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Keywords:

  • cellular condensation;
  • knockout mouse;
  • lacZ reporter;
  • gene trap;
  • OmniBank;
  • organ culture;
  • cadherin11

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The cell adhesion molecule N-cadherin is implicated in many morphogenetic processes, including mesenchyme condensation during limb development. To further understand N-cadherin function, we characterized a new N-cadherin allele containing the lacZ reporter gene under the regulation of the mouse N-cadherin promoter. The reporter gene recapitulates the expression pattern of the N-cadherin gene, including expression in heart, neural tube, and somites. In addition, strong expression was observed in areas of active cellular condensation, a prerequisite for chondrogenic differentiation, including the developing mandible, vertebrae, and limbs. Previous studies from our laboratory have shown that limb buds can form in N-cadherin–null embryos expressing a cardiac-specific cadherin transgene, however, these partially rescued embryos do not survive long enough to observe limb development. To overcome the embryonic lethality, we used an organ culture system to examine limb development ex vivo. We demonstrate that N-cadherin–deficient limb buds were capable of mesenchymal condensation and chondrogenesis, resulting in skeletal structures. In contrast to previous studies in chicken using N-cadherin–perturbing antibodies, our organ culture studies with mouse tissue demonstrate that N-cadherin is not essential for limb mesenchymal chondrogenesis. We postulate that another cell adhesion molecule, possibly cadherin-11, is responsible for chondrogenesis in the N-cadherin–deficient limb. Developmental Dynamics 232:336–344, 2005. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Cellular compaction is a basic morphogenetic process repeated throughout the development of the multicellular organism. In the developing limb, mesenchymal cell condensation is a critical event in the initiation of chondrogenesis and subsequent cartilage formation. Our understanding of limb development has accelerated in the past several years with the identification of signaling molecules necessary for growth and patterning the limb (Hall and Miyake,2000; Tickle and Munsterberg,2001). For example, bone morphogenetic protein-2 (BMP-2) and BMP-4 regulate the size of the mesenchymal cell condensation by recruiting precursor cells. In contrast, the molecular mechanisms underlying the condensation process itself remain largely unknown.

The organization of embryonic cells into limb structures is established through a series of morphogenetic events, involving cell sorting, cell migration, and cell aggregation, processes for which cell–cell and cell–matrix interactions are thought to be important. One of the cell adhesion molecules implicated in this process is N-cadherin, a member of the classic cadherin family, which mediates calcium-dependent cell–cell adhesion (Tepass et al.,2000). Classic cadherins are single-pass transmembrane proteins that interact intracellularly with a group of proteins called catenins, α-catenin, β-catenin, plakoglobin, and p120ctn (Gumbiner,2000). Formation of the cadherin–catenin complex is required for cadherin-mediated cell adhesion, and α-catenin, which binds either cadherin–β-catenin or cadherin–plakoglobin complexes mediates linkage to the actin cytoskeleton. The strength of adhesion is also modulated by p120ctn binding to the juxtamembrane region of the cadherin cytoplasmic domain (Anastasiadis and Reynolds,2000). In addition to providing structural information, N-cadherin may provide signals that influence the differentiation of cells toward the bone lineage. In studies using E-cadherin–deficient embryonic stem (ES) cells, it was demonstrated that transfection of N-cadherin directed their differentiation to cartilage and neuroepithelium when the ES cells form teratomas in mice (Larue et al.,1996). Their adhesive specificity and cellular distribution during embryogenesis suggest an important role for cadherins in morphogenesis and maintenance of the tissue phenotype.

The expression pattern of N-cadherin in the developing limb suggests an important role in mesenchymal cell condensation a prerequisite for chondrogenesis. N-Cadherin displays a diffuse expression pattern in the central core region in the precartilaginous stage followed by a dramatic increase as condensation begins and diminishes as chondrogenic differentiation initiates (Oberlender and Tuan,1994a,b). Mature cartilage does not express N-cadherin, whereas the cells in the condensing perichondrium surrounding the forming cartilage still exhibit high levels of N-cadherin. A similar expression pattern of N-cadherin is observed in high density micromass cultures of limb mesenchyme in vitro (Oberlender and Tuan,1994b; Tavella et al.,1994). Much of our knowledge about how N-cadherin might function during limb development comes from studies using antibodies that block specifically the function of N-cadherin. Introduction of antibody into limb buds of chick embryos during the period of active cellular condensation resulted in significant developmental delays and gross deformities (Oberlender and Tuan,1994a). In similar experiments using micromass cultures derived from limb mesenchymal cells, blocking N-cadherin function resulted in inhibition of chondrogenesis (Oberlender and Tuan,1994a,b). In these investigations, blocking N-cadherin function specifically during the compaction stage had the greatest effect on chondrogenesis. Similar findings were observed in high-density cultures of murine embryonic mesenchymal cells induced to undergo chondrogenesis with BMP-2 (Haas and Tuan,1999). In another study, recombinant expression plasmids encoding wild-type or mutant forms of N-cadherin were introduced into limb mesenchymal cell cultures (Delise and Tuan,2002). Of interest, the overexpression of wild-type N-cadherin enhanced cellular condensation and subsequent chondrogenesis. In contrast the mutant N-cadherins inhibited condensation and chondrogenesis. Other studies have suggested that N-cadherin regulation during the condensation process is controlled by various signaling molecules, including transforming growth factor-β and Wnts (Tufan et al.,2002; Tuli et al.,2003).

The progress zone (PZ) represents the undifferentiated proliferative compartment of the distal limb bud underlying the apical ectodermal ridge. Mesenchymal cells derived from the PZ from different stages of chick limb buds sort out in monolayer culture, suggesting differential cell adhesive properties dependent on their positions along the proximodistal axis. Differences in N-cadherin protein levels are thought to be involved in this cell sorting process (Yajima et al.,1999,2002). N-Cadherin function blocking antibodies were able to inhibit the sorting out of different stage PZ cells in culture suggesting that differences in N-cadherin may provide positional identity along the proximodistal axis. In addition, the accumulation of N-cadherin was inhibited by retinoic acid treatment of distal mesenchyme cells, consistent with changes in positional identity. In summary, over the past 10 years, numerous in vitro and in vivo studies provided evidence that N-cadherin plays an active role in limb morphogenesis.

In this study, we characterized a novel N-cadherin gene trap allele and observed high levels of lacZ expression in precartilaginous tissue in the mouse embryo, which led us to further investigate N-cadherin's role in limb development. Previous studies from our laboratory have shown that limb buds can form in N-cadherin–null embryos expressing a cardiac-specific cadherin transgene; however, these partially rescued embryos do not survive long enough to observe limb development (Luo et al.,2001). To overcome the embryonic lethality, we used an organ culture system to examine limb development ex vivo. We demonstrate that N-cadherin–deficient limb buds were capable of mesenchymal condensation and chondrogenesis resulting in skeletal structures, indicating that N-cadherin is not essential for this process.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Characterization of a New N-Cadherin–Null Allele

Insertional mutagenesis has proved very useful in various model organisms, including the mouse to identify important developmentally regulated genes. This so-called gene trap methodology allows for the insertion of a reporter gene such as lacZ under the transcriptional regulation of the mutated gene (Friedrich and Soriano,1991). By using ES cell technology (Zambrowicz et al.,1998), mice were obtained with a random retroviral insertion into the 34-kb intron one of the N-cadherin gene (Miyatani et al.,1992). To identify the insertion site of the retroviral vector oligonucleotide primer pairs were used to amplify various regions of intron 1 from homozygous and wild-type genomic DNA. After determining the relative position of the insertion site (Fig. 1A), a genomic probe was used to confirm the disruption of the N-cadherin gene by Southern blot analysis (Fig. 1B). After sequencing a flanking junction, it was determined that the retroviral vector inserted 427 bp downstream of exon 1 into the N-cadherin gene. To confirm that the gene trap allele was a null allele, embryonic day (E) 9.5 embryos were obtained from intercross mating and protein lysates were examined by Western blot analysis. No N-cadherin protein was observed in the homozygous embryo, indicating that the gene trap disrupted expression of the N-cadherin gene (Fig. 1C). Consistent with coordinate regulation of the cadherin/catenin complex, the cytoplasmic binding partner, β-catenin, was significantly reduced in the N-cadherin–deficient embryo (Fig. 1C). Furthermore, the gene trap allele did not complement our original N-cadherin–null allele (Radice et al.,1997), demonstrating that the mutations were allelic (data not shown).

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Figure 1. Characterization of an N-cadherin gene trap allele. A: Schematic representation of the insertion site of the gene trap construct. The retroviral gene trap construct contained flanking long terminal repeats (LTR), a splice acceptor sequence (SA), and βgeo fusion gene. A flanking probe used to characterize the insertion site is shown (probe). Restriction sites are abbreviated as follows: E, EcoRI; H, HindIII; P, PstI. The arrows represent the oligonucleotide primers used for polymerase chain reaction genotyping. Exons are represented by closed boxes. B: Southern blot analysis of wild-type (+/+) and heterozygous (−/+) N-cadherin gene trap mice. C: Western blot analysis of wild-type (+/+) and homozygous (−/−) embryonic day 9.5 embryo lysates using N-cadherin and β-catenin antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal shows loading of samples between lanes.

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β-Galactosidase Staining Pattern in N-Cadherin Gene Trap Embryos

To examine the expression of the gene trap allele, embryos were stained for β-galactosidase activity at different gestational ages. At E8.5, a ventral view illustrates the strong expression in the heart, neural tube, and somites of a homozygous embryo (Fig. 2A). At this stage, the mutant embryo appeared morphologically normal, in comparison to its wild-type littermate (not shown). By E9.5, the mutant embryo exhibited the identical phenotype of the previously described N-cadherin–null allele (Radice et al.,1997), with the characteristic cardiovascular defect and the lack of brain development. In the heterozygous gene trap embryo, strong lacZ expression was observed in the heart, somites, optic vesicle, and otic placode. In addition to the heart and brain, at E14.5, strong expression was observed in the developing limbs, vertebrae, and mandible (Fig. 2C).

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Figure 2. Whole-mount analysis of β-galactosidase–stained N-cadherin gene trap embryos. A–C: Embryos were recovered at embryonic day (E) 8.5 (A), E9.5 (B), and E14.5 (C). C: The larger E14.5 embryo was dissected in half sagittally to allow penetration of the staining solution and to show the exterior (left) and interior (right) staining patterns. Note the nonstained region of the midbrain in the E14.5 embryo is due to tissue damage during manipulation. h, heart; m, mandible; nf, neural folds; op, optic vesicle; ot, otic placode; s, somites; ve, vertebrae.

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Histologic analysis of a heterozygous E9.5 embryo demonstrated strong staining in the myocardium, neuroepithelium, and paraxial mesoderm (Fig. 3A,B). In the mutant E9.5 embryo, the malformed heart tube consisted of dissociated myocytes surrounding the endocardium (Fig. 3C,D). Of interest, the endocardium, which was thought to be negative for N-cadherin based on immunohistochemical analysis (Luo et al.,2001), was positive for lacZ expression, albeit less intensely stained compared with myocardium. Upon closer examination, weak N-cadherin staining was observed in the endocardium by immunofluorescence using frozen sections (data not shown). In the adult, strong expression of the lacZ gene was confirmed in the differentiated myocardium (data not shown). We conclude that the lacZ expression pattern recapitulates that of the mouse N-cadherin gene as previously reported (Packer et al.,1997; Radice et al.,1997).

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Figure 3. Histological analysis of whole-mount β-galactosidase–stained N-cadherin gene trap embryos. A–D: Transverse sections through the thoracic region of heterozygous (A,B) and homozygous (C,D) embryonic day 9.5 embryos show the expression pattern of the lacZ reporter gene. D: Note the poorly developed N-cadherin–deficient embryo with rounded dissociated myocytes (my) surrounding the endocardium. h, heart; nt, neural tube. Scale bars = 200 μm in A,C, 50 μm in B,D.

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N-Cadherin Gene Trap Expression in the Developing Limb

Previous studies by others suggested an important role for N-cadherin in condensation of mesenchymal cells during chick limb development. We used the gene trap allele to follow the spatiotemporal pattern of N-cadherin expression during forelimb bud development in heterozygous embryos. At E10.5 days, β-galactosidase staining was diffuse throughout the forelimb bud, with reduced staining in proximal regions corresponding approximately to the anterior and posterior necrotic zones (Fig. 4A,B). Two days later (E12.5), lacZ expression became more restricted with loss of staining in the distal and condensed digit-forming regions (Fig. 4B,E). By E14.5, expression was reduced in the maturing digits, with the central core maintaining high expression, including an outline of prechondrogenic condensations (Fig. 4C,F). The expression pattern of the gene trap allele coincided with changes in mesenchymal cell differentiation with highest expression in the less-differentiated prechondrogenic mesenchymal cells.

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Figure 4. N-Cadherin gene trap expression during limb development. A–C: Dorsal views of β-galactosidase–stained NcadlacZ/+ forelimbs at embryonic day (E) 10.5 (A), E12.5 (B), and E14.5 (C). A: Gene trap expression is reduced in the anterior and posterior necrotic zones (arrowheads). B,C: Note the increased staining in regions of prechondrogenic mesenchymal cell condensations. D–F: Longitudinal sections through the distal limb show strong mesenchymal staining at E10.5 (D) that is down-regulated in the developing digits (E,F) with expression remaining in the interdigit regions (asterisks in E,F) and apical ectodermal ridge (arrowhead in E). Scale bars = 400 μm in A–C, 25 μm in D–F.

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Examination of Limb Development Ex Vivo

Given the dynamic expression pattern of N-cadherin during forelimb development specifically, the increased expression during mesenchymal cell compaction, a prerequisite for chondrogenesis, we used a limb bud organ culture system to directly examine N-cadherin's role in this morphological process. In previous studies, we demonstrated that cardiac-specific cadherin expression was sufficient to rescue the N-cadherin null embryos, allowing further development until the limb bud stage; however, these partially rescued embryos did not survive long enough to observe limb development (Luo et al.,2001). To overcome the embryonic lethality, forelimb buds were cultured on Organotypic Millicells (Millipore) with the tissue placed in a liquid–air interface of culture medium, allowing for further development ex vivo (Fig. 5).

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Figure 5. Outline of experimental protocol. N-Cadherin heterozygotes containing the cardiac-specific transgene αMHC/hE-cadherin were backcrossed to heterozygotes to obtain cardiac-restricted rescued embryos (hE-cad, −/−). Forelimb buds were isolated from wild-type and rescued N-cadherin–null embryonic day 10.5 embryos and cultured on Organotypic Millicells (Millipore). The tissue is placed on the membrane surrounded by culture medium, and nutrients pass freely from the bottom chamber to the tissue specimen above.

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The N-cadherin–deficient forelimb buds dissected from E10.5 rescued embryos appeared similar in size compared to wild-type (Fig. 6A,D). After 7 days in culture, the overall growth and morphology of the limb structures appeared similar between N-cadherin–null (n = 5) and wild-type (n = 6) tissues (Fig. 6B,E). After cellular compaction, the cells differentiate and chondrogenesis occurs, initially forming cartilaginous matrix. The extent of chondrogenic differentiation appeared similar in the N-cadherin–null and wild-type limb structures as depicted by Alcian blue staining (Fig. 6C,F).

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Figure 6. Limb bud development in culture. A,B,D,E: Limb buds were isolated from wild-type (A) and N-cadherin rescued (D) embryonic day 10.5 embryos and cultured for 7 days (B,E). C,F: The resulting skeletal structures were stained with Alcian blue. Scale bars = 800 μm in A–F.

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Histologic analysis of the limb structures showed normal cellular arrangement and morphology in the N-cadherin–deficient tissue compared with wild-type (Fig. 7). The compacted perichondrium appeared similar in mutant and wild-type tissue. In addition, the morphology of the differentiated chondrocytes also appeared normal.

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Figure 7. Histological analysis of limb structures after 7 days in culture. A–D: Sagittal sections of limb structures derived from wild-type (A,B) and N-cadherin–deficient (C,D) limb buds. Scale bars = 400 μm in A,C, 50 μm in B,D.

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Expression of the Cadherin/Catenin Complex in Limb Structures

Cadherin/catenin expression was examined in the limb structures after 7 days in culture. In wild-type tissue, N-cadherin was localized to cell–cell contacts in the condensed perichondrium and absent from differentiated chondrocytes (Fig. 8A). As expected, no N-cadherin expression was observed in the N-cadherin–deficient tissue (Fig. 8E). Beta-catenin was expressed at highest levels in the condensed perichondrium with lower levels found in chondrocytes in both N-cadherin–null and wild-type tissue (Fig. 8B,F). The expression of β-catenin in the mutant perichondrium is consistent with the presence of other cadherin family members. Therefore, cadherin-11 expression was examined in the limb structures (Fig. 8C,G). The expression pattern of cadherin-11 was similar to N-cadherin with high levels in the perichondrium and absent in the differentiated chondrocytes in both mutant and wild-type limb structures. Immunohistochemical detection of collagen type II, a major component of the cartilage extracellular matrix, demonstrated similar expression in the N-cadherin–deficient limb structure compared with wild-type (Fig. 8D,H).

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Figure 8. Expression of the cadherin/catenin complex after 7 days in culture. A–C,E–G: Sections of limb structures after 7 days in culture were analyzed by indirect immunofluorescence for expression of N-cadherin (A,E), β-catenin (B,F), and cadherin-11 (C,G). D,H: Chondrogenic differentiation was confirmed by immunostaining for collagen type II. wt, wild-type. Scale bars = 50 μm in A–H.

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Cellular condensation plays a key role in the chondrogenic process. To determine whether cadherin-11 may contribute to this process in the mutant limb, we examined cadherin-11 expression 48 hr after placing the limb buds in culture. At this early time point, areas of active cellular compaction were observed in the explant. Adjacent sections were immunostained for N-cadherin and cadherin-11. N-Cadherin was absent from the mutant tissue as expected (Fig. 9A), whereas cadherin-11 was strongly expressed at cell–cell contacts in the condensing mesenchyme (Fig. 9B), similar to that of wild-type (data not shown). These data suggest that cadherin-11 may functionally substitute for N-cadherin during the compaction process. To confirm that the limb structure derived from the rescued N-cadherin–null embryo did not express the human E-cadherin transgene, the tissue was immunostained with a species-specific E-cadherin antibody. As expected, based on the cardiac-specificity of the alpha–myosin heavy chain (αMHC) promoter, no human E-cadherin was detected in the N-cadherin–deficient limb structure (Fig. 9C).

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Figure 9. Expression of cadherin-11 in condensing mesenchyme from an N-cadherin–deficient limb. A–C: After 48 hr in culture, adjacent limb sections were analyzed by indirect immunofluorescence for expression of N-cadherin (A), cadherin-11 (B), and human E-cadherin (E-cad, C). D: Transgene expression in the embryonic heart is shown as a positive control. Scale bars = 25 μm in A–D.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In this study, we characterized a new N-cadherin allele containing a lacZ reporter inserted into intron one of the N-cadherin gene. This new N-cadherin allele proved to be a null allele resulting in embryonic lethality similar to the previously described N-cadherin null allele (Radice et al.,1997). The mutant embryos exhibited the characteristic cardiovascular defect at E9 with a malformed heart tube accompanied by severe pericardial swelling. As previously reported, the N-cadherin–deficient myocytes lost their elongated morphology and instead were rounded and loosely arranged around the endocardium. The NcadlacZ allele recapitulates the expression pattern of the endogenous N-cadherin gene, representing a useful tool to follow N-cadherin expression under various experimental conditions. Based on high-level lacZ expression in prechondrogenic condensations compared with other embryonic tissues, we focused our attention on the expression pattern of the NcadlacZ allele during limb development.

Based on in vivo and in vitro studies using primarily the chick limb system, N-cadherin is predicted to play a major if not essential role in mesenchymal cell compaction in the developing limb. Function blocking antibodies injected into the chick limb bud resulted in developmental delays and deformities (Oberlender and Tuan,1994a). In vitro analysis using the same antibodies (Oberlender and Tuan,1994b) or dominant-negative cadherin molecules (Delise and Tuan,2002) indicated that N-cadherin plays an important role in mesenchymal cell condensation and subsequent chondrogenesis. Furthermore, gain-of-function studies indicated that N-cadherin enhances mesenchymal cell condensation and chondrogenesis. In contrast, our present genetic study demonstrates that N-cadherin is not essential for mesenchymal cell condensation and chondrogenesis. These different findings may be explained by differences in the experimental systems. In the previous studies, the mesenchymal cells are presumably actively using N-cadherin as their primary adhesion receptor. Hence, perturbation by either antibody or dominant-negative constructs directly affects the cell's ability to use N-cadherin to promote cell–cell interactions. That cellular condensation occurs in the absence of N-cadherin suggests that another adhesion receptor(s) can functionally substitute for N-cadherin. Compensation is more readily observed in genetic systems, because the organism has never seen N-cadherin and has an opportunity to respond by using an alternative adhesive mechanism. In contrast, perturbing function of a protein being actively used by a cell is more likely to generate a dramatic phenotype as the organism will not have the opportunity to adjust and use another mechanism. We might predict that a conditional genetic knockout of N-cadherin would more closely resemble the limb phenotype observed in these other experimental systems.

We expect that a different cell adhesive system(s) was compensating for loss of N-cadherin in our limb bud cultures. The presence of β-catenin at the cell membrane in the N-cadherin–deficient limbs indicates that other classic cadherins are present. We further demonstrated that the type II cadherin cadherin-11 is expressed at high levels in the mutant limb structures, suggesting it may functionally substitute for N-cadherin. Of interest, loss of both N-cadherin and cadherin-11 resulted in a more severe disruption of epithelial somite structure compared with loss of N-cadherin alone, indicating that these two cadherins likely cooperate to maintain tissue integrity (Horikawa et al.,1999). PB-cadherin is also expressed in the developing limb bud (Kitajima et al.,1999); hence, multiple cadherins may cooperate to facilitate mesenchymal condensation during limb morphogenesis. In addition, the Ig superfamily member neural cell adhesion molecule (NCAM) has also been implicated in limb mesenchymal cell condensation (Widelitz et al.,1993) and, hence, may have overlapping function with N-cadherin. However, it should be noted that animals lacking either cadherin-11 (Manabe et al.,2000) or NCAM (Cremer et al.,1997) are viable with normal limbs, indicating that neither of these cell adhesion molecules by themselves is required for limb morphogenesis.

The transcription factor Sox9, containing a high-mobility group DNA-binding domain, is implicated in commitment of mesenchymal cells to the chondrogenic lineage and to subsequent differentiation. It was demonstrated recently that Sox9 can enhance N-cadherin promoter activity in a chondrocytic cell line and can bind to a consensus Sox9-binding motif found in the N-cadherin promoter (Panda et al.,2001). Of interest, the loss of Sox9 leads to the inability of limb mesenchymal cells to compact in the knockout model (Akiyama et al.,2002). However, it was found that N-cadherin and NCAM levels were normal in the Sox9-deficient limbs at E12.5, suggesting that Sox9 perturbs cellular compaction by disrupting another aspect of the cellular adhesion machinery.

In summary, we describe a new N-cadherin mutant allele containing a lacZ reporter that mimics the expression pattern of the endogenous N-cadherin gene. Furthermore, we provide genetic evidence that N-cadherin is not essential for cellular compaction of limb mesenchymal cells and subsequent chondrogenesis. In future studies, it will be interesting to address the possible overlapping functions of N-cadherin and cadherin-11 in mediating mesenchymal cell compaction during limb formation.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Generation of N-Cadherin Gene Trap Mice

NcadlacZ mice were generated from ES cells corresponding to OST 49160 (Omnibank Sequence Tag; Lexicon Genetics, Houston, TX) targeted by gene trapping. The gene trap vector contained a 5′ retroviral long terminal repeat (LTR), a splice acceptor, βgeo (fusion of β-galactosidase and neomycin phosphotransferase genes), a Bruton tyrosine kinase gene, and a 3′ LTR. Retroviral infection, selection, and screening of ES cells were carried out as described previously (Zambrowicz et al.,1997,1998). ES cells corresponding to OST 49160, containing the first exon of the N-cadherin gene upstream of βgeo, as detected by 5′ rapid amplification of cDNA ends analysis, were selected for blastocyst injection into C57BL/6 mice to produce chimeric mice. The chimeric mice transmitted the gene trap allele to their progeny. The location of the insertion site was confirmed by Southern blot analysis using a genomic probe from intron one of the N-cadherin gene. NcadlacZ mice were genotyped by polymerase chain reaction (PCR) using the following primer set: LTR2, 5′ AAA TGG CGT TAC TTA AGC TAG CTT GC 3′; NC23, 5′ GTA TGG CCA AGT AAT GGG GAC 3′. The resulting PCR product was approximately 350 bp.

X-Gal Staining

Embryos and limb buds were fixed in freshly prepared fixative (0.1 M phosphate buffer, pH 7.3, 0.2% glutaraldehyde, 5 mM ethyleneglycoltetraacetic acid, 2 mM MgCl2) for 30 min at room temperature. Fixed tissues were rinsed 3× in detergent rinse (0.1 M phosphate buffer, pH 7.3, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40) at room temperature for 30 min. Tissues were stained in X-gal staining solution (0.1 M phosphate buffer, pH 7.3, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, X-gal 1mg/ml, 20 mM Tris-Cl2, pH 7.3) at 37°C for 3 hr in the dark. After staining, tissues were washed with phosphate buffered saline (PBS) 3× for 15 min and photographed immediately.

Cardiac-Specific N-Cadherin Rescue Embryos

The generation and genotyping of the N-cadherin knockout (Radice et al.,1997) and αMHC/E-cadherin transgenic mice (Luo et al.,2001) were described previously. Yolk sac DNA was obtained from the embryos and subjected to PCR analysis by using primers specific for the N-cadherin mutation and transgene. Heterozygous N-cadherin mice with or without the αMHC/E-cadherin transgene were interbred to generate litters of embryos for limb bud culture.

Limb Bud Culture

Forelimb buds were excised from E10.5 mouse embryos in PBS, washed in culture medium, and placed on the membrane of Millicell culture plate inserts (OICM ORG 50, Millipore) sitting in six-well dish filled with 2.5 ml of culture medium by using a cut Pasteur pipette, which contains a drop of original culture medium (DMEM+20% fetal bovine serum). Limb buds were cultured in 5% CO2 incubator for 2 or 7 days. After 48-hr culture, medium was changed daily.

Alcian Blue Staining of Cultured Limbs

Limbs were stained as previously described (Gamer et al.,2001). Briefly, limbs were fixed in freshly prepared 4% paraformaldehyde overnight. Limbs were rinsed 4× for 20 min and stained in Alcian blue solution containing 0.02% Alcian blue 8GX (Sigma) in 70% ethanol, 30% glacial acetic acid at room temperature for 5 hr. Limbs were washed in 70% ethanol and distilled water 1 hr each. Limbs were processed through 0.5% potassium hydroxide:glycerol series (3:1, 1:1, 1:3) for 2 hr each. Limbs were stored in 100% glycerol and photographed.

Immunofluorescence

Indirect immunofluorescence was performed on paraffin sections of limb explants as previously described (Luo et al.,2001). The sections were incubated with the following primary antibodies: N-cadherin (3B9), β-catenin (5H10), cadherin-11 (5B2H5), E-cadherin (HECD-1; Zymed, So. San Francisco, CA); and collagen type II (II-II6B3, Developmental Studies Hybridoma Bank, Iowa City, IA). The secondary antibody was Cy3-conjugated goat anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Drs. Christer Betsholtz, Roberto Civitelli, and Henrik Semb for their support of the N-cadherin gene trap project; Yanming Xiong, Melanie Lieberman, and Ericka Anderson for technical assistance; and the University of Pennsylvania Biomedical Imaging Core facility for use of the confocal microscope. The II-II6B3 monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank. Y.L. was supported in part by a postdoctoral fellowship from the PA/DE Affiliate of the AHA. G.R. was funded by the NIH.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES