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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00687

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The concerted action of Meox homeobox genes is required upstream of genetic pathways essential for the formation, patterning and differentiation of somites

Baljinder S. Mankoo^1,*, Susan Skuntz², Ian Harrigan¹, Elena Grigorieva¹, Al Candia³, Christopher V. E. Wright³, Heinz Arnheiter² and Vassilis Pachnis^1,

¹ Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Laboratory of Developmental Neurogenetics, NINDS, NIH, Bethesda, MD 20892, USA
³ Department of Cell Biology, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA

View larger version (125K): [in a new window]   Fig. 1. Meox1;Meox2 mutants have profound axial skeleton defects. Alcian Blue/Alizarin Red skeletal preparations of neonates. The forelimbs and shoulder girdle have been removed for ease of viewing. In contrast to control littermates (Meox1+/-;Meox2+/- A and E, n>6), animals with mutations in both Meox1 and Meox2 genes have defects in the development of the axial skeleton (B-H, n>6). Meox1-/-;Meox2+/- display rib fusions and deformations, and vertebral bodies at the lumbar level are split, and tail vertebrae are fused (B,F). Meox1+/-;Meox2-/- are less severely affected, there are no rib defects and lumbar vertebrae appear normal, but tail vertebrae are malformed and fused (C,G). Meox1-/-;Meox2-/- animals lack an axial skeleton (D,H), there are no ribs and, while ossified, deformed vertebrae are formed at the cervical and thoracic level; more posterior lumbar vertebrae are present only as cartilage condensations at the position expected of the neural arches (arrow in H) and tail vertebrae are completely absent. The sternum develops, albeit abnormally, in the absence of the ribs (arrowhead in D). Normally developed neural arches (na) and vertebral bodies (vb) are identified in E.

View larger version (84K): [in a new window]   Fig. 2. Skeletal muscle abnormalities in Meox double mutants. Transverse paraffin wax-embedded sections of control (A,B) and mutant (C,D) foetuses at E16.5, at the level of the tongue (A,C) and forelimbs (B,D) demonstrating that most skeletal muscles are absent or reduced in size in the mutant. These include: the prevertebral muscles of the neck (black arrowhead in A and C), the epaxial (black arrow) muscles of the trunk and also the hypaxial muscles of the trunk including those of the abdominal wall (white arrows) and intercostal muscles (white arrowhead). The brown fat overlying the shoulder muscles in control foetuses (* in B) was also absent in mutants. t, tongue; l, lung; h, heart.

View larger version (117K): [in a new window]   Fig. 3. Defective somitogenesis in the absence of Meox gene function. (A,B) Thin resin sagittal sections of the caudal region of E9.5 embryos. Unlike those of control Meox1+/-;Meox2+/- embryos (A), newly formed somites from double mutant Meox1-/-;Meox2-/- embryos (B) are irregularly shaped and sized (compare sizes of bars), not organized into epithelial spheres and the basal lamina that normally surrounds each somite is no longer evident between somites (black arrows), although it is present dorsal and ventral to somites (white arrows). (C,D) Transverse rostral sections of E10.5 embryos. The epithelial dermomyotome (arrow), characteristic of mature differentiated somites in controls (C), is absent in Meox1;Meox2 double homozygous mutants (D). n, neural tube, sc, sclerotome. (E,F) Longitudinal sections of E10.5 embryos. The segmented organization of adjacent sclerotomes (dashed lines) in controls (E) is absent in double Meox mutants (F). Furthermore, the anteroposterior polarity of each sclerotome, consisting of a rostral half (r) and denser caudal half (c) is not apparent. The epithelial dermomyotome in controls (E, arrows) is again not evident in mutants (F). (G,H) Para-sagittal sections of control and mutant embryos at E10.5. In control embryos, the dorsal root ganglia (DRG) are regularly sized and shaped (G, arrows); by contrast, they are uneven in size and spacing and often fused in mutants (H, arrows).

View larger version (50K): [in a new window]   Fig. 4. Somite epithelialisation and patterning requires both Meox genes. In situ hybridization analysis of Mesp2 (A,B), paraxis (C-F) and Dll1 (G-J) expression in control (A,C,E,G,I) and double Meox1-/-;Meox2-/- mutant (B,D,F,H,J,) embryos at E9.5. In A,B,E-N the caudal end of the embryo is towards the right. (A,B) Dorsal aspects of Mesp2 expression in the rostral presomitic mesoderm. Control (A) and mutant (B) embryos have similar expression profiles. Expression of paraxis in whole-mount preparations (C,D) and para-sagittal sections (E,F). In controls (C,E), paraxis is expressed throughout the epithelial somites, but double mutant embryos (D,F) express paraxis in a dorsally restricted domain. Furthermore, this expression is not maintained in older, more rostral, somites. Expression of Dll1 in caudal (G,H) and rostral (I,J) somites. In control embryos (G), Dll1 is expressed at high levels in presomitic mesoderm (black arrowhead), the caudal halves of newly formed somites (arrows) and the neural tube (white arrowhead). In mutants (H), Dll1 is expressed at high levels in the presomitic mesoderm (black arrowhead), but its expression in somites is virtually extinguished, whereas the neural tube expression remains (white arrowhead). (I) Dll1 is expressed in the myotome (arrows) of anterior differentiated somites of control embryos, and in the neural tube (white arrowhead). In mutants (J), the myotome expression is fused ventrally (arrows). (K-N) In situ hybridization analysis of Epha4. (K) In control embryos, Meox1+/-;Meox2+/-, dorsal views of Epha4 expression show a broad stripe (arrowhead) in the most rostral presomitic mesoderm (in the next somite to form) and a narrow stripe of expression (arrow) in the rostral half of the most recently formed somite. In embryos with one wild-type Meox allele, Meox1+/-;Meox2-/- (L) and Meox1-/-;Meox2+/- (M), the rostral somite stripe of expression (arrow) is less refined. In Meox1-/-;Meox2-/- mutants (N) the rostral half-stripe of Epha4 expression is absent and only the posterior stripe (arrowhead) is visible.

View larger version (118K): [in a new window]   Fig. 5. Disrupted sclerotomal differentiation in Meox1;Meox2 mutants. Whole-mount preparations of in situ hybridization analysis of markers expressed at E9.5 in the sclerotome: Pax1 (A-F), Pax9 (G-J), Twist (K-N) and Foxc2 (O-R). Representative transverse cryosections of whole-mount preparations of control (C,H,L,P) and mutant (F,J,N,R) embryos are shown. (A-C) Pax1 is expressed at high levels in the sclerotome of control embryos (arrowheads), but is not detected in the somites of Meox double mutants (D-F), although branchial arch and limb bud expression persists (arrows). The expression of Pax1 is first seen shortly after epithelial somites form in control embryos (B,C; arrowhead), but is not induced in Meox double mutants (E,F; arrowhead). (G-J) Pax9 is also expressed at high levels in the sclerotome of control embryos (G,H), especially in the caudal half-somites (arrowheads). By contrast, double Meox mutant embryos show greatly reduced Pax9 expression, most evident in the caudal half-somites (I,J). The residual Pax9 expression is restricted to the sclerotomal cells closest to the neural tube (J). (K-N) Twist RNA is detected throughout epithelial somites, and in the sclerotome and dermomyotome of differentiated somites (K,L). In Meox double mutants (M,N), Twist expression is greatly reduced in somites, while expression persists in branchial arches and limb buds. (O-R) Foxc2 is expressed in control embryos in sclerotomal cells (O) preceding Pax1 and Pax9, while its expression in the posterior half of somites of mutant embryos is reduced dramatically in the mutant (Q). The distribution of the Foxc2 signal on sectional analysis is, however, similar in control (P) and mutant (R) embryos.

View larger version (88K): [in a new window]   Fig. 6. Regulation of skeletal myogenesis in Meox1;Meox2 double mutants. In situ hybridization analysis of gene expression at E9.5 for: Pax3 (A,B), Pax7 (C,D), Myf5 (E-H) and myogenin (I,J). Control embryos (A,C,E,I), Meox mutants (B,D,F,G,H,J). (A) Pax3 is expressed in the dermomyotome of differentiated somites (arrowheads) and the neural tube (arrow). In Meox1-/-;Meox2-/- embryos (B), however, Pax3 is expressed at very reduced levels in the ventrolateral region of the dermomyotome of somites (arrowheads), while neural tube expression remains normal (arrow). The dermomyotome Pax7 expression seen in control embryos (arrowheads) (C) is extinguished in Meox double mutants (D). Myf5 mRNA is localised to the ventrolateral dermomyotome of control embryos (arrowhead, E). In double mutant embryos, Myf5 expression is not detected in caudal somites, and only at reduced levels in rostral somites (F). Myf5 expression is limited along the dorsoventral axis in mutants, compared with controls. Embryos with only one wild-type Meox allele have an intermediate phenotype; those with one Meox1 allele (G), Meox1+/-;Meox2-/-, were less severely affected than those with one Meox2 allele (H), Meox1-/-;Meox2+/-. (I,J) In the absence of Meox gene function, the expression of myogenin was reduced and limited to inter-limb somites (J).