Abnormal patterning of the pharyngeal arch arteries and aortic sac in Alk5/Wnt1Cre mutants
During cardiovascular development, the PAAs undergo a complex set of sequential asymmetric remodeling steps resulting in the left-sided aortic arch. To determine, whether ALK5-mediated signaling was involved in remodeling of PAAs, we performed intracardiac India ink injections at different developmental stages. While at E10, Alk5/Wnt1-Cre mutants did not show obvious differences in the PAAs, abnormal remodeling became obvious in mutants a day later at E11 (Fig. 2). The controls displayed the well-formed 3rd, 4th and 6th PAAs. Moreover, the carotid duct (the dorsal aorta between the 3rd and 4th PAAs) was already regressing as demonstrated by the reduced size (Fig. 2A). In Alk5/Wnt1-Cre mutants, the 3rd and 4th pairs of PAAs were bilaterally hypoplastic, whereas the 6th pair of PAAs was notably hyperplastic (Fig. 2B). Furthermore, the carotid duct was remarkably large, when compared to controls. While the controls displayed an interruption of the carotid duct at E12 and E13 as expected (Fig. 2C), the mutants demonstrated an uncharacteristic break of the dorsal aorta between the 4th and 6th pairs of PAAs (Fig. 2D).
Figure 2  Abnormal patterning of the PAAs in Alk5/Wnt1-Cre mutants. Left lateral view after intracardiac ink injections to visualize the developing PAAs at E11.0 (A,B), E12.0 (C, D) and E13.0 (E, F) in controls (A, C, E) and Alk5/Wnt1-Cre mutants (B, D, F). Arrow in A points to the regressing carotid duct. Asterisk in B depicts the corresponding structure in the mutant with no signs of regression. Asterisk in D illustrates the aberrant regression of the dorsal aorta between the 4th and 6th PAAs. PT, pulmonary trunk; Ao, Aorta; TA, truncus arteriosus. Around E11.5, the aortic sac normally forms a distinctive T-shaped structure, as seen in frontal sections of the control sample in Fig. 3(A,C). Subsequently, the right horn of this structure transforms into the prospective brachiocephalic artery, while the left horn together with the left 4th PAA gives rise to the definitive aortic arch [23]. In Alk5/Wnt1-Cre mutants, the T-shaped aortic sac failed to form (Fig. 3B,D). Instead, the truncus bifurcated to a left and right arm, which further branched to the PAAs, particularly to the predominant pair of 6th PAAs (Fig. 3B,D). The observed phenotype is consistent with the absence or severe hypoplasia of structures derived from the aortic sac in late stage embryos (E17), e.g., the missing brachiocephalic artery and severe shortening of the ascending truncus as shown in the Figure 1.
Figure 3  Abnormal Aortic Sac in Alk5/Wnt1-Cre mutants. Alk5/Wnt1-Cre mutants (B, D) fail to form the typical T-shaped structure of the aortic sac seen in controls at E11.5. (A, C). A-B, frontal image of ink-injected embryos; C-D, frontal sections (H&E staining). Arrows in A and B point to the level of section shown in C and D (red arrows in C and D point to the aortic sac of the control and mutant, respectively).