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Keywords:

  • anterior cardinal vein;
  • bone morphogenetic protein;
  • chordin;
  • DiGeorge syndrome;
  • aortic arches;
  • pharyngeal development;
  • persistent truncus arteriosus;
  • outflow tract;
  • vein patterning;
  • vascular defect

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Classic dye injection methods yielded amazingly detailed images of normal and pathological development of the cardiovascular system. However, because these methods rely on the beating heart of diffuse the dyes, the vessels visualized have been limited to the arterial tree, and our knowledge of vein development is lagging. In order to solve this problem, we injected pigmented methylsalicylate resins in mouse embryos after they were fixed and made transparent. This new technique allowed us to image the venous system and prompted the discovery of multiple venous anomalies in Chord−/− mutant mice. Genetic inactivation of Chordin, an inhibitor of the Bone Morphogenetic Protein signaling pathway, results in neural crest defects affecting heart and neck organs, as seen in DiGeorge syndrome patients. Injection into the descending aorta of Chrd−/− mutants demonstrated how a very severe early phenotype of the aortic arches develops into persistent truncus arteriosus. In addition, injection into the atrium revealed several patterning defects of the anterior cardinal veins and their tributaries, including absence of segments, looping and midline defects. The signals that govern the development of the individual cephalic veins are unknown, but our results show that the Bone Morphogenetic Protein pathway is necessary for the process. Developmental Dynamics 236:2586–2593, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Ink injection has been used for at least a century to visualize the cardiovascular system (Evans,1909). We (Délot et al.,2003) and many others have successfully used this technique to visualize anomalies of the developing cardiovascular system in mouse embryos. The occurrence of resins, either in liquid form or polymerizing as casts, has allowed considerable refinement and has yielded amazingly detailed images, in particular of the remodeling of aortic arches, both normal and pathological (e.g., Merscher et al.,2001). However, because these methods rely on the beating heart to diffuse the dyes, the vessels visualized have been mostly limited to the arterial tree, and our knowledge of mammalian vein development has been lagging. We modified the technique, using injection of pigmented methylsalicylate resins into mouse embryos after they had been fixed and cleared. The arterial tree can be visualized with this method by injecting into the descending aorta in the tail/trunk region. In fact, the images of the pericardiac vessels were very clear because the heart itself is generally not stained. In addition, varying the point of injection allowed us to visualize the venous system. We illustrate the technique using wild-type embryos at various stages of development, as well as homozygous mutants for the Chordin gene.

Genetic inactivation of Chordin, a gene encoding an extracellular inhibitor of the bone morphogenetic protein (BMP) signaling pathway (Sasai et al.,1994), results in lethality at birth with multiple defects, localized to organs of the peripharyngeal region of the mutants (Bachiller et al.,2003). The anomalies include hypoplasia or absence of thymus and parathyroid glands, and cardiac outflow tract (OFT) defects, and anomalies of several skull bones. All affected tissues are derivatives of the pharyngeal endoderm or of tissues that require signals from the pharyngeal endoderm during embryogenesis. Chordin is expressed during early development in the dorsal mesendoderm of the mouse embryos (Bachiller et al.,2000). Later on, the expression is restricted to the dorsal part of the pharyngeal endoderm, where its role appears to be at least twofold. It is required for pharyngeal development per se, but also for the patterning of adjacent mesoderm and neural crest migrating through it. Absence of Chrd first results in deficiencies in the formation of derivatives of the pharyngeal endoderm (thymus, parathyroids), defects that are associated with a decrease in expression of the transcription factor Tbx1 in the pharyngeal endoderm and are also observed in Tbx1 mutants (Jerome and Papaioannou,2001; Lindsay et al.,2001; Merscher et al.,2001). In addition, the spectrum of defects strikingly resembles that seen in models of cephalic neural crest ablation in chick (Kirby et al.,1983). Several aspects of the phenotype, such as total absence of the aortico-pulmonary septum (persistent truncus arteriosus) or abnormal remodeling of the aortic arches (retroesophageal subclavian arteries), are attributable to a failure of neural crest to reach the heart region and are similar to the defects observed in the human DiGeorge/Velocardio-facial syndrome spectrum (OMIM #188400 & #192430). Gene expression studies in Chrd−/− embryos (Bachiller et al.,2003) showed that Chordin is upstream of two other genes involved in the pathogenesis of DiGeorge syndrome models, Tbx1 and Fgf8 (Abu-Issa et al.,2002; Frank et al.,2002; Macatee et al.,2003; Xu et al., 2004). As Chrd expression is restricted to the dorsal endoderm in the neck region at embryonic day (E) 9.0, we hypothesized that absence of Chordin in this tissue resulted in the lack of a signal for neural crest cells to use mesendoderm as a substrate for migration and, in consequence, a lack of neural crest cells in the organs they normally populate (Bachiller et al.,2003). It should be noted, however, that not all the neck region defects seen in Chrd−/− fetuses can be attributed to endodermal or neural crest derivatives. In particular, defects in bones of the base of the skull suggested that absence of Chordin also affects specification, migration, or differentiation of cells of mesodermal origin (Bachiller et al.,2003).

To better understand how the cardiac and aortic arches defects observed in Chrd−/− mice arise during embryogenesis, we injected pigmented methylsalicylate into the descending aorta in the tail of midgestation embryos. The resin diffused to the arterial system, and allowed us to image how the persistent truncus arteriosus of Chrd−/− mutants arises from a very severe phenotype of the developing aortic arches. Because it does not rely on dispersion of dye by the beating heart, this novel injection method also allowed us to visualize the developing venous system. Multiple anomalies of the development of the anterior cardinal vein and its tributaries were uncovered in the anterior region of Chrd−/− mutants. Although the signals that govern the assembly of individual cephalic veins are currently unknown, our results suggest that the BMP pathway plays an important role in this process. Identifying the genetic interactions involved will be important not only to understand cephalic venous development and its anomalies, but also for the study of the lymphatic system, which is initiated by the budding of lymph sacs off the anterior cardinal veins (ACVs; Blum and Pabst,2006).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The use of colored resins injected in the beating hearts of mouse embryos yields exquisitely refined images and has greatly enhanced our ability to describe vascular phenotypes, in particular those of aortic arches remodeling defects. However, in mouse embryos, these have been mostly used for cardiac ventricular injection, limiting the reach of the dye to the arterial system. We have refined the technique by injecting the pigment after the embryos were fixed and cleared. This modification permits the selection of dye entry points other than the heart, and consequently, allows for the labeling of the venous tree. Double injection into each of the ventricles of the heart of wild-type embryos at E14.5, that is, after ventricular septation is complete, yielded dual-color images of the OFT vasculature with good detail, including ramifications of the pulmonary arteries into the lung lobes (Fig. 1A). The resolution at these late stages is limited by the opacity of the embryo. In earlier embryos, either the venous (Fig. 1B) or arterial (Fig. 1C) vessels could be visualized by varying the point of dye injection.

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Figure 1. Different injection sites label different parts of the cardiovascular system in wild-type mouse embryos. A: Injection into the left and right ventricles of an embryonic day (E) 14.5 embryo with red and blue pigments, respectively. The branching of the left pulmonary artery (PA) in blue, the origins of the coronary arteries in red (Cor), and the ductus arteriosus (DA), a normal structure during embryonic development, are clearly visible. B,C: At E9.0–E9.5, injection into the heart resulted in labeling in the venous system (B) while injection in the tail marked the arterial system (C). Injection sites are marked with an asterisk.

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Arterial Development of the Wild-Type OFT

At early E9.5 stage, the first two branchial arches are formed and are irrigated by bilateral aortic arches (AA) 1 and 2. AA3 is seen sprouting from the ventral edge of AA2 (arrow in Fig. 2A). At a later E9.5 stage (see shape change of first branchial arch), aortic arches 1 to 3 are now formed and the leading edge of AA4 is visible (Fig. 2C). AA1 has changed shape and is starting to resolve into a capillary bed in the first branchial arch. The next day (Fig. 2E), all aortic arches have formed, and the pulmonary arteries are seen extending from the ventral edge of AA6. AA2 is forming a capillary bed, a process that is now complete for AA1. Penetration of dye into this capillary bed is generally difficult and may (Fig. 2G) or may not (Fig. 2E) succeed. At E11.5, the OFT has started septating and two separate blood flow compartments are highlighted by the resin (magnified in Fig. 2H). The change of shape of aortic arches 3, 4 and 6 is also visible, as a result of elongation of the aortic sac segment between AA4 and AA6. At E12.5, aortic arch remodeling is well under way, starting posteriorly: pulmonary trunk and aorta are now fully septated (Fig. 2J) and the left sixth aortic arch has regressed and is no longer communicating with the OFT (Fig. 2K).

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Figure 2. Development of aortic arches and persistent truncus arteriosus in Chrd−/− embryos. A,C,E: Wild-type embryos. B,D,F: Chrd−/− embryos. Embryos were staged-matched according to first branchial pouch shape, which is unaffected in Chrd mutants (Bachiller et al.,2003). Aortic arches are numbered with roman numerals. H, heart; OV, otic vesicle; CA, cephalic artery; TA, truncus arteriosus. Blue resin was injected in the tail artery. At each stage, one embryo is shown representative of the phenotype, which was fully penetrant and showed very little variability. A,B: Early embryonic day (E) 9.5. In the wild-type (A), branchial arches 1 (BA1) and 2 are formed, and the leading edge of forming aortic arch (AA) 3 is indicated by an arrow. B: Only the first aortic arch is patent at this stage in the mutant. C,D: Late E9.5. C: In the wild-type, AA1-3 are now formed and the leading edge of the forming AA4 is indicated by an arrow. D: In the mutant, AA1 has already remodeled and broken into a capillary bed. Only one arterial arch forms posterior to AA1. E,F: E10.5. In the wild-type, all arterial arches have now formed and AA1 and 2 are remodeled. Pulmonary arteries (PA) are forming off AA6. In the mutant, there is still only one aortic arch posterior to AA1 and the leading edges of the PAs have not formed yet. G–I: At E11.5 in the wild-type (G), the outflow tract (OFT) has started septating between left and right blood flows (magnified in H), while the mutant OFT remains a single artery (I). Pulmonary arteries (black arrowheads in G,H) are not visible in the mutant. J–L: E12.5. In the wild-type (J, left view, and K, right view), the OFT is now fully remodeled into aorta (Ao) and pulmonary trunk (PT), and the right AA6 is regressing (highlighted in dotted red line). The pulmonary arteries have now formed in the mutant (arrowhead in L). The single vessel of the outflow tract (OFT) constitutes a persistent truncus arteriosus (TA). dAo, descending aorta.

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Development of the Persistent Truncus Arteriosus in Chrd−/− Embryos

Chordin homozygous mutants develop a fully penetrant OFT defect, with very little variability in its manifestation. It is characterized, at birth, by a total absence of the aortico-pulmonary septum (or persistent truncus arteriosus) and the lack of large portions of the great vessels derived from the pharyngeal arch arteries (Bachiller et al.,2003). To understand how such a phenotype arises during embryogenesis, we followed the development of the aortic arches in Chrd−/− embryos between E9.0 and E12.5.

Differences between mutant and wild-type littermates were seen at the earliest stage examined. At early E9.5, only AA1 is visible, and no posterior communication between the aortic sac and the dorsal aortae exists (Fig. 2B). The cephalic arteries were not detectable in the mutants, but these vessels were visible in only one (Fig. 2A) of four wild-type embryos at this stage, indicating that they become amenable to dye injection around this time of development. Furthermore, the cephalic arteries can be seen in late E9.5 mutants (Fig. 2D), which suggests that their early absence is most likely due to a minor developmental delay. Later that day, when 3 pairs of aortic arches ensure cardio-aortic communication in wild-type embryos, all the blood in the mutants circulates through a single (bilateral) vessel at the level of the otic vesicle (Fig. 2D). In addition, AA1 has remodeled and broken into a capillary bed in the mutants slightly earlier than in wild-type siblings.

At E10.5, there is still only one vessel posterior to AA1 (Fig. 2F). The shape of this vessel suggests that the aortic sac is directly attached to the aortae and that the whole territory where AA3-6 should have grown is absent. The leading edges of the pulmonary arteries, which are clearly visible in the wild-type at this stage (Fig. 2E), have not formed (or are not patent) yet in the mutant. A day later, while the wild-type OFT has started septating left and right blood flows, the mutant OFT remains a single artery (Fig. 2I). The pulmonary arteries are still not visible in the mutant. At E12.5, the OFT is still unseptated, as it will remain, but the pulmonary arteries are now patent (Fig. 2L).

Anterior Cardinal Veins Display Multiple Anomalies in Chrd−/− Embryos

The earliest phenotype observed in veins of Chordin homozygous mutant embryos was abnormal looping of the ACV in the region around the otic vesicle. The pattern of this was variable, usually unilateral. In wild-type embryos, at E9.5, the ACV is a robust straight vessel, which extends from the heart to the eye (Fig. 1B). Two very short branches are visible just posterior to the otic vesicle (Fig. 3A). In the mutant shown in Figure 3B, these two branches are anastomosed and connect at a third level with the ACV anterior to the otic vesicle. At later developmental stages, the normally straight ACV could follow variable, complex, three-dimensional patterns, as in the embryo seen in Figure 3C, or make side loops as in the E12.0 embryo in Figure 3D (arrowheads). In one E12.0 mutant (Fig. 3E,F), an abnormal loop was formed between the ACV and the post-otic vein, originating at the position of the pre-otic vein (in all other embryos at that stage, no preotic vein was found, see below).

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Figure 3. Loops in the anterior cardinal veins (ACVs) of Chrd−/− embryos. A: Wild-type embryo. B–F: Chrd−/− embryos. A,B: Dye injection in the atrium (at) of embryonic day (E) 9.5 embryos revealed loops in the ACV in the region around the otic vesicle (OV) in mutants (B), an anomaly never seen in wild-type siblings (A, a close-up of Fig. 1B). As can be seen in transparency, this defect was unilateral and the left vein was not affected in this embryo. C: The right ACV (blue) of this E11.0 mutant follows a complex three-dimensional path. This vessel did not form a loop or show any “holes.” D: The right ACV (purple) of this E12.0 mutant makes two side loops (highlighted with white dotted lines) as well as small “holes.” This embryo was also injected with methylsalicylate in the tail artery and shows the truncus arteriosus (TA) and the ascending aorta in blue. E,F: In this E12.0 mutant, an abnormal anastomosis was formed between the left ACV and post-otic vein, originating at the position of the pre-otic vein. This loop was also perforated by small “holes.” F: To help visualize the loop in the almost perpendicular plane, E was stylized using the emboss feature of Adobe Photoshop using the following parameters: 100%, 135° angle, 6 pixels. All views are lateral, with embryos facing to the right in A–D, and to the left in E–F. BA1, first branchial arch. PostOt, post-otic vein. Vein branches nomenclature is after Blechschmidt (1961).

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Several smaller “holes” are visible in this loop, as well as in Figure 3D, a phenomenon that could be due to ectopic intussusception. Alternatively, this perforated aspect of the vessels is reminiscent of primitive anastomosing vascular networks seen in some vessels undergoing vasculogenesis and could represent a delay in maturation of these veins.

In addition to (or maybe in some cases resulting from) the loopings, patterning defects became evident in older embryos. In wild-type embryos, the otic vesicle sits between the vena preotica and the vena postotica, both of whom drain into the ACV (Fig. 4A). In mutants, the preotic branch did not develop (Fig. 4B). This phenotype was highly penetrant (5/6 embryos analyzed at E11.5 and 5/6 at E12.5). In addition, in a seventh E12.5 embryo, described above in Figure 3EF, a tributary that could correspond to the preotic vein abnormally looped onto the post-otic vein.

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Figure 4. Abnormal patterning of the neck veins in Chrd−/− embryos. A–F: Purple resin was injected into the atria of wild-type (A,C,E) and Chrd−/− (B,D,F) embryos. A,B: Absence of the pre-otic vein in Chordin mutants. At embryonic day (E) 11.5, the otic vesicle sits between the vena preotica (PreOt) and the vena postotica (PostOt) in wild-type embryos (A). In mutants, the pre-otic branch does not develop (B). C,D: Missing segment of the anterior cardinal vein (ACV) results in abnormal origin of the mandibular venous branch (blue #) in the mutants (D). Compare position with the origin of the post-otic vein (white asterisk). E,F: Midline defect (E12.5). The thyroid veins normally originate bilaterally from the ACVs but do not cross the midline (dotted blue line). F: In mutants, one branch was predominant over the other and crossed the midline. The smaller branch never crossed the midline and was less amenable to dye injection, which could be due to delay in patency or actual hypoplasia. (The heads of injected embryos were separated from the body and the mandibles removed for these ventral views.)

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Another, fully penetrant (7/7 E11.5 embryos), phenotype that occurred bilaterally in the mutants was the lack of the segment of the ACV that normally lies posterior to the origin of the vena postotica. The absence of this segment results in the abnormal positioning of the venous branch that drains the mandible (Fig. 4D; compare distance between the blue pound sign and the origin of the post-otic vein, marked by a white asterisk).

Finally, a midline defect was seen at E12.5 in veins draining the neck region of three of three embryos studied at E12.5. The thyroid veins normally originate bilaterally from the respective ACV but do not cross the midline (Fig. 4E). In mutants, although the anatomy was remarkably similar, one branch (the left or the right in different embryos) was predominant over the other and crossed the midline (Fig. 4F), although it never contacted the contralateral ACV. The other branch appeared shorter and never crossed the midline.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

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.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Collection and Genotyping of Embryos

Heterozygous Chrdrtm1DR mice (Bachiller et al.,2003) in the B6SJLF1 background were mated to generate Chrd−/− and wild-type siblings at embryonic days E9.5 to E14.5. Genomic DNA was extracted from the yolk sacs and the genotype was determined by polymerase chain reaction using the following primers: (5′-GTT CCA CAT ACA CTT CAT TCT CAG-3′), (5′-GGT AGG AGA CAG AGA AGC GTA AAC T-3′), and (5′-GAG TTA GGA GGT GGA GCT CTA CAC-3′), which yield bands of 400 and 600 bp for the mutant and wild-type alleles, respectively.

Vessel Injections

Embryos were dissected and fixed in 4% paraformaldehyde overnight. Embryos at E11.5 and older were left to bleed in 1× phosphate buffered saline (PBS) for 30 min before fixation to eliminate blood cells and help even dye spread. Fixed embryos were dehydrated through a methanol series (25%, 50%, 75%, 100%, in 1× PBS) and stored at −20°C. Right before injection embryos were cleared in methylsalicylate (Fisher, catalogue no. O3695-500) from 20 min to 2 hr, depending on embryo size. Injections were performed as soon as the embryos settled at the bottom of the dish as longer times in clearing solution gave excessive hardening of tissues. Embryos were injected under a bottom-lit binocular dissecting microscope, using a fine-tipped glass capillary needle while immerged in methylsalicylate. The resin used for injections was 1% solution of blue or red pigment (Polysciences, Inc.) in methylsalicylate. The point of needle entry to visualize veins of late E9.5 embryos and older was the atrium or sinus venosus. For early E9.5 embryos, veins can also be visualized by injection into the left ventricle. For arteries, the point of injection was the descending aorta in the tail region.

Outgrowth of the aortic arches (E9.0–E10.5) was analyzed in detail in 15 wild-type embryos and 4 mutants, and remodeling of the posterior-most three aortic arches (E11.0–E12.5) in 11 wild-type and 6 mutants. At E9.5–E10.5, loops were seen in the anterior cardinal veins of 5/5 successfully injected embryos but not in 5/5 wild-type siblings. No loops were seen in 7/7 and 12/12 wild-type embryos at E11.5 and E12.5, respectively. At E11.5, 2/3 mutants had loops, 1/3 an abnormal branching pattern. At E12.5, 6/10 mutant embryos had closed loops, while the other 4 displayed anterior cardinal vein tortuosity. In addition, in wild-types, the preotic veins were fully patent in 3/5 embryos at E11.5 and 11/11 at E12.5. In mutants, preotic veins were absent bilaterally in 5/6 mutants at E11.5 and, at E12.5, were absent bilaterally in 5/6 and unilaterally in 1/6.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

D.B. was funded by a Victor Goodhill Endowment, and E.D. was funded by the Children's Heart Foundation and the Laubisch Research Fund.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES