gain weight as efficiently as their wild-type littermates. Between P16 and P19 their weight plateaued and then progressively decreased. Shortly before death the body weight amounted to merely 60 to 70% of the weight of their normal siblings (Fig. ). Heterozygous mice showed a slight weight reduction but no obvious anatomical or behavioral defects (Fig. ).
FIG. 2. Phenotype and mortality of Nfix−/− mice. (A) Wild-type (Nfix+/+) and knockout (Nfix−/−) mice at P24; Nfix−/− mice were 25 to 30% smaller and showed a domed head shape and a hunchback. (more ...)
To further characterize the defects in Nfix−/− mice, histological examinations of inner organs were performed, but apart from a generalized reduction in size, almost all organs appeared normal. However, in all affected animals the smaller digestive tract appeared pathological, showing thinning of the intestine walls and reduced blood supply and containing a yellowish/brownish fluid (Fig. ). In histological preparations, myenteric neuronal innervation appeared normal, as well as the mucosa, submucosa, muscle layers, and serosa (data not shown). In addition to the intestinal anomalies, a general loss of muscle tissue was observed, but histological examination of muscle samples showed no evidence of a myopathy (data not shown). Taken together, death of Nfix−/− mice is most likely a consequence of the spectrum of neurological and intestinal problems which result in an inability to thrive.
NFIX-deficient mice develop hydrocephalus and a deformation of the spine.
For further analysis, magnetic resonance imaging was performed with 3-week- and 3-month-old NFIX-deficient animals. Hydrocephalus was revealed in 3-week-old Nfix−/− mice which was characterized by dilatation of the lateral brain ventricles and the third ventricle. The fourth brain ventricle showed a normal size (Fig. ). Analysis of 3-month-old mice showed that hydrocephalus progressed with increasing age (Fig. ).
FIG. 3. MRI and skeletal analysis of Nfix−/− mice. Sagittal (A and C) and coronal (B and D) MRI analysis of 3-week-old (A and B) and 3-month-old (C and D) NFIX-deficient mice (−/−) and wild-type littermates (+/+). (more ...)
Imaging of the spine revealed a progressive skeletal pathology. The mouse spine has two physiological curvatures in the ventral axis. There is a cervico-cervical lordosis and thoraco-thoracical kyphosis. Angles of both vertebral bowings were measured using the Cobb method (8
) in 3-week-old and 3-month-old Nfix−/−
mice and compared to those of wild-type control mice. The cervical angle in 3-week-old control mice was 74.9°, and the thoracic angle was 96.4°. In NFIX-deficient mice of the same age, corresponding angles were 52.8° and 81°, respectively (Fig. ). At the age of 3 months, both angles in control mice became wider, measuring 88.2° for the cervical angle and 115° for the thoracic angle. In the 3-month-old Nfix−/−
mouse the opposite effect was observed, as both angles became acuter and measured 41° and 52°, respectively. These changes caused severe cervico-cervical hyperlordosis and thoraco-thoracical hyperkyphosis. Because of the increased kinking of the spine, the truncal length of Nfix−/−
mice appeared significantly reduced (Fig. ). Scoliosis, which is characterized by an abnormal lateral curvature of the spine, was also observed.
To examine the presence of primary skeletal abnormalities, whole-mount Alizarin red and Alcian blue skeletal preparations of 3-week- and 3-month-old Nfix−/− and control mice were compared. No alterations in the number of vertebral bodies, no bone deformations, and no generalized skeletal dysplasias were observed (Fig. ). Other skeletal bones, like the femur and tibia, were 25 to 30% shorter compared to wild-type animals (data not shown), which contributed to the general reduction in body size of Nfix−/− mice. Alterations in cranial bones appeared to be caused by the progressive hydrocephalus and were characterized by macrocephaly, thinning of the cranial bones, and less-pronounced cranial sutures (Fig. ).
Hydrocephalus is associated with partial agenesis of the corpus callosum in Nfix−/− and Nfix+/− mice.
To further characterize the hydrocephalic phenotype, serial coronal sections of brains of adult Nfix−/−, Nfix+/−, and wild-type mice were performed. Severe dilatation of the lateral brain ventricles and the third ventricle was noted in Nfix−/− mice compared to sections of wild-type brains. The fourth ventricle of Nfix−/− mice was not altered. In Nfix+/− mice a slight enlargement of lateral and third ventricles was also observed (Fig. ). Analysis of sagittal sections and intraventricular dye injections indicated that communication between the lateral and fourth ventricle was not inhibited, and so stenosis of the aqueduct as a possible cause for the hydrocephalus can be excluded (data not shown). Another defect observed in Nfix−/− and Nfix+/− mice was a partial agenesis of the corpus callosum. In coronal sections of rostral brain regions the callosal body was present but rather thin compared to wild-type controls (Fig. ), whereas in more-caudal regions it was completely absent in NFIX-deficient (Fig. ) and heterozygous (data not shown) animals. To analyze the expression pattern of the Nfix gene in the brain, we stained coronal and sagittal sections of Nfix−/− brains with X-Gal to identify the NFIX::LacZ fusion protein. Expression was detected in the hippocampal dentate gyrus, in the ependymal cell layer of the ventricular system, and in the cortex (Fig. ). Sagittal sections also revealed strong staining in the cerebellum (Fig. ).
FIG. 4. Histological analysis of the brains of 23-day old mice. (A to H) Hematoxylin- and eosin-stained 6-μm coronal paraffin sections of rostral (A to F) and caudal (G and H) brain regions. Wild-type (+/+) mice revealed a normal phenotype (more ...)
To determine whether the observed callosal defect was already evident during development, we examined serial brain sections of E17.5 embryos. In rostral Nfix−/− brain regions the corpus callosum was present but thinner than in wild-type animals (Fig. ), whereas more caudally NFIX-deficient mice exhibited complete agenesis of the callosal body (Fig. ). A slight reduction of callosal fibers was also observed in Nfix heterozygous mice compared to wild-type controls (Fig. ). In addition an enlargement of lateral brain ventricles was evident at this early developmental stage in brains of Nfix−/− mice (Fig. ).
FIG. 5. Histological phenotypes of the brain at E17.5. Hematoxylin- and eosin-stained 6-μm coronal paraffin sections of the rostral brain (A and B) and in the plane of the anterior commissure (C to H). In the rostral portions of the brain a very thin (more ...)
We determined by RNase protection assays the expression of a series of neuronal marker genes, including myelin basic protein, neurofilament (NF-68), and glial fibrillary acidic protein (GFAP). The analysis revealed no alterations in the expression of these genes in brains of NFIX-deficient mice compared to control mice (data not shown).
Impaired ossification in vertebral bodies of Nfix−/− mice.
Besides the observed brain malformations, NFIX-deficient mice also exhibited severe skeletal abnormalities. In order to investigate the observed deformation of the vertebral column in more detail, paraffin sections of spines of 14-day-old, 23-day-old, and 7-month-old NFIX-deficient and control mice were examined (Fig. and Fig. ). Histological analysis revealed that in 14-day-old Nfix−/− mice larger amounts of cartilaginous material were present in the vertebral bodies in comparison to wild-type animals, indicating that the ossification process was delayed (data not shown). Sections of spines of 23-day-old Nfix−/− mice showed more pronounced morphological alterations. In regions of the most severe cervical and thoracic bowing, we observed that enlarged cartilaginous endplates associated with chondrocyte-like cells reached into the marrow cavity of the vertebral bodies, while control mice showed complete ossification of these structures (Fig. ). In addition, trabecular and cortical bone was thinner in bones of Nfix−/− mice than in wild-type animals (Fig. ). The intervertebral disks showed pathological alterations as well. They appeared irregular in shape, and the nucleus pulposus was smaller and sometimes fragmented (Fig. ). In some disks the lamellar orientation of the collagen fibers in the annulus fibrosus was also disturbed. X-Gal staining of cryosections of spines of Nfix−/− mice revealed Nfix gene expression in chondrocytes of the cartilaginous endplate and in trabecular bone (Fig. ). To see if the impaired ossification was overcome during maturation, the spine of the one surviving 7-month-old mouse was analyzed and compared with a wild-type mouse (Fig. ). Vertebral bodies were completely ossified, and no remaining chondrocyte-like cells were found in the vertebral marrow in the Nfix−/− mouse, though cartilaginous endplates were still abnormally enlarged (Fig. ). In addition, the anterior part of the intervertebral disks seemed compressed and the nucleus pulposus exhibited degenerative changes when compared to disks of the wild-type mouse (Fig. ).
FIG. 6. Histological analysis of the vertebral columns of 23-day-old mice. Hematoxylin- and eosin-stained 6-μm sagittal paraffin sections (A to F) and X-Gal-stained 20-μm cryosections (G) are shown. (A) Sections of thoracic spine of a wild-type (more ...)
FIG. 7. Histological analysis of spines of 7-month-old mice. Hematoxylin- and eosin-stained 6-μm sagittal paraffin sections of vertebral bodies and intervertebral disks of an Nfix+/+ mouse (A and B) and an Nfix−/− mouse (more ...) Enlarged epiphyseal growth plates in the femurs of Nfix−/− mice.
To analyze whether ossification was also impaired in other bones, femoral sections of 22-day-old Nfix−/− and wild-type mice were histologically analyzed (Fig. ). Hematoxylin- and eosin-stained paraffin sections revealed an enlargement of the epiphyseal growth plate in Nfix−/− femurs compared to wild-type mice (Fig. ). This enlargement was due to an increase of the number of chondrocytes within the resting zone. In contrast, the zone of columnar proliferating chondrocytes was severely reduced and the zone of hypertrophic chondrocytes exhibited also a slight reduction (Fig. ). Trabecular bone mass was diminished, and an altered appearance of the bone marrow was evident compared to control mice (Fig. ). NFIX expression was detected in epiphyseal chondrogenic cells and also in the trabecular bone-forming cells, which corresponded to the observed defects (Fig. ). To analyze whether the impaired ossification and reduced mass of trabecular bone were associated with reduced bone density, radiographic analyses were performed. CT analysis revealed a mineralization deficiency in the hind limbs and jawbones of Nfix−/− mice compared to wild-type mice (Fig. ). These findings indicate that NFIX plays an important role during endochondral ossification and mineralization.
FIG. 8. Histological analysis of femurs of 22-day-old mice. Hematoxylin- and eosin-stained 6-μm longitudinal paraffin sections (A to F) and X-Gal-stained 20-μm cryosections (G and H) of NFIX-deficient (−/−) and wild-type (+/+) (more ...)
3D visualization using volume-rendering techniques of Nfix−/− and Nfix+/+ 23-day-old mice. Arrowheads indicate differences in bone densities in the femur (A) and jawbone (B).
Altered expression of tetranectin in Nfix−/− mice.
In order to elucidate the molecular mechanism of delayed endochondral ossification and decreased mineralization in Nfix−/− mice, we analyzed specific skeletal marker genes. Semiquantitative RT-PCR analysis of RNA from bones of adult Nfix−/− and wild-type mice revealed no significant alterations in collagen 1α1, collagen 2α1, collagen 10α1, and osteocalcin gene expression (Fig. and data not shown).
FIG. 10. Semiquantitative RT-PCR analysis of skeletal marker genes. mRNA from adult organs of wild-type (+/+) and NFIX-deficient (−/−) mice was reverse transcribed, and cDNA was analyzed by PCR with col2a1-specific (A), osteocalcin-specific (more ...)
Furthermore, we analyzed the expression of the plasminogen-binding extracellular matrix protein tetranectin, because it had been postulated earlier that tetranectin plays a crucial role in mineralization during osteogenesis (68
). Furthermore, tetranectin deficiency in the mouse leads to kyphosis due to an asymmetric development of growth plates and intervertebral disks (30
). We detected a significant decrease in the level of tetranectin mRNA transcripts in lung, skeletal muscle, and femurs of adult Nfix−/−
animals compared to wild-type tissues (Fig. ). These data indicate that NFIX is involved in the regulation of the activity of the tetranectin gene.