Année 2006-2007
Université de la Méditerranée
Maîtrise 1er semestre
Biologie du Développement
Prof. Christophe Marcelle
marcelle@ibdm.univ-mrs.fr
Formation des membres chez les vertébrés
1) Origine embryonnaire des tissus formant les membres
antérieurs et postérieurs
Domains of somitic myogenesis in the early chick embryo. (a) Schematic view
of a three-day chick embryo (stage 18, 36 somites) with somites (shown in gray) on
either side of the neural tube. (b–e) Cross sections of somites and axial tissues on
the right-hand side of the embryo at levels indicated in (a). (b) Epithelial somite
(level I). Newly formed somite receives signals from the neural tube (nt),
notochord (ntc), surface ectoderm (se), and lateral plate mesoderm (lpm). Lineage
tracing experiments have shown that the dorsal part of the somite is fated to form
the dermomyotome (blue). (c) Somite at level VII. Dermomyotome (dm, blue) has
differentiated from the dorsal region of the somite and expresses Pax-3 and Pax-7.
The dorsomedial lip (dml) of the dermomyotome forms next to the neural tube and
expresses MyoD and high levels of Myf-5. Ventral region of the somite undergoes
an epithelium to mesenchyme conversion and forms the sclerotome (scl) that will
later give rise to the vertebrae and ribs. (d) Thoracic/interlimb level somite. Cells
from the dermomyotome translocate through the medial and lateral lips to form the
epaxial and hypaxial myotome (both shown in red). The lateral lip of the
dermomyotome grows ventro-laterally and will later comprise the ventral body
wall muscles (gray arrows). (e) Forelimb level somite. Myotome (red) continues to
form subjacent to the dermomyotome (blue). Long-range migrating MPCs
delaminate from the hypaxial dermomyotome and invade the limb bud. During
their migration they express Pax-3. Upon differentiation in the limb bud, they will
express MyoD and Myf-5.
2) Régulation moléculaire de la migration des progéniteurs
musculaires dans le membre
KI Lacz dans le locus de Myf5. Coloration ßgal. A:
Heterozygote Myf5 LacZ; B. Splotch (-/-) / Heterozygote
Myf5 LacZ
Poulet 3 jours, section transversale, anticorps anti Pax3. Remarquer l'expression de Pax3
dans la partie dorsale du système nerveux, dans le dermomyotome, ainsi que dans les cellules
migrant dans le mésenchyme de l’aile.
KO Pax3. Observer (flèches) l'absence de tous les progéniteurs
Musculaires migratoires (progeniteurs de la langue, des membres
Et –non indiqués- du diaphragme). En plus, on remarque des défauts
au niveau des DRG.
Fig. 3 Aberrant migration of limb muscle precursor cells lacking Lbx1h. ß-gal
staining of Lbx1h –/– (a,c,e,g,i,k) and Lbx1h +/– (b,d,f,h,j,l) embryos at different stages of
development. Lateral views of E10.5, 34-somite (a,b) and E13.5 (i,j) whole-mount
preparations and a dorsal view of cleared E11, 38-somite embryos (c,d) are shown.
Transversal vibratome sections through forelimbs (e,f) and hindlimbs (g,h) of E10.5, 34somite embryos are shown. k,l, Transversal vibratome sections through the shoulder region
of E13.5 embryos. In Lbx1h +/– embryos, migration of ß -gal + cells into limb buds and
formation of individual muscles is normal. In contrast, dorsal muscle precursor cells of
forelimbs and all muscle precursor cells of hindlimbs have lost their orientation and are
dislocated in the intermediate and lateral plate mesoderm. The dislocation is particularly
evident in (a; red arrowhead) and (e,g; black arrow). ß -gal + , Lbx1h –/– muscle precursor
cells that form flexor muscles of forelimbs are less affected and follow their original path
(black arrows in c and e), although their migration is slightly retarded. Precursor cells of
flexor muscles are visible on the ventral side of the forelimb in (i). Lbx1h –/– mice lack
precursor cells of extensor muscles of forelimbs (red arrowheads in c,e,i). No ß -gal + ,
Lbx1h –/– muscle precursor cells are present in hindlimbs of Lbx1h –/– mice (red arrowhead
in g, black arrowhead in i).
Fig. 2 Absence of extensor muscles in forelimbs of Lbx1h –/–
neonates. Comparable transverse sections of forelimbs of wild-type
(a,c,e,g) and Lbx1h –/– (b,d,f,h) mice were stained with antibodies
against Myhc. Sections through the shoulder region (a,b), the forearm
proximal (c,d) and distal (e,f) of the elbow, and the metacarpals (g,h) are
shown. Arrows in (d,e) indicate the complete absence of extensor
muscles in Lbx1h –/– mice. Formation of flexor muscles is virtually normal in the proximal forelimb. The arrowhead in (h) points to the absence
of all muscles including mm. flexores digitorum in the metacarpal
region.
3) Croissance des membres: rôle de l'AER, rôles des FGFs
A-F: TUNEL; G-H: BrdU après ablation de l'AER.
Figure 1 Timing of Fgf4 and Fgf8 inactivation in hindlimb and forelimb buds. a, Diagram
of mutant alleles carried by Msx2-cre; Fgf4; Fgf8 (double knockout, KO) animals. Cre
recombinase, expressed in limb bud ectoderm under the control of Msx2 promoter
elements, mediates intragenic recombination of loxP sites flanking Fgf4 and Fgf8
sequences. This deletes essential exons in the floxed alleles, converting them to null
alleles. b–m, Analysis of gene expression and protein levels in hindlimb (b–g) and
forelimb (h–m) buds. Limb bud developmental age is reported in terms of total number
of somites (som). Intact limb buds (b–e, h–k) are shown in dorsal view, with anterior on
the left and posterior on the right. Assays were performed for Fgf4 (b, c, h, i) and Fgf8
(d, e, j, k) expression by RNA in situ hybridization in whole mount, and for FGF8
protein (f, g, l, m) by immunofluorescence. The arrows in i and k point to precocious
Fgf4 expression and the abnormally small domain of Fgf8 expression, respectively. n,
Diagram illustrating the timing of Fgf4 and Fgf8 expression and the timing of Msx2-cre
function.
Figure 2 Limb skeletal phenotypes. a–h, Skeletal preparations of hindlimbs (a, b) and forelimbs
(c–h) from normal and double knockout mice at birth, stained for cartilage (alcian blue) and bone
(alizarin red). Arrows in b point to vestigial cartilaginous elements present in place of the hindlimb
skeleton in double knockout mice. Note that the pelvic girdle, which is not derived from the
hindlimb bud, appears normal. Forelimbs of two double knockout mice are shown, illustrating the
most severe (d) and mildest (e) phenotypes obtained. The arrow in d points to the olecranon process,
which identifies the single zeugopod element as an ulna. Autopods of the skeletons in c, d, e, are
shown at higher magnification in f, g, h, respectively.
Figure 3 AER morphology and gene expression in hindlimb buds. a, b, Transverse sections through the
AER of limb buds at 34 som, stained with anti-E-cadherin (red) and Hoechst dye (blue) to illustrate AER
morphology. c–x, Gene expression assayed by RNA in situ hybridization in whole mount. c–h, AER-FGF
genes: Fgf8 (35 som), using an Fgf8 exon-1-specific complementary DNA probe containing sequences not
deleted by Cre (320 base pairs (bp) including 84 bp of 5' untranslated region); Fgf9 (36 som); Fgf17 (34 som).
i–p, Genes whose expression was not detected in mutant mesenchyme: Bmp4 (36 som); Ptch (34 som);
Hoxd13 (36 som); Fgf10 (35 som). The arrow in i points to the mesenchymal expression domain of Bmp4 in
the normal limb bud. q–x, Genes whose expression was detected in mutant mesenchyme: gremlin (40 som);
Msx1 (35 som); Meis1 (34 som); Alx4 (35 som).
4) Polarité dorso-ventrale du membre des vertébrés:
E) Polarité antéro-postérieure du membre
P