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

First published online 1 June 2005
doi: 10.1242/dev.01877

This Article Summary Full Text Full Text (PDF) Supplementary Material 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 Lallemand, Y. Articles by Robert, B. Search for Related Content PubMed PubMed Citation Articles by Lallemand, Y. Articles by Robert, B. Social Bookmarking                         What's this?

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development

Yvan Lallemand, Marie-Anne Nicola^*, Casto Ramos^*,, Antoine Bach, Cécile Saint Cloment and Benoît Robert

Unité de Génétique Moléculaire de la Morphogenèse, Institut Pasteur, URA 2578 du CNRS, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France

View larger version (89K): [in a new window]   Fig. 1. Limb phenotype of Msx1-/-; Msx2-/- double-null embryos at 14.5 dpc. (A-F) Alcian blue-stained skeletal preparations. (A,B) Forelimb of a control (A) and a double homozygous (B) embryo. There is an overall reduction in size of the limb in the mutant and the radius is absent (asterisk). (C,D) Higher magnification of the autopods of the embryos shown in A and B, respectively. Only one phalange has developed on digits II to V in the double mutant (asterisk) and the interdigital webbing has not regressed. The arrow indicates the presence of an abnormal distal mesenchyme condensation between digit II and III. The metacarpal (m), the first (p1) and second (p2) phalanges are indicated only for digit III, with the missing phalange indicated by an asterisk for the double mutant. (E,F) Autopods of two polydactyl double mutant embryos. In both cases, six digit formations are present (arrowheads) and the anterior-most digit is longer than a normal thumb. (E) The radius is completely absent whereas it is almost normal in F. CI-IV, carpal I to IV; cc, central carpal; h, humerus; ic, intermedioradial carpal; r, radius; s, scapula; u, ulna; uc, ulnar carpal. (C-F) Anterior is towards the right.

View larger version (60K): [in a new window]   Fig. 2. AER maturation, but not initiation, is impaired in Msx1-/-; Msx2-/- double-null embryos. (A,B) Initiation of the AER is not delayed in the double mutants as revealed by Fgf8 expression at the 14-somite stage. In both panels, arrowheads delimitate the forelimb territory. (C,D) At 10.5 dpc, the AER in the double-null embryo appears shorter and thicker than in the control. (E-H) At 11.5 dpc, the AER remains thicker than normal as revealed by Bmp7 expression (E,F) or histological analysis of transverse sections (G,H). (I,J) The level of Fgf4 expression in the AER at 10.5 dpc is lower in the double mutants. All panels display forelimbs and anterior is towards the right in A-F,I,J.

View larger version (51K): [in a new window]   Fig. 3. The anteriormost part of the limb buds fails to develop in Msx1-/-; Msx2-/- double mutant embryos. (A,B) Double in situ hybridisation with Fgf8 (red) and Fgf4 (purple) probes on control (A) and Msx1-/-; Msx2-/- double mutant (B) embryos. At 11.5 dpc, in control embryos, the AER displays an anterior domain expressing Fgf8 (red staining) but not Fgf4 (purple staining), whereas in the double mutant the anterior expression limit is the same for both genes. Note the sharper aspect of the anterior border of the limb bud in the double mutant. White arrows indicate the anterior limit of Fgf8 and black arrows indicate the anterior limit of Fgf4 expression domains. (C,D) Left-side view of 10.5 dpc embryos showing both forelimbs (FL) and hindlimbs (HL). The Alx4 expression domain, corresponding to the anterior third of the limb bud mesenchyme, is normal at this stage in the double mutant. (E,F) At 10.75 dpc, the anterior Hoxd11-negative domain, corresponding to the anterior third of the normal limb bud (E), is virtually absent in the mutant (F), whereas the posterior Hoxd11-expression domain has globally the same size as in the control. (G,H) Analysis of Hoxd11 expression at 11.5 dpc confirms that the anterior domain is missing from the limb buds of double mutants. (I,J) Whole-mount analysis of apoptosis in limb buds at 10.5 dpc (ventral view). TUNEL staining shows no increase of apoptosis in the AER of the double mutant at E10.5. (A,B,E,F,I,J) Forelimbs. (G,H) Hindlimbs. Anterior is towards the right in all panels.

View larger version (42K): [in a new window]   Fig. 4. Dorsoventral specification is altered in the double-null limb buds. (A-D) En1 expression in control (A,C) and double-null mutants (B,D): limb buds are viewed from their distal tip with their ventral side facing upwards. Anterior is towards the right. En1 expression level is reduced in the limb field at 9.5 dpc (24 somites) (A,B) and is undetectable anteriorly at 11.5 dpc (C,D) in the double mutant (D, arrow). (E-I) Lmx1b expression in control (E,G) and double mutant (F,H,I) embryos. (E,F) Limb buds are viewed from their distal tip with the ventral side facing towards the upper leftward side. Anterior is uppermost. The broken line indicates the normal dorsoventral boundary of the limb bud. At 10.5 dpc, the Lmx1b expression domain extends ventrally at the anterior aspect of the limb bud in the double mutant (arrow). (G,H) Views of the anterior border of limb buds at 11.5 dpc showing the sharp expression limit of Lmx1b at the level of the AER in the control (G), whereas the Lmx1b expression domain extends ventrally in the double mutant (arrowhead) (H). The black arrow indicates the anterior limit of the AER. (I) A section of the limb bud shown in H, according to the plane indicated by the white arrows, that confirms the presence of an ectopic Lmx1b expression domain in the ventral mesenchyme (arrowhead). The broken line indicates the dorsoventral boundary. Dorsoventral orientation is indicated by D and V.

View larger version (49K): [in a new window]   Fig. 5. AER regression is delayed in Msx1-/-; Msx2-/- double mutant embryos. Comparison of Fgf8 expression in control (A,C,E,G) and double mutant (B,D,F,H) embryos. (A-D) At 12.5 dpc, the AER has disappeared at the level of the interdigital spaces in control embryos (A). In double-null embryos, it still appears continuous (B) and thicker than normal (C,D). (E-H) At 14.5 dpc, the AER is normally absent (E,G) but still observed at the digit tips of double-null embryos (F,H, arrowheads). At this stage, the Fgf8 expression pattern confirms the variability of the limb phenotypes ranging from oligodactyly (F) to polydactyly (H). (A-D,G,H) Forelimbs. (E,F) Hindlimbs. (C,D) Anterior views of A,B, respectively. Anterior is towards the right in A,B,E-H.

View larger version (45K): [in a new window]   Fig. 6. Anteroposterior polarisation of the limb buds is affected in Msx1-/-; Msx2-/- double mutant embryos after 11.5 dpc. (A,B) At 11.5 dpc, in normal embryos, Fgf4 is expressed in the posterodistal part of the AER, and excluded from the anterior fourth of the AER. In the double mutants, Fgf4 expression level is normal but expression domain is shifted anteriorly (A,B, arrowheads). (C,D) At 12.5 dpc, Fgf4 is still detectable at the anterior tip (white arrow in D) of the AER in double mutant embryos. (E-H) Hoxd11 is expressed only in the region corresponding to the future digits II to V in both normal hind- (E) and forelimbs (G). In the double mutants (F,H), it is expressed along the whole distal mesenchyme regardless of whether the limb bud (generally hindlimbs) is truncated (F) or, conversely (essentially forelimbs), overgrown anteriorly (H). (I,J) In a few double mutant embryos, Shh is faintly expressed ectopically at the anterior margin of the forelimbs (J, arrow). The posterior domain is slightly shifted distally (arrowheads). (A-D,G-J) Forelimbs. (E,F) Hindlimbs. In all panels, anterior is towards the right.

View larger version (41K): [in a new window]   Fig. 7. Bmp signalling in double mutant limbs. (A) At 10.5 dpc, in control embryos, Bmp4 is expressed in the AER, in the posterior mesenchyme and in a faint anterior domain of the limb bud. (B) Bmp4 expression is similar in the double-null limb bud. The anterior limit of the Bmp4 expression domain in the ectoderm corresponds to the anterior limit of the AER (black arrow). (C,D) At 11.5, owing to the anterior truncation of both the AER (arrow) and mesenchyme, the anterior Bmp4 expression domain is almost completely absent in the mutant. A reduced Bmp4 expressing domain is still present at the proximal anterior border (arrowhead). In the rest of the limb bud, Bmp4 expression is normal. (E,F) At 13.5 dpc, Bmp4 expression is observed in the finger tip mesenchyme of control embryos (E, arrowheads) but not in the mutants (F). By contrast, expression is observed in the remaining AER of the double-null mutants (F) which has regressed at this stage in control embryos. (G,H) At 13.5 dpc, Bmp2 expression is similar in the control and the double mutants, except that no negative anterior domain is observed. In the control (G), the arrowhead indicates a posterior domain of the limb bud that expresses Bmp2 at a higher level. (I,J) Bmp7 is expressed similarly to Bmp2 in control and mutant limbs. (K,L) Gremlin expression persists slightly longer in the interdigital webbing of the mutants. The dark proximal domain in K is an artefact due to the thickness of the specimen at this site. (A-J) Forelimb buds, (K,L) hindlimb buds. In all panels, anterior is towards the right.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter