Very Severe Aortic Arch Defect in Chrd−/− Embryos: Individual AA2-6 Never Form
Injection into the wild-type arterial system allowed observation of the stages of formation of the aortic arches. Each new aortic arch sprouts ventrally from the leading edge of the immediately anterior one, probably by angiogenesis, although the existence of local foci of vasculogenesis in the newly formed branchial arches cannot be ruled out. Interestingly, it appears that pulmonary arteries are formed in the same way. To understand whether the abnormal OFT observed in newborn Chordin mutants (Bachiller et al.,2003) results from impaired formation or from abnormal remodeling of the aortic arches, we injected dye in stage-matched sibling pairs of E9.5 to E12.5 wild-type and homozygote mutant embryos. These experiments demonstrated a severely impaired aortic arch development, with only two arteries allowing communication between the aortic sac and the paired dorsal aortae, at different times. AA1 forms normally, but appears to resolve into a capillary bed earlier in mutants than in wild-types. In consequence, we did not observe concurrent patent states of AA1 and the single posterior aortic arch (although it could occur at stages not analyzed). Posteriorly to AA1, only one vessel formed in lieu of AA2-4. This finding is associated with an absence of the matching branchial arches. The shape of the unique vessel suggests that a whole cube of tissue is missing in this region where the branchial arches should develop, with impaired growth along both the antero–posterior and dorso–ventral axes. In mutants, the existence of ventral angiogenic sprouts that lead to the formation of the following aortic arches in wild-types may be guessed in some pictures, but they do not extend, perhaps because they do not find the cues to join a matching aortic arch. The angiogenic process, however, is not blocked as the pulmonary arteries will form from the appropriate place in the mutant, although with a significant delay. This finding results in the normal formation of pulmonary arteries in the absence of a sixth aortic arch.
Multiple Defects in the Anterior Cardinal Veins of Chrd−/− Embryos
Injection into the heart at E9.5 or into the atria at later stages, uncovered several vein patterning defects in the neck and head region of Chordin mutant embryos, including absence of venous branches, abnormal patterning, and a midline defect. The earliest detected anomaly was the presence of “rings” in the anterior cardinal veins, at a stage where they are still composed exclusively of endocardial cells, before their colonization by smooth muscle cells (Takahashi et al.,1996). These rings were either vessels anastomosing on themselves (loops) or following complex three-dimensional paths never observed in wild-type embryos. In addition, one of the three main venous trunks draining the embryonic head, the pre-otic vein, was absent (or at least not patent) in mutants. Also, a segment of the ACV posterior to the origin of the post-otic vein was missing, resulting in the abnormal location of the venous trunk that drains the mandibula. Finally, the thyroid/thymic trunk, which normally originates bilaterally from the anterior cardinal veins and follow the jaw line until both branches face each other close to the midline (where they eventually anastomose), was asymmetrical in mutants, with the dominant branch crossing the midline. While the genetic distinction between arteries and veins is now known to arise very early (reviewed in Adams,2003), the signals that govern patterning of veins and the developmental mechanisms leading to the formation of cerebral veins are largely unknown. The defects we observed were restricted to the neck and head regions, and never appeared in the trunk or tail regions, in keeping with the sites of expression of Chordin in the embryo (Bachiller et al.,2003). Thus our results show that the BMP signaling pathway is critical for the formation of the ACVs and some of their tributaries.
All vessels are first composed of an endothelial layer, which is derived from angioblasts. Using antibodies against quail angioblasts, it has been shown that the main systemic vessels form by at least two different mechanisms. Early vessels such as the posterior cardinal veins or the aorta form by coalescence of angioblasts (Coffin and Poole,1988; Pardanaud et al.,1989), a process termed vasculogenesis (Risau et al.,1988). The precise mechanism of vasculogenesis leading to the formation of the primary embryo vasculature is species-dependent (Coffin and Poole,1991; Cleaver and Krieg,1998). For example, formation of posterior cardinal veins in frogs and the dorsal aorta in quail does not require cell migration (vasculogenesis type I). Conversely, angioblasts migrate before coalescing (vasculogenesis type II) to form the posterior cardinal veins of quail and the dorsal aorta in Xenopus. Only mesoderm in contact with endoderm contains angioblasts, and the role of endoderm appears to be twofold: first to induce angioblast in the adjacent mesoderm, and later to pattern the mesoderm, creating regional differences (Pardanaud et al.,1989). The primary vessels assembled by vasculogenesis are then thought to extend throughout the embryo by angiogenesis, a process that requires cell proliferation and migration. While patterns of expression of the zebrafish homologue of Fli-1, a marker of the developing endothelium, suggested that head vessels form differently than trunk vessels (Brown et al.,2000), vein formation is not described in detail in the head, even in chicken where most studies were performed. Very early ink injection experiments in chick suggested that the ACVs form from the remodeling of a thin capillary network, connected to the aortae (Evans,1909), but experimental data were scarce. Since then, most mapping experiments have focused on the angiogenic potential of identified populations of mesenchyme, rather than on the origin of the angioblasts that form individual head vessels (Noden,1988,1990; Couly et al.,1995). Therefore, it is unclear whether mammalian ACVs and their tributaries are formed by angiogenesis, vasculogenesis, or by a combination of both. In chicken, vasculogenesis mostly occurs in organs composed of both endoderm and mesoderm, while tissues composed of mesoderm and ectoderm are sites for angiogenesis (Pardanaud et al.,1989), although there are some exceptions to this principle, such as the formation of the coronary vessels (Kattan et al.,2004). Whether this happens also in mice is not fully demonstrated, and certainly in the case of neck veins both mechanisms could occur. On one hand, grafting experiments in quail/chick chimeras demonstrated the ability of angioblasts to aggressively migrate through head tissue (Noden,1991) and a parallel between the behavior of neural crest cells and angioblasts has been postulated (Coffin and Poole,1991). On the other hand, the ACVs form adjacent to (chordin-expressing) endoderm, and could derive by vasculogenesis from angioblasts resident in the paraxial cephalic mesoderm, which has extensive angiogenetic potential (Noden,1990; Couly et al.,1995).
Regardless of the mechanism of assembly of the angioblasts, Chordin deficiency in the endoderm could affect ACV formation by at least three different mechanisms. Absence of chordin in the cephalic mesendoderm could result directly in the absence of cues for angioblastic migration. This could explain the loops and generally abnormal paths followed by the ACV, as well as the absence of the vena preotica. Absence of chordin could also result in missing inductive signals to pattern regional differences, which may lead to the absence/reduction of segments along the antero–posterior axis. In addition, the various anomalies of ACV development seen in Chrd mutants could be an indirect result of the inability of neural crest cells to migrate through this region of the neck (Bachiller et al.,2003). While the cephalic neural crest itself does not have angiogenic potential, it is possible that interaction between neural crest and migrating angioblasts is necessary for the latter to find their geographical cues. Relations and mutual developmental influence between cranial mesoderm and neural crest is well documented in the case of musculoskeletal structures formation (reviewed in Noden and Trainor,2005).
The last anomaly of vein patterning observed in Chrd−/− embryos was a midline defect, where the veins draining the thymic/thyroid region develop asymmetrically and one of them crossed the midline. Interestingly, while nothing is known of the signals that govern the formation of this particular vein, Chordin was recently suggested to be involved in vascular midline patterning in a different context (Reese et al.,2004). In the embryonic disc, the midline remains an avascular zone, bordered by the paired dorsal aortae, a phenomenon dependent on the presence of the notochord, a Chordin-expressing structure. Implantation of Chordin-expressing COS cells in the paraxial or lateral plate mesoderm is sufficient to create a vascular-free zone in quail. In addition, Chordin and another BMP antagonist, noggin, were shown to inhibit endocardial cell migration in vitro. These data have suggested that chordin and/or other BMP inhibitors could contribute to forming the median avascular zone of the embryo (Reese et al.,2004). Chrd−/− mouse embryos do not (Bachiller et al.,2003) appear to have lost that avascular region, which could be due to species differences, or to molecular redundancy. However, it is possible that the midline defect we observed in mutant cephalic veins is a more subtle manifestation of a similar molecular mechanism.
In conclusion, our data show that Chordin, a member of the BMP signaling pathway, is critical for the early development of the neck and head vasculature. In particular, we were able to demonstrate the existence of severe defects in the formation of the aortic arch arteries and of the anterior cardinal veins and some of their tributaries. A novel approach to the dye injection technique allowed us to visualize the new phenotypes in early mouse mutant embryos. This innovative technique, applied to other mouse mutant strains, will help to uncover the morphological abnormalities produced by alterations in other regulatory pathways, and open the possibility for further studies into the molecular basis of such defects.