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Loss of Eph-receptor expression correlates with loss of cell adhesion and chondrogenic capacity in Hoxa13 mutant limbs

H. Scott Stadler^*, Kay M. Higgins and Mario R. Capecchi

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

View larger version (14K): [in a new window]   Fig. 1. Hoxa13GFP targeting vector, genotype analysis and expression. (A) Partial restriction map of the Hoxa13 locus. White and black boxes represent exons 1 and 2 respectively. Bar represents the DNA fragment used as a flanking probe in the Southern analysis. S, SphI; X, XhoI; RV, EcoRV; R, EcoRI. (B) 10 kb Hoxa13GFP targeting vector. Forward and reverse PCR primer sites are denoted by the black arrows. (C) Southern transfer analysis of homologous recombinants and germline transmission with SphI DNA digested from wild-type Agouti offspring (+/+), and Agouti offspring showing germline transmission of the Hoxa13GFP allele (+/-). (D) PCR genotyping of embryonic yolk-sac DNAs derived from a heterozygous intercross.

View larger version (70K): [in a new window]   Fig. 2. Expression characterization of Hoxa13GFP allele in the heterozygous embryos. (A) Expression in the distal forelimb at E 10.5. Arrowhead denotes no expression in the AER. (B) Hoxa13GFP expression expands within the progress zone at E11.5. Localization of Hoxa13GFP-expressing cells to the condensing digit regions in E 13.5 forelimbs (C) and hindlimbs (D). (E) Condensing vascular mesenchyme in the umbilical arteries (UA) also expresses Hoxa13. (F) Expression of Hoxa13GFP is also readily detected in the genital ridge (GR) and tissues surrounding the urogenital sinus (US). (G) Higher magnification image of E 11.5 heterozygous umbilical artery showing Hoxa13GFP expression in the condensing mesenchymal (arrow) and endothelial (arrowhead) layers. Vascular lumen (L). (H) Higher magnification image of an E11.5 homozygous mutant UA showing a lack of mesenchymal/endothelial cell layer stratification. Inset is the normal expression of ephrin A3 in a mutant umbilical artery demonstrating that the lack of stratification does not globally affect gene expression patterns. L, vascular lumen. Scale bar: 50 µm for G,H.

View larger version (94K): [in a new window]   Fig. 3. In vitro analysis of cell attachment and aggregation of dissociated limb mesenchyme. FACS-enriched Hoxa13 mutant homozygous cells demonstrated marked reduction in cell adhesion and mesenchymal aggregate formation compared with sorted cells from heterozygous littermates. (A,B) Heterozygous and homozygous mutant cultures 12 hours after plating. Although unattached to the plates, the homozygous mutant cells appeared viable as measured by Trypan Blue exclusion (data not shown). (C) Aggregation of heterozygous mutant cells in the developing mesenchymal aggregates (arrowhead) 24 hours after plating. (D) Homozygous mutant cultures typically exhibited an approximate eightfold reduction in cell attachment and mesenchyme aggregate (arrowhead) formation. (E,F) Combined mutant (-/-) and wild-type (+/+) cells demonstrate that Hoxa13GFP -/- cells will aggregate efficiently in the presence of wild-type (GFP nonexpressing) cells, forming a large chimeric aggregate. (G,H) Higher magnification reveals that homozygous mutant mesenchymal cells contribute to chimeric mesenchyme aggregation by binding to wild-type cells, which attach more efficiently to the tissue culture dish. Arrowheads indicate the position of an attached wild-type (non-fluorescent) cell. (I) Hoxa13GFP +/- cells contributing to a developing cartilage nodule 72 hours after plating (arrow). Inset shows positive staining of the nodules for Alcian Blue (arrowhead) and collagen type II (J). (K) Micromass culture of FACS-enriched Hoxa13GFP +/- cells 6 days after plating, demonstrating the capacity to form digit-like structures in vitro.

View larger version (123K): [in a new window]   Fig. 4. Expression of EphA7 in E 13.5 limbs and micromass cultures as detected by in situ hybridization and immunohistochemistry. (A) Heterozygous mutant left forelimb. Arrows denote EphA7 expression in phalangeal condensations P1 and P2 for digit IV and the condensing digit V mesenchyme. (B) Homozygous mutant left forelimb. Arrow denotes the presumptive digit V region. (C) Heterozygous mutant left hindlimb. Arrow denotes expression in the digit V region. (D) EphA7 expression in the hindlimb of a homozygous mutant littermate. (E) Hoxa13GFP expression in the distal P2 anlage of digit II in heterozygous forelimbs. Arrow denotes localization of Hoxa13GFP-expressing mesenchyme to the mesodermal/epidermal boundary. Arrowhead denotes the position of mesenchymal condensation proximal to the epithelial/mesodermal boundary. (F) Localization of mesenchyme expressing Hoxa13GFP in a mutant digit II anlage. Arrows denote mesenchymal cells present in the epidermal layer. Arrowhead indicates the distally shifted mesenchymal condensation of the mutant digit anlage. (G) EphA7 expression in the mesodermal and epidermal layers of the same heterozygous digit II region. (H) Reduced EphA7 expression in the same mutant digit II region. (I) Combined image of Hoxa13GFP and EphA7 in the same heterozygous digit II section. Arrow indicates the precise delineation of the boundary separating mesodermal cells expressing Hoxa13GFP from the overlying epidermis that expresses EphA7. (J) Combined expression of Hoxa13GFP and EphA7 in the same mutant digit II region. Note the complete absence of a defined mesodermal/epidermal boundary, as well the more distal localization of the condensing digit mesenchyme (arrow). (K) Bright-field image of the same heterozygous digit II region. Note the thin uniformly defined layer of cells comprising the epidermal layer (arrowhead). (L) Bright field image of the same mutant digit II region. Note the thickened, irregularly shaped epidermal layer (arrow). (M) Aggregating limb mesenchyme expressing Hoxa13GFP isolated from limb buds heterozygous for the Hoxa13 mutation. Note the dense configuration of cells forming the central mesenchymal condensation (arrow). (N) Aggregating limb mesenchyme isolated from limb buds homozygous for the Hoxa13 mutation. Note the reduced cell density in mesenchymal condensations derived from homozygous mutant cells (arrowhead). (O) EphA7 is highly expressed in mesenchymal condensations prepared from cells heterozygous for the Hoxa13 mutation. (P) EphA7 expression is missing in condensations prepared from mesenchyme homozygous for the Hoxa13 mutation. Scale bars: 100 µm for E-L; 50 µm for M-P.

View larger version (73K): [in a new window]   Fig. 5. Ephrin A3 expression in the developing autopod of Hoxa13GFP mutant homozygous and heterozygous littermates at E 13.5. (A) Perichondrial expression of Hoxa13GFP in digits II and III in a heterozygous limb section. (B) Expression of Hoxa13GFP in a mutant littermate section, showing decreased maturation of the digit anlage as well as poor delineation of the perichondrial border. (C) Expression of ephrin A3 in the developing digit II and III perichondrial borders of the same heterozygous limb section. Note the lack of ephrin A3 expression within the condensing digit anlage (arrow). (D) Diffuse expression of ephrin A3 in the same mutant limb section reflecting the undifferentiated status of the digit mesenchyme. Arrow denotes the site of a poorly delineated digit II perichondrium. (E) Co-localization of Hoxa13GFP expressing mesenchyme and ephrin A3 in the perichondrium of the developing heterozygous digit anlage. Note that the strong delineation of the outermost portion of the perichondrial border is demarcated by the highest levels of Hoxa13GFP and ephrin A3 expression (yellow cells). (F) Co-localization of Hoxa13GFP and ephrin A3 in the mutant digit II-III region. Noticeably absent are yellow cells depicting the delineation of the outer perichondrial border. (G) Bright field image of the condensing digit mesenchyme and perichondrial border in the same heterozygous section. Note the clear demarcation of the perichondrial border (arrowheads) from the condensing digit II and III mesenchyme (arrows). (H) Bright field image of the same mutant limb section. Note the poor separation between the putative perichondrial region (arrowhead) and the condensing digit anlage (arrow). Scale bar: 100 µm.

View larger version (134K): [in a new window]   Fig. 6. In vitro application of an EphA7 antibody disrupts mesenchyme aggregation and chondrogenic nodule formation. (A,B) Micromass cultures of E 12.5 Hoxa13GFP heterozygous (+/-) mesenchymal cells 48 hours after incubation with DMEM media. Arrows denote normal aggregation and development of chondrogenic nodules. (C) Micromass culture of an E12.5 Hoxa13GFP homozygous mutant (-/-) limb mesenchyme plated at the same cell density as (A,B) and treated with DMEM media. Arrow denotes minimal cell aggregation and condensation. (D,E) Parallel cultures of E 12.5 Hoxa13GFP (+/-) mesenchymal cells 48 hours after incubation with normal rabbit serum, indicating that serum lacking EphA7 antibodies had no effect on the capacity of the heterozygous mesenchyme to aggregate and form chondrogenic nodules (arrows). (F) Homozygous mutant mesenchyme is also unaffected by incubation with the rabbit serum as minimal condensations are still formed (arrow). (G,H) Parallel cultures of heterozygous cells incubated with an EphA7 antibody exhibited marked reductions in cell attachment, aggregation, and chondrogenic nodule formation (arrow). (I) Incubation of homozygous mutant cultures with the same concentration of EphA7 antibody had only a minimal affect on the adhesion and condensation properties of these mutant cells. Scale bar: 50 µm.

View larger version (110K): [in a new window]   Fig. 7. Proximal-distal cell sorting of wild-type and Hoxa13GFP limb mesenchyme. (A) Combinations of proximal wild-type and distal heterozygous mesenchyme cells 19 hours after inoculation. Note the sorting of heterozygous and wild-type mesenchyme cells into proximal (arrows) and distal (arrowheads) mesenchyme cell condensations. (B) Combinations of proximal wild-type and distal mutant mesenchyme cells 19 hours after inoculation. Note the inclusion of mutant cells into the wild-type condensations (arrowheads). (C) TO-PRO-3 iodide staining of wild-type and heterozygous mesenchyme cell condensations showing separation of proximal (arrows) and distal (arrowheads) condensations. (D) TO-PRO-3 iodide staining of proximal wild-type and distal mutant cells showing inclusion of mutant cells into proximal condensations (arrowheads). (E) Multiwavelength analysis showing sorting of distal heterozygous cells (green) from proximal wild-type cells (red). (F) Multiwavelength analysis showing a lack of sorting between distal mutant cells (green) and wild-type proximal cells (red). Scale bar: 100 µm.

View larger version (66K): [in a new window]   Fig. 8. Characterization of Msx-1 expression and interdigital apoptosis in limbs heterozygous and homozygous for the Hoxa13GFP mutation. (A) Normal digit separation in wild-type adult hindlimb. (B) Digit II-III fusion (arrowhead) in a heterozygous adult hindlimb. (C) E13.5 mutant hindlimb showing no initiation of digit separation. (D) TUNEL assay of apoptosis between digits II and III in a wild-type E13.5 hindlimb. (E) Reduced apoptosis between digits II and III in a heterozygous hindlimb. (F) Absence of apoptosis between digits II and III in a mutant homozygous hindlimb. (G) Cryosections of a normally developing E 13.5 heterozygous hindlimb reveal low levels of ectopic interdigital cells (arrowhead) that express Hoxa13, as well as predominantly normal regression of GFP-expressing cells to the condensing digit mesenchyme (arrow). (H) Cryosections of homozygous Hoxa13GFP mutant hindlimbs reveal a persistence of cells (arrowhead) in the interdigital regions, reflecting a failure of these cells to sort from the interdigital zone to the condensing digit mesenchyme. (I,J) Forelimb and hindlimb expression of Msx-1 in the interdigital tissues in a Hoxa13GFP heterozygous embryo. (K,L) Forelimb and hindlimb expression of Msx-1 in homozygous mutant embryos. (K) Arrowhead indicates the interdigital region between digits IV and V where consistently higher levels of Msx-1 expression were detected in mutant forelimbs. By contrast, consistently lower levels of Msx-1 expression were also detected in mutant forelimbs in the posterior margin of digit V (arrow). Scale bars: 100 µm.

View larger version (110K): [in a new window]   Fig. 9. EphA4 and EphA7 expression in the umbilical arteries (UAs) of E11.5 Hoxa13GFP heterozygous and mutant homozygous littermates. (A,B) Hoxa13GFP expression in the developing vascular wall of heterozygous and mutant homozygous embryos. (C,D) Hoxa13GFP expression in the developing vascular wall of heterozygous and mutant embryos, vascular lumen (vl). (E) Expression of EphA4 in the developing UA vascular wall of heterozygotes. (F) Loss of EphA4 expression in the developing UA vascular wall of mutant homozygotes. (G) Expression of EphA7 in the developing wall of the UA of heterozygotes. (H) Loss of EphA7 expression in the developing wall of the UA in Hoxa13GFP mutant homozygotes. (I) Multiwavelength analysis detects coexpression of EphA4 and Hoxa13GFP in same cells within the developing UA walls of heterozygotes. (J) Multiwavelength analysis from a mutant homozygote UA demonstrates a loss of EphA4 expression in cells expressing Hoxa13GFP. (K) Multiwavelength analysis of EphA7 and Hoxa13GFP expression reveals co-expression in the cells of the developing UA wall in heterozygotes. (L) Multiwavelength analysis of a mutant homozygote reveals a loss of EphA7 expression in cells that express Hoxa13GFP in the developing UA wall. Scale bar: 50 µm.