QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Beverdam, A. Articles by Meijlink, F. Search for Related Content PubMed PubMed Citation Articles by Beverdam, A. Articles by Meijlink, F.

Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice

Hubrecht Laboratory, Netherlands Institute for Developmental Biology, PO Box 85164, 3508AD Utrecht, The Netherlands

View larger version (18K): [in a new window]   Fig. 1. Targeted mutagenesis of Alx3. (A) Structure of the Alx3 locus: exon 2, 3 and 4 are depicted by open rectangles. The homeobox and the aristaless box (encoding the aristaless/OAR domain) are coloured. (B) Targeting vector: a 6 kb lacZ-loxP-pGK-TKNEOpA-loxP cassette was inserted in sequences of exon 2 upstream of the homeobox. 5' 1.9 and 3' 5.5 homologous sequences are indicated. Arrows in cassette indicate direction of transcription. (C) Targeted allele. The NcoI-XhoI probe corresponds to a region not present in the targeting construct, whereas the SstI-StuI probe detects the short homology arm. Relevant restriction sites and probes are indicated. Restriction fragments for Southern analysis are indicated with broken arrows above wild-type Alx3 allele and under targeted Alx3 allele. Coloured boxes: black, probes; red, loxP sites; blue, lacZ cassette; yellow, pGK-TKNEOpA cassette; green, homeobox; pink, aristaless-box. Restriction sites: B, BamHI; N, NcoI; P, PstI; RI, EcoRI; S, StuI; Sa, SalI.

View larger version (86K): [in a new window]   Fig. 2. Essentially correct Alx3/lacZ expression in knock-in mice; comparative expression analysis of Alx3 and Alx4 in E10.5 embryos. (A) Detection of Alx3 mRNA (whole-mount in situ hybridisation) in whole embryo. (B) lacZ expression in Alx3+/- mouse embryo. Expression of Alx3 and lacZ are qualitatively identical, confirming proper targeting of the Alx3 locus. Staining in brain vesicles in A is background staining. (C,D) Radioactive in situ hybridisation showing overlapping expression of Alx3 and Alx4 in frontonasal processes (FNP) at E9.5. (G,H) Expression of Alx3 and Alx4 in medial nasal process (MNP) and mandibular arch (BA) at E10.5. (E,I) Schemes indicating approximate orientation of sections. (F,J) Whole-mount in situ hybridisation of E10.5 embryos showing overlap of expression in cranium and anterior mesenchyme of limbs.

View larger version (52K): [in a new window]   Fig. 3. Forelimb and pectoral girdle defects in Alx mutants. Alizarin Red (bone) and Alcian Blue (cartilage) stained forelimbs and pectoral girdles. Genotypes: (A,D) Alx3+/-/Alx4+/+; (B,E) Alx3+/+/Alx4lst-J/lst-J; (C,F) Alx3-/-/Alx4lst-J/lst-J. Arrowheads indicate the clavicle and arrows indicate (normal position of) deltoid crest. Asterisk marks extra digit in B,C. (D-F) Dissected clavicles; double arrows and L-M indicate lateral-medial orientation of the clavicles.

View larger version (60K): [in a new window]   Fig. 4. Cranial phenotype of newborn Alx3/Alx4lst-J double mutant mouse. Lateral (A-D) and frontal (E-H) views on crania of Alx3+/- (A,E), Alx4lst-J/lst-J (B,F), Alx3+/-/Alx4lst-J/lst-J (C,G) and Alx3-/-/Alx4lst-J/lst-J (D,H) newborn mice. The anterior region is truncated, the nose is split and the eyes are open in all mutants. Note abnormal appearance of skull vault, owing to reduced parietal frontal and nasal bones in D. The severity of the cleft nose phenotype is highly variable.

View larger version (73K): [in a new window]   Fig. 5. Craniofacial abnormalities in Alx3/Alx4lst-J double mutant newborn mice. Alizarin Red (bone)/Alcian Blue (cartilage) stained skulls are shown. First column shows a dorsal view on the vaults of the skulls. Second column shows dorsal views on skulls from which vaults were dissected. Third column shows a ventral view on skulls from which mandibles and vaults were dissected. Fourth column shows the dissected mandibles. Genotypes are indicated to the left. Green arrows indicate the basipresphenoid, which constitutes the border of neural crest-derived skeletal elements and cephalic and somitic mesoderm derived skeletal elements. White arrows indicate the nasal septum.

View larger version (58K): [in a new window]   Fig. 6. Craniofacial abnormalities in Alx3-/-/Alx4lst-J/lst-J double mutant newborn mice. Photos and corresponding diagrams of control and Alx3/Alx4lst-J double mutant skulls. Dorsal view on vaults of control (A,B) and Alx3/Alx4lst-J double mutant (C,D) skulls. Ventral views on skulls from which mandible was dissected of control (E,F) and Alx3/Alx4lst-J double mutant (G,H) mice. All skeletal elements are represented by a colour in the diagrams as indicated in the legend. Asterisks in E,G indicate positions of pterygoid processes. Abbreviations: As, alisphenoid bone; Bo, basioccipital bone; Bs, basisphenoid bone; M, maxilla; Ns, nasal septum; P, palatine; Ps, basipresphenoid bone; S, squamosal bone; So, supraoccipital bone.

View larger version (76K): [in a new window]   Fig. 7. Histological analysis of cranial phenotype. Transversal sections of wild-type (A,C,E,G) and Alx3-/-/Alx4lst-J/lst-J (B,D,F,H) embryos. Stage (embryonic day) is indicated in lower left-hand corner. Asterisks, nasal septum precartilage condensation; arrows, nasal cavity; arrowheads, Jacobson’s (vomeronasal) organ. Schemes below, with stages indicated, show approximate plane of section. Abbreviations: ps, palatal shelves; to, tongue.

View larger version (106K): [in a new window]   Fig. 8. Aetiology of the split nose phenotype. The nasal processes of Alx3/Alx4lst-J double mutant embryos fail to fuse around E11.5. Shown are frontal views on control (A-D; either Alx3+/- or wild type) and stage-matched Alx3/Alx4lst-J double mutant embryos (E-H) of E10.5 to E11.5 as indicated above photos. Posterior regions of most of the embryos were cut off to allow free view on the nasal processes. In embryos at E11.5, the Alx3 expression domain was visualised by X-Gal staining to detect ß-galactosidase. Arrowheads point to medial edges of the medial nasal processes, demonstrating the improper outgrowth and fusion of the nasal processes in double mutant embryos. Note the increasing difference in distance between the processes of control and Alx3/Alx4lst-J double mutant embryos.

View larger version (66K): [in a new window]   Fig. 9. Study of phenotype by analysis of expression patterns of marker genes in serial sections. Probes used are specified to the right. (A,E,G-I,M-O) Control embryos (either Alx3 heterozygotes or wild type); (B,C,F,J-L,P-R) Alx3-/-Alx4lst-J/lst-J. (A-F) Serial sections of two E10.5 embryos and expression of ectodermal markers, where D is a scheme to indicate approximate plane of sections in the embryos used in this experiment. (G-R) Nearby serial sections of two control and two double mutant embryos and expression of two mesenchymal markers. (G,J,M,N) E10.5 embryos; (H,I,K,L,N,O,Q,R) Rather advanced E11.5 embryos. Orientation of sections of this series of embryos is depicted in lower row of panels. Arrows (in A-C) indicate Shh expression in ectoderm of the medial nasal processes. Abbreviations: BA, branchial (mandibular) arch; FB, forebrain; FNP frontonasal process; HB, hindbrain; HE, heart; Man, max, mandibular and maxillary process, respectively, of first branchial arch; MNP, medial nasal process; MP, maxillary process; NC, nasal cavity; NS, nasal septum; O, otocyst; Ps, palatal shelf (part of maxilla); To, tongue.

View larger version (81K): [in a new window]   Fig. 10. Apoptosis in Alx3/Alx4lst-J double mutant embryos. In a restricted region of the frontonasal process of Alx3/Alx4lst-J double mutant embryos, a significant number of mesenchymal cells undergo apoptosis compared with stage-matched control embryos. (A,D,F,H,K) control embryos (wild-type or Alx3+/-); (B,E,G,I,L) embryos of double mutant embryos; (C,J,M) schemes depicting plane of sections of embryos shown to their left. TUNEL assays were carried out to detect apoptosis. Positive cells appear red in this assay, as seen in brightfield images (A,B,F,G). Because of difficulty with visualising TUNEL staining simultaneously with histology, we show also darkfield images where positive cells appear as white to orange spots. The yellow squares in D,E mark the crucial area where we found differences in apoptosis in E10.0 embryos. These areas are shown enlarged, and in brightfield, in F,G. No or very few apoptotic cells were detected in the vicinity of the nasal processes of embryos of E10.5 and E11.5. The yellow arrows in panels H,I indicate apoptopic areas in the maxillary component of the first branchial arch, confirming that the negative result in this experiment was not due to technical failure.