Fig. 4. X-Gal and GFP expression in heterozygous (A,C,E,G,I,K,M,O,Q,S,U,W,Y) and homozygous mutant animals (B,D,F,H,J,L,N,P,R,T,V,X,Z) at E9.5 (A-D), E10.5 (E-P), E11.5 (Q-V) and E12.5 embryos (W-Z), revealing Six4-GFP expression and Six1-lacZ expression. (A-D) Six1 expression is detected in the head mesenchyme (asterisk in A), otic vesicle (white arrow in A), branchial arches (black arrow in A) and pharyngeal clefts (white arrows in C) in heterozygous embryos. In dKO embryos, Six1 expression at the head level is severely reduced (asterisk in B) and lost in the otic vesicle (white arrow in B); although mesodermal Six1 expression is still detected (D), most Six1 expression is lost in pharyngeal pouch endoderm and surface ectoderm in dKO embryos. Fusion between the first and second branchial arches is observed at this stage (white arrow in D). At the thoracic level, the nephrogenic chord expression of Six1 is detected in the heterozygote (black arrowhead in A) but not detected in the dKO embryo (black arrowhead in B). At the somitic level, Six1 is expressed at comparable levels in the heterozygous (A) and homozygous (B) animals. (E-P) Six1 is still highly expressed in the ventral otic vesicle (white arrow in E,G) and trigeminal ganglion (white arrowhead in E,G) in heterozygotes, but not in the dKO embryos (white arrow and white arrowhead, respectively, in F and H). (G,H) Enlargement of control (G) and KO (H) embryos at the head level. (I,J) Enlargement of control (I) and KO (J) embryo at the interlimb level. At the somitic interlimb level, Six1 expression is detected in the myotome (E,I). In the dKO, Six1 expression is more diffuse, while still expressed in myotomes (F,J). (K-P) Vibratome sections. At the hindlimb level (K,L), Six1 is detected in the nephrogenic chord (arrow in K) but is not in the dKO, while many ß-gal-positive cells invade this ventral region of the embryo (L). Six1-positive cells enter the limb bud in the heterozygous embryos (K), but not in the dKO embryo, where somitic ß-gal-positive cells are not confined to the somite but are found more ventromedially (L). At the interlimb level (M,N) Six1-positive cells are diffuse and do not extend ventrally in the dKO embryo (N), while they invade ventral region in the heterozygous embryos (M). Six1 expression in DRG is preserved, but a few ß-gal-positive cells are also seen in the neural tube in the dKO (N,P; data not shown). At the forelimb level (O,P), Six1-positive cells are present in the limb bud in the heterozygous embryos (O), while in dKO embryos most cells accumulate less ventrally, and are not found in the limb bud, but are also detected beneath the ectoderm more medially (P). (Q-V) Six1 (Q, R) and Six4 (S-V) are mainly detected in limbs, and somites, where they are co-expressed in the DRG, myotomes, and ventral (black arrows) and dorsal dermomyotomal lips. In dKO, somites still express these two genes (R,T). Myotomes are disorganized, their ventral extensions are reduced (white double headed arrow), and GFP and ß-gal are no longer detected in the ventral lips (black arrows). A population of GFP and ß-gal-positive cells is found in the limbs of dKO embryos but they are not myogenic cells (Bonnin et al., 2005). (W-Z) Six1 is expressed at higher levels in heterozygous (W,Y) than in homozygous mutant embryos (X,Z). At the thoracic level, ventral extension of the dermomyotome, normally marked by Six1 expression, is lost in dKO embryos (white arrows). Posterior expression in the limbs is maintained.