Mice were backcrossed into the selected genetic backgrounds for at least 14 generations and analyzed throughout the different stages of development in all three backgrounds. We analyzed a minimum of six embryos of each genotype obtained from three or more litters for each time point tested. Since the CD1 and Dba/1 mice displayed identical skeletal and muscular developmental defects (unpublished results), the remaining experiments described were carried out in the CD1 background, unless indicated otherwise.
Phenotypes of Noggin Null Mice in Selected Mouse Strains
At 10.5 dpc, the Noggin null mouse embryos were readily identifiable with craniofacial, somatic, and apparent vascular defects (Fig. 1A). The defects were similar to those already described (McMahon et al., 1998). An interesting exception was the limb bud phenotype, which was less pronounced in the CD1 background (Fig. 1A, inset) as compared to published observations (McMahon et al., 1998). Specifically, the observed size of the limb buds was similar between Noggin wild type and null mice in the CD1 mouse strain, whereas in 129X1/SvJ/C57BL/6, the hind limb buds were reduced in size (see Fig. 8 in McMahon et al., 1998).
Figure 1. Morphology of Noggin null embryos at different developmental stages. A: 10.5 dpc; the insets show magnification of developing hind limb bud. S, somite. B: 12.5 dpc; the inset shows a magnification of the tail phenotype. C: 14.5 dpc; two Noggin null embryos are shown, on the left an embryo with exencephalic head (white arrow), on the right a mutant embryo with closed head. Dotted arrows point to edemas. C′: 14.5 dpc in C57BL/6 genetic background. D: Newborn embryos in CD1 and (D′) Dba/1 backgrounds. All embryos within each panel were photographed at the same magnification.
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At 12.5 dpc, the most striking consequence of Noggin inactivation was the degeneration of the tail structure, frequently accompanied by a large hematoma (Fig. 1B). No tail remnants were recovered at later developmental stages.
At 14.5 dpc, dorsal edemas as well as hematomas in other parts of the body were detected (Fig. 1C, dotted arrows). Two distinct head phenotypes were observed (also reported by McMahon et al., 1998), including exencephaly (Fig. 1C, white arrows). The frequency of the exencephalic phenotype was lower in the DBA/1 background (25%) than in CD1 background (60%).
Inactivation of Noggin in the C57BL/6 mouse strain led to midgestation embryonic lethality, and at 14.5 dpc, the embryos were completely necrotic (Fig. 1C′). Since we were interested in the midgestation and perinatal limb phenotypes, analysis of the Noggin null mutation in this background was not pursued further.
Both CD1 and Dba/1 mouse strains could carry the Noggin null embryos to term. In both cases, however, the embryos died perinatally (Fig. 1D and D′, respectively). The lungs of newborn embryos could, in some cases, float on water, indicating that the newborns were able to take at least one breath. The newborn Noggin null embryos were shorter along the rostral-caudal axis than the wild type littermates, had stubby limbs, and lacked tails.
Analysis of the consequences of Noggin inactivation on skeletal development revealed novel features. The effect on bone mineralization during development was different depending on bone type (Fig. 2A and A′). In some cases, mineralization was apparently accelerated, as in the cervical vertebrae (Fig. 2A and A′, yellow ellipse), while in other cases, the absence of Noggin had an opposite effect or no effect on ossification. For instance, the ossification of the mandible was comparable between wild type and knockout embryos, but zygomatic and parietal bones were not ossified in the knockout animals at 14.5 dpc (Fig. 2A and A′). In contrast, and as previously reported (Brunet et al., 1998), the bones of the developing forelimbs displayed extensive chondrogenesis followed by ossification spanning the stylopod and zeugopod, which resulted in elbow fusions at 14.5 dpc. The ossification of the hind limbs in Noggin null mice was, however, delayed (Fig. 2A and A′). Upon birth, the extent of the skeletal ossification was comparable between CD1 and DBA/1 mouse strains (Fig. 2B and B′ and data not shown).
Figure 2. Comparison of skeletal development using Alcian Blue/Alizarin Red staining at 14.5 dpc (A,A′) and newborn (B,B′) mice. A,B: Wild type. A′,B′: Knockout. F, forelimbs; H, hind limbs; M, mandible; Z, zygomatic bone; P, parietal bone. The yellow dotted ellipse in A and A′ indicates the cervical vertebrae. C: Results of quantitative PCR for Noggin and (D) for Gdf5 mRNAs. The Y axis represents the relative quantity of transcripts, normalized to mouse GAPDH mRNA. E–G: Results of dual in situ hybridization (DISH). E,F: 12.5- and 13.5-dpc limbs, respectively. The left column shows the results of hybridization on the wild type and the right column on the Noggin null mouse limbs. On the merged panel, the cell nuclei were counterstained with DAPI. White arrowhead points to the carpal expression of Gdf5 mRNA in the Noggin null mice. G: 14.5-dpc wild type limbs (top) and knockout limbs (bottom) hybridized with probes for collagen type II (green) and Ihh (red) mRNAs, counterstained with DAPI. All limbs have been oriented with the anterior side pointing upwards and proximal pointing leftwards.
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Noggin null mice did not produce detectable Noggin mRNA (Fig. 2C), while Gdf5 mRNA level was reduced 5-fold as measured by quantitative PCR (Fig. 2D). Analysis of the Gdf5 mRNA expression pattern using DISH (Tylzanowski et al., 2003) revealed that at 12.5 dpc there was no detectable difference between wild type and knockout embryos (Fig. 2E), whereas at 13.5 dpc., this difference was readily visible (Fig. 2F). One of the striking features of Gdf5 mRNA expression at 13.5 dpc was its prominent expression in the carpal region (Fig. 2F, arrowhead). The expression of Indian hedgehog (Ihh) was maintained in the central part of the monophalangeal digit in the Noggin null mice (Fig. 2G).
Muscle and Skin Development
Next, we investigated the myogenic differentiation in the developing limbs of Noggin null mice. One of the striking features observed was the severe reduction of muscle tissue in newborn mouse embryos (Fig. 3C–E′). The onset of myogenesis in the developing limb was not affected as demonstrated by normal expression of a marker gene for the myogenic commitment, Pax3 (McMahon et al., 1998; and our unpublished data). Since the onset of myogenic differentiation was unaffected, but terminal muscle differentiation was impaired in the absence of NOGGIN protein, we have extended our investigation to determine at which point muscle differentiation was affected.
Figure 3. Muscle and skin phenotype in limbs of Noggin null mice. A,B: Wild type and (A′,B′) knockout sagittal frozen sections of 14.5-dpc limbs stained with antibodies against MyoD (A and A′) or Myosin Heavy Chain (B and B′). The antibody green staining was merged with blue DAPI counterstain. The limbs have been oriented with the anterior side pointing leftwards and proximal pointing upwards. C–E′: Histological analysis of limbs from wild type (C–E) and knockout (C′–E′) newborn mice stained with hematoxilin and eosin. D and D′: Magnification of muscle tissue while E and E′ are the same section seen in polarized light. F,G: Wild type, and (F′,G′) knockout skin phenotypes in the developing limbs. F,F′: A result of staining with antibodies against βcatenin (red) of 14.5-dpc embryos. The limbs have been oriented with the anterior side pointing upwards and the proximal part pointing leftwards on the picture. G,G′: A histological section through a limb of a newborn mouse. Ep, epidermis; der, dermis; hf, hair follicle; pcm, panniculus carnosus muscle.
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To address this issue, we determined the expression patterns of selected proteins associated with muscle development. Markers of the intermediate stages of muscle development, such as MyoD (Fig. 3A,A′) or myogenin (data not shown), were detected at the same anatomical locations in wild type and knockout embryos. The expression of myosin heavy chain, the marker for terminal muscle differentiation associated with the formation of myocytes and myotubes (Andres and Walsh, 1996), was, however, different in Noggin null mice. The expression of this marker at 14.5 dpc was detected in both the wild type and Noggin null mice. In the wild type mice, the staining had the typical elongated appearance associated with formation of multinucleated myotubes. In contrast, in the Noggin null mice, the staining had a more rounded appearance suggestive of cells that failed to fuse into myotubes at this stage of development (compare Fig. 3B and B′).
At birth, the myofibers of the knockout embryos formed loose structures, unlike the wild type muscles that formed densely packed fibrils with visible striations. The nuclei of the cells in the muscle fibers of Noggin null mice also failed to migrate to the cell wall (Fig. 3D and D′).
One of the ways to investigate thickness of muscle fibers is to analyze their birefringence on H&E stained sections using polarized light. The thick fibers tend to shift the light towards the more reddish color, while thin fibers maintain the white coloring (Chow et al., 2001). The muscle fibers of Noggin null mice were apparently thinner and somewhat disorganized as indicated by their appearance in polarized light (Fig. 3E and E′).
Skin of the knockout animals, as reported for the 129X1/SvJ strain (Botchkarev et al., 1999), was affected in the same manner in CD1 (Fig. 3F–G′) and Dba/1 genetic backgrounds (not shown). At 14.5 dpc, the expression of β-catenin was prominent in the wild type mice, but in the Noggin null mice, it was not detected by immunofluoresence (Fig. 3F,F′). At 18.5 dpc, unlike in 129X1/SvJ mice, rudimentary hair follicles did form (Fig. 3G,G′).
Several previous publications reported a lack of Noggin haploinsuficiency phenotype in mice (Brunet et al., 1998; McMahon et al., 1998; Botchkarev et al., 1999; Groppe et al., 2002). Since we generated Noggin heterozygous mice in different genetic backgrounds, we reexamined the Noggin heterozygous animals.
Quantitative PCR analysis revealed that the heterozygous mice produced Noggin mRNA at levels slightly above 50% (Fig. 2C). Examination of the developing limbs of newborn Noggin heterozygous mice revealed carpal and tarsal fusions in 100% of animals in all three backgrounds investigated: CD1, Dba/1, and C57BL/6 (Fig. 4A–F”; and data not shown). These fusions formed two morphological classes, independent of the strain used (data not shown). In one, carpals 2, c, and 3 were fused, while in the second, only carpals c and 3 were fused (Fig. 4B–D).
Figure 4. Haploinsufficiency in the Noggin mice. A,A′: Alcian blue/Alizarin red staining of the forelimbs of wild type (wt) and heterozygous (HZ) embryos, respectively, at 18.5 dpc. The yellow oval demarcates the area of joint fusions. B–D: Hematoxilin/eosin staining of sections through the wild type (B) and HZ (C,D) carpal regions of 18.5-dpc mouse limbs. Roman numerals I–V denote metacarpals; 1,2,3,4/5, distal carpal bones; c, central carpal bone; r, radiale; u, ulnare; R, radius; U, ulna. B′–D′: Expression of collagen type II (green) and Gdf5 (red) mRNAs in wild type (B′) and HZ (C′,D′) carpal region at 14.5 dpc. Arrows point to joints with differential expression of Gdf5 mRNA (see text for details). E,F”: Alizarin red/Alcian Blue staining of four weeks old animals. E: Wild type and (E′,E”) HZ forelimbs; F (wild type), and (F′,F”) hind limbs. 1,2,3,4/5, distal carpal bones (or tarsal in F–F”); c, central carpal bone; r, radiale; u, ulnare; R, radius; U, ulna; TA, talus; CA, calcaneus. Alizarin red/Alcian Blue staining of (G–I) wild type and (G′–I′) HZ 14.5-dpc mouse embryos. I,I′: Rib cage seen along cranial/caudal axis while J and J′ show area along ventral/dorsal axis. The arrows in H′ point to kinks in the vertebral column.
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Next, we investigated the developmental onset of these fusions taking into consideration the possibility that they could be primary or secondary in nature. To accomplish this, we analyzed the expression patterns of Gdf5 mRNA, a molecular marker associated with joint formation, and collagen type II mRNA, a molecular marker for developing cartilage. At 12.5 dpc, the expression patterns of Gdf5 and collagen type II mRNAs were the same in wild type and heterozygous animals (data not shown). At 14.5 dpc, the fusions were detected by the analysis of the expression pattern of Gdf5 mRNA. As shown in Figure 4B′–D′, the expression of Gdf5 mRNA was not detectable in parts of the developing carpal region (compare arrows in Fig. 4B′ and 4C′–D′).
The carpal/tarsal fusions remained in the limbs upon birth and throughout the life of the animal, and no additional joint fusions were detected at later stages of development in all three genetic backgrounds: CD1 (Fig. 4E–F″) as well as C57BL/6 and Dba/1 (data not shown).
In addition, the Noggin haploinsufficiency in the CD1 background exhibited kyphosis, detectable in 30% of the animals (Fig. 4G–I′). The ribcage of the embryos were reduced in size due to malformed ribs (Fig. 4H,H′ and I,I′). Also, the embryos had tails that were kinked at the lumbar bone (arrows in Fig. 4G and G′).