Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, UT 84112-5331, USA
* Present address: Department of Molecular and Medical Genetics, Oregon Health Sciences University and Shriners Hospital for Children, Portland, OR 97201, USA
Author for correspondence (e-mail: mario.capecchi{at}genetics.utah.edu)
Accepted July 23, 2001
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SUMMARY |
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Key words: Hox genes, Limb development, Ephrin receptors, Mesenchymal condensations, Mouse
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INTRODUCTION |
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MATERIALS AND METHODS |
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Generation of targeted ES cell lines and germline transmitting mice
The targeting vector was electroporated into R1 embryonic stem cells (Nagy et al., 1993) and the cells subjected to positive and negative selection (Mansour et al., 1988). Genomic DNA from 144 colonies was digested with SphI and analyzed by Southern transfer analysis to identify two homologous recombinants. Cells from both recombinant clones were microinjected into C57BL/6 blastocysts to generate chimeric mice. Chimeric males were crossed to C57BL/6 females and DNA from Agouti progeny was tested by Southern blotting and PCR analysis to confirm germline transmission of the mutant Hoxa13GFPneo allele (Fig. 1C). Embryos from heterozygous intercrosses were genotyped by PCR using yolk-sac DNA. PCR conditions consisted of 35 cycles of 94°C for 15 seconds, 62°C for 15 seconds and 72°C for 35 seconds. Mutant (378 bp) and wild-type (177 bp) amplification products (Fig. 1D) were produced using the Hoxa13 forward primer (GTCGTCTCCCATCCTTCAGAC), the GFP reverse primer (GCACTGCACGCCGTAGGTCA) and the Hoxa13 reverse primer (TGTTCTGGAACCAGATTGTGAC; Fig. 1B). The neomycin resistance cassette was removed by crossing Hoxa13GFP heterozygous females to Cre deleter mice (Schwenk et al., 1995).
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Micromass cultures
Staged limb buds (Wanek et al., 1989) from mutant and heterozygous embryos at gestational age E11.5 were collected in 4°C Ca2+- and Mg2+-free phosphate-buffered saline (PBS; Gibco/BRL). Enrichment for Hoxa13GFP expressing cells was achieved either by microdissection of the fluorescent regions of the limb bud using fine tungsten needles and a fluorescence equipped Leica MZ12 stereoscope or by cell sorting using a Becton Dickinson FACS ADVANTAGE cell sorter. The two enrichment methods produced similar results. Dissected tissues were dissociated at 37°C for 10 minutes in Ca2+- and Mg2+-free PBS (Gibco/BRL) containing 0.1% trypsin and 0.1% collagenase as described (Owens and Solursh, 1982), followed by pipetting three to five times through a sterile 70 µm cell basket (Costar). The cell suspension was washed twice using 5 ml of serum free Dulbeccos MEM media (Gibco/BRL) and counted on a hemocytometer. The cell suspensions were diluted to a final concentration of 2x107 cells/ml with Dulbeccos MEM media containing 15% FBS supplemented with non-essential amino acids, 50 U/ml penicillin and 25 ug/ml streptomycin. Cell suspensions were inoculated onto 60 mm Falcon tissue culture dishes as described (Ahrens et al., 1977) and placed in a 37°C incubator with a 6% CO2 atmosphere for 1 hour to allow for cell attachment. After 1 hour the plates were flooded with 8 ml of media which was changed daily. Mesenchyme aggregations were recorded 19 hours after plate inoculations, whereas cartilaginous nodules were analyzed 3-4 days later.
Antibody blocking assay
Hoxa13GFP-expressing tissues from the autopods of E12.5 embryos were dissected and dissociated with 0.05% trypsin-EDTA and 0.05% collagenase followed by passage through 70 µm Netwell filters (Costar) to generate single-cell suspensions. The single cell suspensions were plated at a density of 2x107 cells/ml in 20 µl aliquots in 60 mm tissue culture dishes. A second 20 µl aliquot containing either 4 µg of a rabbit EphA7 polyclonal antibody dialyzed for 4 hours in 1x PBS (Santa Cruz Biotechnology Cat. No. SC-917), pre-immune rabbit serum, or DMEM media was immediately added to the micromass cells which were then incubated at 37°C for 1 hour. After incubation, 7 ml of DMEM media supplemented with 10% fetal calf serum and 50 U/ml penicillin, and 25 µg/ml streptomycin was added to each dish. A second aliquot of the EphA7 antibody, or rabbit serum was added to the respective dishes in conjunction with the 7 ml of media. The cells were grown in a standard 37°C incubator at 5% CO2. Micromass cultures were photographed 48 hours after plating under phase contrast using a Leica DMIL microscope fitted with a Nikon Coolpix 990 digital camera.
Cell sorting assay
Proximal limb bud tissues (400-500 µm from the AER) from stage matched E11.5 Swiss Webster embryos were dissected and processed for micromass culture as described above. At the same time, the fluorescent regions of stage matched E11.5 limb buds from Hoxa13GFP mutant and heterozygous embryos were dissected and processed for micromass culture. Stock suspensions of wild-type Swiss Webster cells, Hoxa13GFP mutant cells and heterozygous cells were adjusted to equivalent concentrations of 4x107 cells/ml. From these stock suspensions, micromass plates were inoculated with equal concentrations of wild-type and either mutant or heterozygous cells as described (Ahrens et al., 1977; Owens and Solursh, 1982; Ide et al., 1994). Nineteen hours after inoculation, the attached cells were stained with TO-PRO-3 iodide (Molecular Probes) and analyzed for proximal-distal sorting of wild-type and Hoxa13GFP-expressing cells using the previously described BioRad Confocal Imaging System.
Immunohistochemistry
E11.5 limbs were fixed at room temperature in Carnoys fixative for 2 hours followed by rinses in 1x PBS containing 0.5% Triton X-100 (PBX) for an additional 2 hours. Tissues were then placed in a PBX blocking solution containing 4% skim milk and 2% donkey serum, and rocked slowly at 4°C for 4 hours. NCAM (5B8) and collagen type II (II-II6B3) mouse antibodies (Developmental Hybridoma Bank, University of Iowa, Iowa City, IA) were diluted in PBX containing 2% skim milk and 2% donkey serum, and incubated with the limb tissue overnight at 4°C. The primary antibody was removed and the limbs were washed for 3 hours in PBX at room temperature. After washing, the limbs were incubated overnight at 4°C with a donkey anti-mouse secondary antibody conjugated with either Texas Red or Cy5 (Jackson Immunological). Confocal analysis was performed as described above.
For micromass cultures, the media was removed and the anchored cells were washed briefly in 1x PBS followed by fixation in 4% formaldehyde for 10 minutes. After fixing, the cells were again rinsed in 1x PBS for 20 minutes, followed by permeabilization and blocking in PBX containing 2% skim milk and 2% donkey serum for 1 hour. After blocking, primary antibodies, including EphA2, EphA4 and EphA7 (Santa Cruz Biotechnology), ephrin A1, ephrin A2 and ephrin A3 (Santa Cruz Biotechnology), N-Cadherin (Transduction Labs), ß-catenin (Transduction Labs), Paxillin (Transduction Labs), E-Cadherin (Transduction Labs), antiphosphohistone H3 (Upstate Biotechnology), and D1C4 (a kind gift from Anthony Capehart), were incubated with the micromass cultures in blocking solution overnight at 4°C. The next day, the primary antibody was removed, and the micromass cultures were washed for 3 hours at room temperature in 1x PBS, followed by an overnight incubation at 4°C in blocking solution with a species specific Cy5-conjugated donkey secondary antibody.
For cryosections, embryos were fixed for 2 hours in 4% paraformaldehyde and briefly rinsed in 1x PBS for 10 minutes. Cryoprotection of the embryos was achieved using a sequential series of 10, 20 and 30% sucrose/PBS solutions. The embryos were oriented in OCT (Tissue Tek) filled molds and rapidly frozen. The embryos were sectioned on a Zeiss Cryostat at a thickness of 20-30 µm. Sections were mounted on Superfrost plus slides (Fisher) for microscopic and immunohistochemical analysis.
RNA in situ hybridization
Plasmids containing the EphA7 (Mdk1) and Msx1 genes generously provided by Drs T. Mori and Y. Chen, respectively, were used to produce antisense riboprobes. Embryo preparation, hybridization and analysis were performed as previously described (Manley and Capecchi, 1995). BM-Purple (BMB-Roche) was used for the alkaline phosphatase color reactions, which were extended for 17 hours at room temperature for the EphA7 riboprobe.
TUNEL analysis of apoptosis
Terminal UTP nick end labeling (TUNEL) of DNA was performed as a modification of the technique described by Maden et al. (Maden et al., 1997). Limbs from E11.5-13.5 embryos were fixed at room temperature for 2 hours in 4% paraformaldehyde. After fixing, the limbs were washed for 3 hours at room temperature with several changes of 1x PBS containing 1% TritonX-100. The limbs were placed in 1.5 ml microfuge tubes and pre-incubated at 37°C for 30 minutes in PBS containing 1x terminal transferase buffer (Boehringer Mannheim Biochemical), 1% Triton X-100 and 2.5 mM CoCl2.. The preincubation buffer was replaced with a 1x PBS solution containing 1x terminal transferase buffer, 10 µM dUTP (2:1 dUTP:dUTP-biotin), 2.5 mM CoCl2, 1% Triton X-100 and 0.5 U terminal transferase/µl buffer, and incubated at 37°C for 3 hours. The limbs were then washed for 3 hours in PBS and incubated overnight at 4°C with streptavidin conjugated with Texas Red (Jackson Immunological). After washing for 1 hour in PBS, confocal analysis of the limbs was performed as described above.
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RESULTS |
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Hoxa13GFP expression was characterized in E10.5-E13.5 embryos to confirm accurate recapitulation of the published Hoxa13 mRNA expression pattern (Haack and Gruss, 1993; Warot et al., 1997). As early as E10.5, strong expression can be seen in the distal forelimb mesenchyme, but not in the overlying apical ectodermal ridge (AER) (Fig. 2A). By E 11.5, Hoxa13GFP expression is readily detected in the progress zone (Fig. 2B) of the elongating limb bud. By E13.5 limb expression of Hoxa13GFP appears to be restricted to the condensing digit mesenchyme, at the sites where many of the chondrogenic defects occur in Hoxa13 homozygous mutant mice (Fig. 2C,D) (Fromental-Ramain et al., 1996). Finally, the GFP-tagged allele of Hoxa13 identifies the population of mesenchymal cells expressing Hoxa13 in the developing umbilical arteries (Fig. 2E,G,H) and genitourinary regions, including the genital ridge and tissues surrounding the urogenital sinus (Fig. 2F). Our Hoxa13GFP-null homozygotes die in utero between E11.5 and E15.5, exhibiting limb and umbilical vascular defects similar to those in mice homozygous for the Hoxa13 mutant alleles as described previously (Fromenthal-Ramain et al., 1996; Warot et al., 1997). Examination of umbilical arteries from homozygous mutants (Fig. 2H) revealed nearly a complete loss of mesenchymal/endothelial cell layer stratification relative to heterozygous littermate controls (Fig. 2G).
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Although care was taken to plate identical numbers of Hoxa13GFP heterozygous and homozygous mutant cells, it is apparent that after 12 hours in culture, fewer mutant homozygous cells adhere to the culture dish and initiate aggregation, compared with Hoxa13GPF heterozygous cells (Fig. 3A,B). Eighteen to 24 hours after plating, the Hoxa13 heterozygous mesenchymal cells aggregate into multiple dense precartilaginous condensations (Fig. 3C). Seventy-two hours later, these aggregates differentiate into cartilaginous nodules (Fig. 3I) that stain positively with Alcian Blue (Fig. 3I, inset) and express collagen type II (Fig. 3J). With further in vitro culture, the Hoxa13-expressing cells become restricted to the periphery of the condensate, forming digit-like structures (Fig. 3K). This restriction in Hoxa13GFP expression to the periphery of the digit-like elements in vitro is noteworthy, because during the later stages of normal chondrogenesis in the developing limb bud, 5'-Hox gene expression becomes restricted to the perichondrium, a layer of cells that surrounds each condensate and controls its pattern of growth (Davis and Capecchi, 1994). The same sequence of differentiation is observed with unsorted mesenchymal cells isolated from wild-type embryonic limbs. However, approximately 24 hours of additional culture time is needed with the wild-type, unsorted mesenchymal cells isolated from limb buds to reach the equivalent state of differentiation. The precocious behavior of the Hoxa13GFP heterozygous cells relative to wild-type cells may be due to the former cells being a more purified and distinct (i.e. FACS-purified and Hoxa13-expressing) population of mesenchymal cells.
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To determine whether Hoxa13 homozygous mutant cells can participate in the production of condensates in the presence of wild-type cells, equal numbers of dissociated wild-type cells (GFP negative) and homozygous mutant cells (GFP positive) were combined and plated onto culture dishes. The combined cells produced large mesenchymal condensates containing fluorescent (/) and nonfluorescent (+/+) cells (Fig. 3E,F). Examination of these mixed aggregates revealed that the mutant (/) cells contributed to the condensing mesenchyme by attaching to anchored wild-type cells (Fig. 3G,H). Both mutant and wild-type cells within these aggregates subsequently express collagen II. Thus, in the presence of wild-type cells, Hoxa13/ cells are capable of forming aggregates and differentiating along the chondrogenic pathway. This suggests that a major deficit of Hoxa13/ mesenchymal cells is in their inability to self-aggregate and adhere to the culture dish.
Hoxa13 function correlates with EphA7 expression in the limb mesenchyme
In homozygous mutant and wild-type limbs, no quantitative differences in the expression of the following cell surface and pro-adhesion molecules were detected at E11.5-E13.5: paxillin, ß-Catenin, E-cadherin, EphA2, EphA4, ephrin A1, ephrin A2, D1C4, desmoglein, NCAM, fibronectin, catenin, collagen Type II and integrin B1 (data not shown).
In E13.5 limbs, EphA7 mRNA is localized to the condensing digit and carpal/tarsal mesenchyme (Fig. 4A-D). In mutant fore- and hindlimbs, levels of EphA7 expression were consistently lower in all digits, with even lower levels of expression in the regions where digit I and digit V are derived. In the forelimb, an additional region of EphA7 expression was also present in the dorsalmost aspect of each digit, delineating the region where tendons differentiate (Fig. 4A). However, in mutant forelimbs, only digit II appeared to demonstrate EphA7 expression in the dorsal tendon region (Fig. 4B).
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Consistent with the above observations, the marked reduction in EphA7 expression is also observed in vitro, comparing micromass mesenchymal cultures prepared from mutant homozygous and heterozygous control embryonic autopods (Fig. 4M-P).
Ephrin A3 expression is altered in Hoxa13GFP mutant limbs
The distribution of the ephrin ligand A3, which binds with high affinity to the EphA7 receptor (Janis et al., 1999), was examined in the developing autopod of homozygous Hoxa13GFP mutant and heterozygous embryos. By E13.5 the expression of ephrin A3 and Hoxa13GFP in heterozygotes is restricted to the perichondrial region (Fig. 5A,C,E). However, in mutant limbs, this perichondrial restriction is not apparent, as both HoxaA13GFP and ephrin A3 are expressed throughout the developing digit and interdigit regions (Fig. 5B,D,F). Notably, the cells contributing to the digit condensations were discernable in the mutant limb sections under bright field microcopy (Fig. 5G,H), and the cells defining the perichondrial boundary were not visible, suggesting that Hoxa13 is involved in establishing the boundary between the perichondrium and the adjacent condensing mesenchyme.
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Hoxa13 is necessary for EphA7 and EphA4 expression in condensing vascular mesenchyme
Hoxa13GFP homozygous mutant embryos demonstrate a consistent narrowing of the umbilical arteries as early as E10.5, with complete ablation of the lumen of one of the vessels occurring by E13.5 in all (4/4) mutant embryos examined (data not shown). Confocal microscopy of sections containing the umbilical arteries revealed a high level of Hoxa13GFP expression in the condensing mesenchyme and differentiating endothelial layers (Fig. 2E,G,H). At higher magnification, cells forming the vascular wall appear highly disorganized in Hoxa13GFP mutant homozygotes (Fig. 2H). Indeed, the stratification of vascular mesenchyme and endothelium was absent in mutants, whereas heterozygous littermate controls demonstrated normal mesenchymal and endothelial layer formation (Fig. 2G).
Immunohistochemical examination of proteins involved in vasculogenesis revealed a significant reduction in EphA4 and EphA7 expression in the condensing vascular mesenchyme and endothelium of Hoxa13GFP mutant homozygotes (Fig. 9). These data demonstrate the levels of EphA4 and EphA7 expression are reduced in the mutant tissue, and again that the abnormal cellular organization of the mesenchyme and endothelium that form these vessels. It is important to note the changes in morphology exhibited in the mutant umbilical arteries does not create a generalized reduction in UA marker expression as even the ligand for EphA4 and EphA7, ephrin A3, is normally expressed in the mutant vessels (Fig. 2H, inset). Interestingly, both EphA4 and EphA7 are still expressed normally in the gut, ventral neural tube and dorsal root ganglia in these same mutant homozygous embryos (data not shown).
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DISCUSSION |
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We have shown that Hoxa13GFP -expressing cells, which lack functional Hoxa13 protein, are unable to attach efficiently to culture dishes and to form large condensates. They remain detached, cease to divide and die after a few days in culture. By contrast, Hoxa13GFP -expressing cells that retain Hoxa13 function (i.e. are heterozygous for the Hoxa13 mutation) form robust prechondrogenic condensations, which in culture progress along the chondrogenic differentiation pathway.
Hoxa13GFP -null mesenchymal cells can form mixed prechondrogenic condensates with wild-type mesenchymal cells and then undergo chondrogenic differentiation. However, when Hoxa13GFP -null cells are plated in the presence of wild-type mesenchymal cells isolated from a more proximal region of the limb bud, they do not sort themselves from the proximal cells. Thus, Hoxa13 may regulate genetic pathways that control the adhesive properties of the distal limb mesenchyme and in the process, confer positional value along the proximodistal axis to this population of cells. Previous studies in chick (Yokouchi et al., 1995) support this conclusion, as misexpression of Hoxa13 in the proximal limb bud alters proximal mesenchymal cell identity in the opposite manner, appearing to confer more distal limb character onto cells that normally do not express Hoxa13.
Alterations in the expression of EphA7 and its ligand may explain the aberrant cell adhesion, sorting and boundary formation of the distal autopodal mesenchyme
In the condensing digit mesenchyme, EphA7 expression closely parallels the expression of Hoxa13GFP . At E11.5 both Hoxa13GFP and EphA7 are expressed strongly throughout the dorsal mesenchyme (this work) (Araujo et al., 1998). By E13.5, the expression of Hoxa13GFP and EphA7 remains coincident in the perichondrium of the condensing forelimb digits. Interestingly, ephrin A3 expression also overlaps with Hoxa13GFP and EphA7 in both E11.5 and E13.5 limbs, with expression finally restricted to the perichondrial region (Fig. 5C). This co-localization suggests a role for Hoxa13 in mediating perichondrial boundary formation by positively regulating EphA7 expression, which in turn interacts with ephrin A3 to delineate the perichondrial boundary. The Eph receptors and their membrane-bound ligands are ideally suited to establish such boundaries, as their interactions provide the repulsive signals necessary to restrict intermingling between heterogeneous mesenchymal cell populations (Ciossek et al., 1995; Valenzuela et al., 1995; Wada et al., 1998). A similar role for this interaction may be evident in the reinforcement of the boundaries between rhombomeres of the vertebrate hindbrain (Taneja, 1996; Mellitzer et al., 1999; Xu et al., 1999).
In the autopod, reduced EphA7 expression in conjunction with alterations in the expression of ephrin A3 provides a molecular mechanism to explain both aberrant cell adhesion and defects in limb patterning associated with loss of Hoxa13 function. In the distal limb, differential cell sorting is required to organize the undifferentiated mesenchyme into morphologically distinct musculoskeletal domains. Finally, antibody blocking of EphA7 demonstrates that this receptor has the capacity to regulate autopod mesenchymal cell aggregation and chondrogenic nodule formation in vitro, providing a mechanistic link between the regions affected by loss of Hoxa13 function and the reduction of EphA7 expression.
Because Hox genes have the capacity to regulate genes at multiple levels of a developmental cascade (Weatherbee et al., 1998), it is possible that other pro-adhesion molecules involved in autopod patterning are regulated by Hoxa13. Although we did not see a reduction in EphA4 expression in the mutant autopod, other EphA family members may be involved in this process. The residual capacity of homozygous mutant cells to attach and form some chondrogenic nodules, as well as our observation that EphA7 expression is not completely lost in intact Hoxa13GFP homozygous mutant embryos, leaves open the possibility that additional proteins, such as Hoxd13, function in parallel with Hoxa13 to pattern the autopod. Finally, our observation that EphA7 expression is reduced in homozygous mutant limbs may suggest an indirect role for Hoxa13 in mediating EphA7 expression. However, our in vitro characterization of EphA7 expression in FACS-enriched homozygous mutant cells, indicates that EphA7 expression is absent. This finding, in conjunction with normal levels of Msx-1 expression in intact mutant limbs, suggests that changes in EphA7 expression in the mutant autopod are more likely the result of direct regulation in specific tissues.
Limb defects in Hoxa13GFP mutants: the role of programmed cell death
TUNEL assays of homozygous mutant limbs at E11.5 and 12.5 did not reveal significant increases in programmed cell death relative to control embryos (data not shown) which indicates that the reduced capacity to form the appropriate autopod prechondrogenic condensations observed in Hoxa13 mutant homozygotes is not explained by increased apoptosis. On the contrary, this study revealed a loss of normal apoptosis in the interdigital regions of the autopod in Hoxa13 mutant homozygotes and reduced apoptosis between digits II and III of embryos heterozygous for the Hoxa13 mutation. The reduced level of apoptosis provides an explanation for the persistence of interdigital tissue in heterozygous and homozygous Hoxa13 mutants. Normally, Hoxa13 expression becomes restricted to the digit perichondrium by E13.5. However, in both heterozygous and homozygous Hoxa13 mutant embryos, Hoxa13-GFP labeled cells are observed in the interdigital tissue (Fig. 7G,H). This observation provides another example of the reduced ability of Hoxa13 mutant cells to sort themselves properly, resulting in the failure to establish an appropriate boundary between Hoxa13-expressing and nonexpressing tissues. The reduction or absence of normal apoptosis in the interdigital region of these mutant embryos might in turn follow from a failure of the ectopic Hoxa13-expressing cells to respond to apoptotic signals. Alternatively, the persistence of interdigital tissues could also result from changes in cell proliferation. An examination of mitotic cells in interdigital tissue using a mitosis-specific marker, anti-phosphohistone H3, did not reveal differences in the number of mitotic cells (data not shown).
Loss of EphA4 and EphA7 expression in the umbilical vasculature of Hoxa13GFP mutants
The high level of Hoxa13 expression in the condensing mesenchyme and endothelial layer of the UA suggests a major role for Hoxa13 in the formation of these vessels. Narrowing of the UA is observed as early as E10.5 in Hoxa13GFP homozygous embryos, with complete unilateral closure being seen in all of the E13.5 embryos examined. It is important to note that during murine gestation, the exiting UAs fuse into a single vessel outside the embryo proper (Kaufman and Bard, 1999), whereas humans typically maintain two UAs during gestation. This difference might explain why stenosis of a single UA may not be life threatening in midgestation to humans (Pavlopoulos et al., 1998), whereas stenosis of the UA in mice would severely impact the volume of blood that reaches the placenta in mice, particularly if the occlusion occurred proximal to or outside of the umbilical ring.
Analysis of the vessel walls of E13.5 embryos reveals distinct mesenchymal and endothelial cell types in heterozygous and wildtype littermates. The UA walls of Hoxa13 mutant homozygotes, on the other hand, are disorganized with no stratification into morphologically distinct cell types. The loss of EphA4 and EphA7 expression in the developing UA wall of Hoxa13 mutant homozygotes by E11.5 may well explain the loss of cellular organization in the formation and maintenance of the boundaries between vascular mesenchyme and endothelium. Numerous studies have suggested the interaction of ephrins and their receptors during vasculogenesis and angiogenesis (Pandey et al., 1995; McBride and Ruiz, 1998; Wang et al., 1998; Adams et al., 1999, Ogawa et al., 2000).
In summary, a prominent feature common to both the limb and umbilical artery defects observed in Hoxa13 mutant homozygous embryos is a failure to organize and properly pattern the mesenchyme within these developing tissues. We have provided evidence that mesenchymal dysfunction, in part, be understood as defects in mesenchymal cell aggregation and adhesion. A loss of ephrin receptor expression was common to both tissues. These observations suggest that Hoxa13 directly or indirectly controls the aggregation, adhesion and sorting capacity of the mesenchymal cells within the autopod in part by regulating the ephrin/Ephrin receptor signaling system. Modulation of this signaling system would ensure that the appropriate cellular boundaries are formed and maintained during morphogenesis of these tissues. This role of Hoxa13, though not necessarily its only role in the formation of the distal limb skeletal elements and umbilical vasculature, nevertheless begins to explain many of the cellular defects observed in these mutant animals.
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ACKNOWLEDGMENTS |
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