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Mesp1 Expression Is the Earliest Sign of Cardiovascular Development
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Mesp1 Expression Is the Earliest Sign of Cardiovascular Development Yumiko Saga,* Satoshi Kitajima, and Sachiko Miyagawa-Tomita
Understanding the molecular mechanism leading to formation of the heart and vasculature during embryogenesis is critically important because malformation of the cardiovascular system is the most frequently occurring type of birth defect. While the hearts of all vertebrates are derived from bilateral paired fields of primary mesodermal cells that are specified to the cardiac lineage during gastrulation, the mechanism for lineage restriction, and the origin of the myocardium and endocardium have not been defined. Recently, we found that a transcription factor, Mesp1, is expressed in almost all precursors of the cardiovascular system and plays an essential role in cardiac morphogenesis. Mesp1 may play a key role in the early specification for cardiac precursor cells. (Trends Cardiovasc Med 2000;10:345–352). © 2001, Elsevier Science Inc.
Wieland F, Harter C: 1999. Mechanism of vesicle formation: insights from the COP system. Curr Opion Cell Biol 11:440–446. Wiggins D, Gibbons GF: 1996. Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid. Biochem J 320:673–679. Williams KJ, Brocia RW, Fisher EA: 1990. The unstirred water layer as a site of control of apolipoprotein B secretion. J Biol Chem 265:16,741–16,744. Wu X, Zhou M, Huang L-S, et al.: 1996. Demonstration of a physical interaction between microsomal triglyceride transfer protein and apolipoprotein B during the assembly of apoB-containing lipoproteins. J Biol Chem 271:10,277–10,281. Xie Z, Wise H: 1997. Cyclic AMP-independent activation of neutrophil like HL-60 cells by prostaglandin E2. Cell Signal 7:531–537. Yeung SJ, Chen SH, Chan L: 1996. Ubiquitinproteasome pathway mediates intracellular degradation of apolipoprotein B. Biochemistry 35:13,843–13,848. Zhou M, Fisher EA, Ginsberg HN: 1998. Regulated co-translational ubiquitination of apolipoprotein B 100. J Biol Chem 273: 24,649–24,653. Zhou M, Wu X, Huang LS, Ginsberg HN: 1995. Apoprotein B 100, an inefficiently translocated secretory protein, is bound to the cytosolic chaperone, heat shock protein 70. J Biol Chem 270:25,220–25,224. PII S1050-1738(01)00071-8
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The critical phase of embryonic development is known as gastrulation. The immediate outcome of gastrulation is the formation of the definitive germ layers and the organization of a general body plan, which serves as a blueprint of the embryonic body. The developmental fate of cells in the epiblast has been analyzed within the last decade with the use of a transgenic lacZ marker, fluorescent dyes, chick-quail transplantation chimeras, or intracellular labels. The resulting fate map is essentially the same for the chick as for the mouse and urodeles, and indicates that, despite geometrical differences,
Yumiko Saga and Satoshi Kitajima are at Cellular & Molecular Toxicology Division, National Institute of Health Science, 1-18-1, Kamiyohga, Setagaya-ku, Tokyo 158, Japan. Sachiko Miyagawa-Tomita is at Department of Pediatric Cardiology, The Heart Institute of Japan, Tokyo Women’s Medical University, 8-1, Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. * Address correspondence to: Dr. Yumiko Saga, Cellular & Molecular Toxicology Division, National Institute of Health Sciences, 1-18-1, Kamiyohga, Setagaya-ku, Tokyo 158, Japan. Tel.: 81-3-3700-9652; fax: 81-3-37009647; e-mail address: [email protected]. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
topological fate relationships appear to be conserved among the vertebrates (Lawson and Pedersen 1992, Tam et al. 2000). The heart is the first functional organ formed during organogenesis. Cells destined to become cardiac mesoderm are fated, induced, and specified prior to and during gastrulation. The cardiac mesoderm cells invaginate through the primitive streak, and migrate and spread with the cranial mesoderm. Subsequently, the bilaterally symmetric cardiac precursors migrate and converge at the midline of the embryo to form the cardiac crescent, which then forms a linear, single heart tube. The tube formed by an outer myocardium and an inner endocardium undergoes rightward looping. The looped heart tube then undergoes septation to generate the mature, four-chambered cardiac structure. Many molecules are associated with cardiogenesis after formation of the crescent-shaped cardiac primordium. Nkx2.5 (Lints et al. 1993), Flt1 (Yamaguchi et al. 1993), Mef2B/C (Molkentin et al. 1996), Gata4 (Heikinheimo et al. 1994, Kuo et al. 1997), and dHand/ eHand genes (Srivastava et al. 1995) are expressed in the crescent-shaped cardiac primordium, myocardium and/or endocardium, respectively. Furthermore, many
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Figure 1. Mesp1 expression and the phenotype of the Mesp1-null embryo. (A) Mesp1 expression starts in the newly formed mesodermal cells at the onset of gastrulation (6.5 dpc). The expression continues until 7.5 dpc and is then quickly down-regulated. The axial and paraxial mesoderm does not express Mesp1. This early expression of Mesp1 completely disappeared before heart tube formation. Lateral view. A, anterior; P, posterior; Hf, head fold. (B) Cardiac malformation observed in Mesp1-null embryos at 9.5 dpc. Instead of a single heart tube generated in the wildtype, various types of malformations were observed in the mutants (from a single to two separated tubes). Each tube is sometimes separated into two small chambers. Frontal view. (C) Expression of Mesp1-LacZ indicating normal migration of the cranial-cardiac mesoderm in a heterozygous embryo at 9.5 dpc, but delay of migration in the homozygous embryo. Lateral view. Em, embryonic region; Ex, extraembryonic region.
signaling molecules are involved in the later cardiac morphogenesis (Fishman and Chien 1997, Olson and Srivastava 1996). Prior to the crescent-shaped cardiac primordium formation, however, little is known about the molecular signals that guide the cardiac mesoderm. In this article, we summarize a series of molecular and genetic analyses on the Mesp genes, Mesp1 and Mesp2, which are implicated in the early specification of cardiac precursor cells. • Mesp1 Is Expressed in the Early Mesoderm and Involved in Cardiac Morphogenesis Mesp1, belonging to the bHLH transcription factor family, is expressed in the early mesoderm at the onset of gastrulation (Figure 1 and Saga et al. 1996). The expression of the transcript is restricted to a part of mesoderm ingressed through the primitive streak of the early stage embryo (6.5–7.0 dpc) and quickly downregulated after 7.5 dpc and no expression has been observed in the paraxial or axial mesoderm. Only the slight expression observed at a later stage embryo (8.5 dpc in Figure 1) might represent a part of the cranial-cardiac mesoderm. The indication of involvement of Mesp1 in cardiac morphogenesis comes from a gene knockout study. Homozygous Mesp1 null mice exhibit various anomalies of heart tube formation and looping (Figure 1B). The heart tubes range from two completely sepa-
rated tubes to a single tube. The separated tubes show various degrees of cardia bifida, from full to partial bifurcations (Saga et al. 1999). This anomaly is most likely to be caused by a failure of ventral fusion of the pericardial mesoderm. However, these hearts show well-formed myocardium and endocardium. Therefore, cellular differentiation accompanied by heart tube formation occurs normally even in Mesp1-null embryos. Because Mesp1 is expressed in the nascent mesoderm at the onset of gastrulation, it is assumed that the abnormality is due to a precursor cell defect. To monitor the cell lineage, a knockin mouse (Mesp1LacZ) which has the b-galactosidase gene under the control of the Mesp1 promoter was analyzed (Saga et al. 1999). As the bgal activity is retained for a longer period than the mRNA signal, the cell movement can be traced from the onset of gastrulation to a later stage. The most anterior mesodermal cells expressing Mesp1-LacZ migrated to generate the future cardiac field and this migration was markedly delayed in the Mesp1-null embryo (Figure 1C), indicating that the cardiac anomaly is caused by the delay in mesodermal migration. However, since the b-gal activity was down-regulated before heart tube formation, it was not clear whether Mesp1 was expressed in the cardiac precursors or in other cells supporting cardiac morphogenesis. To define the cell lineage of Mesp1-expressing mesodermal cells from the beginning of the ex-
pression to the final destination, the knockin mouse (Mesp1-cre) which has Cre recombinase under the control of the Mesp1 promoter-enhancer was generated (Saga et al. 1999). • Mesp1-Expressing Cell Lineage Cell type-specific genomic alternations mediated by the Cre/loxP system constitute a powerful approach to the study of cell lineages (Trainor et al. 1999, Yamauchi et al. 1999, Zinyk et al. 1998). On crossing heterozygous Mesp1-Cre and reporter mice CAG-CAT-Z, one quarter of the progeny should consist of double transgenics (Figure 2A). Only cells that express Cre from the Mesp1 allele should undergo recombination between the loxP sites of the reporter construct, excising the CAT gene and permitting LacZ expression. As the Cre-mediated excision is cell-heritable, the marked cells and all their progeny should express LacZ at later stages, even after Cre is no longer expressed. Thus, LacZ staining in the double-transgenic embryo should reveal the progeny of all cells that express Cre transiently during development under the control of Mesp1 promoter and enhancer. Intercrossed embryos examined at 7.5 dpc revealed that the extraembryonic mesoderm and cranial-cardiac embryonic mesoderm in the lateral plate mesoderm was positive for LacZ staining (Figure 2B), while the paraxial and axial mesoderm were negative, indicat-
Figure 2. Lineage analysis of Mesp1-expressing cells. (A) Schematic presentation of the strategy for lineage analysis. Mesp1-cre which has the Cre recombinase gene under the Mesp1 promoter, is mated with a reporter mouse, CAG-CAT-Z, which has an inactive LacZ gene in the absence of Cre-mediated recombination (Sakai and Miyazaki 1997). Once Mesp1 is expressed, Cre recombinase eliminates the CAT gene which leads to activation of the LacZ gene. Because CAG promoter is ubiquitously active, the cells which expressed Mesp1 are expected to express LacZ continuously. (B) LacZ staining pattern of the 7.5 dpc intercrossed embryo, which exhibits strong staining in the extraembryonic mesoderm and progenitors of the cranial-cardiac mesoderm (CCM) (lateral view on left panel). Posterior view (right panel) shows no expression in paraxial (PM) and axial mesoderm (AM). A anterior; P, posterior; Ex, extraembryonic region. (C) LacZ staining of the 9.5 dpc intercrossed embryo, which exhibits strong staining in the heart (H). AL, anterior limb; M, mandibular arch; Otv, otic vesicle; OV, optic vesicle. (D) Cross-section of C (heart region), showing strong staining in both myocardium and endocardium including the cono-truncal and atrioventricular cushions. LacZ staining is seen in the endocardium, myocardium, and mesenchymal cells of the cushion at the atrioventricular canal, and the epicardium as shown in the upper right corner. Some cranial vessel primordia also show the LacZ staining. IC, inferior atrioventricular endocardial cushion; OFT, outflow tract; PRV, primitive right ventricle; SC, superior atrioventricular endocardial cushion. (E) Cross-section of C (caudal region) showing staining in the endothelial cells of the dorsal aorta (DA). (F) A section of the extraembryonic yolk sac showing staining in the endothelial cells (ET) but not in the endodermal cells (ED).
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Figure 2
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Figure 3. Comparison of gastrulating embryos (7.5 dpc) of wild-type and dKO. Photographs taken from the lateral view and schematic drawings are shown. In the wildtype, early ingressed groups of mesodermal, extraembryonic and cranial-cardiac cells have migrated out from the primitive streak. The paraxial mesoderm has also migrated anteriorly and the lateral mesoderm follows. Axial mesoderm has emerged from the most anterior part of the node and is elongated along the midline. In contrast, in the dKO embryo, no clear mesodermal layer was generated in the embryonic region although the primitive streak was formed and extraembryonic mesoderm was placed relatively normally. Axial mesoderm was initially generated normally but no further extension was observed in the dKO embryo.
Figure 4. Chimera analysis. (A) Schematic presentation of the strategy for chimera analysis. The double KO mouse line was genetically marked by mating with the Rosa26 mouse which shows ubiquitous b-gal activity in all cells. A dKO (2/2) 8-cell stage embryo obtained by intercross of the dKO mouse was aggregated with an 8-cell stage wild-type embryo and the blastocyst thus formed was transferred to the foster mother. The embryo was recovered at 9.5 dpc and stained for detection of b-gal activity. Only dKO (2/2) cells show b-gal activity and wild-type cells should not have any b-gal activity. (B) A chimera embryo which was generated by aggregating a dKO and a wild-type embryo. Note that the heart is composed of only wild-type white cells and lacks the LacZ-positive dKO cells. Lateral view. (C) A transverse section of B, confirming that heart tissues are exclusively composed of wild-type cells. The epicardium also consists of wild-type cells. PLV, primitive left ventricle; PRV, primitive right ventricle; OFT, outflow tract.
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ing that Mesp1-expressing cells contribute to the extraembryonic mesoderm and the cranial-cardiac mesoderm. Later embryos (9.5 dpc) were examined to further trace the lineage of cells expressing Mesp1. We previously reported that only the myocardium in the heart was derived from Mesp1-expressing cells (Saga et al. 1999). More detailed and careful analyses, however, revealed the b-gal activity not only in the heart tube but also in the vascular system, that is, the dorsal aorta, intersomitic vessels, and cranial vessels (Figure 2C–F). A cross-section showed the LacZ staining in the endocardium as well (Figure 2D), indicating that both cell types (myocardium and endocardium) were derived from Mesp1expressing cells. At the atrioventricular canal and outflow regions, the b-gal activity is seen in the endocardium, myocardium, and mesenchymal cells forming cardiac cushions. Furthermore, the epicardium and proepicardium show the LacZ staining. The strong LacZ staining indicates that the endothelial cells in the yolk sac are also derived from Mesp1-expressing cells (Figure 2F). These observations indicate that Mesp1 is activated at first in the extraembryonic mesoderm and then in the common precursor cells of myocardium, endocardium, and the vascular system. Therefore, Mesp1 may represent the earliest molecular marker for these cell lineages. Thereafter, flk-1 (an early molecular marker of endothelial precursor cells) (Yamaguchi et al. 1993) and Nkx-2.5 (an early molecular marker of cardiac progenitor cells) (Lints et al. 1993) transcripts become detectable in the 7.0 dpc and 7.5 dpc mouse embryo, respectively. In view of the endocardial cell lineage, in chick embryo, one hypothesis is that the endocardial cells originate from the two different precursors, that is, cardiac mesoderm and non-cardiac mesoderm (Eisenberg and Markwald 1995). We propose that the pre-myocardial and pre-endocardial cells have a common precursor (i.e., the Mesp1-expressing mesoderm). It is noted that no somitic mesoderm was stained, although intersomitic vessels were clearly stained. Although we are not sure of the origin of the intersomitic vessels, it is not likely to be the paraxial mesoderm. We previously showed that endogenous Mesp1 was expressed in the presomitic mesoderm just before segmentation (Saga et al. 1996). However, Cre is not expressed in the presomitic
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mesoderm from this Mesp1-cre allele since no b-gal activity was detected in any progenitor of the paraxial mesoderm. The reason is not clear but it could be due to interference by a pgk-neo cassette which was included in the targeting vector. This was fortunate for us because it enabled us to distinguish the cranial-cardiac mesoderm from the paraxial mesoderm.
(Figure 3). The embryos died at 9.5 dpc and exhibited only a head-like structure. They lacked any caudal structures behind the anterior head region, such as a posterior head region, heart or somites. These observations indicate that Mesp1 and Mesp2 function cooperatively during gastrulation and are essential for development of the embryonic mesoderm.
• Mesp1, Mesp2 Double Knockout Mouse
• Chimera Analysis Reveals Cell-autonomous Function of Mesp1 and Mesp2 in Cardiac Morphogenesis
The abnormal heart morphogenesis in Mesp1 (2/2) embryos resulting in cardia bifida is caused by delayed migration of the cardiac precursor cells. However, the initial suppression of cell migration is rescued at the later stages and the Mesp1 (2/2) embryo eventually generates an abnormal heart tube. The involvement of Mesp2, another gene of the same family, is indicated because of the similar expression pattern. An early expression study did not reveal Mesp2 expression at the early stage embryo, and Mesp2 knockout mice did not show any defect in cardiac morphogenesis (Saga et al. 1997). However, a prolonged in situ reaction revealed Mesp2 expression at 6.5–7.0 dpc in a pattern very similar to that of Mesp1. Furthermore, Mesp2 expression was retained longer in Mesp1 (2/2) embryos than in wild-type embryos. Accordingly, Mesp1- and Mesp2-doubleknockout mouse (Mesp1,p2-dKO) was generated to clarify the function of Mesp2 during gastrulation and to understand the cooperative functions of the two transcription factors (Kitajima et al. 2000). Mesp1,p2-dKO embryos exhibited abnormal gastrulation, because no clear embryonic mesodermal cell layer was observed between the endoderm and ectoderm in the embryonic region. The dense cell accumulation in the primitive streak indicated the initiation of gastrulation and the ingression of ectodermal cells into the primitive streak. However, the mesodermal cells appeared unable to depart from the primitive streak. On the other hand, extraembryonic mesoderm was generated and differentiated to generate blood and endothelial cells. Detailed analysis, using several mesodermal molecular markers, revealed that Mesp1,p2-dKO embryos lacked embryonic non-axial mesodermal cell layers, although the axial mesoderm initially generated also showed a defect during later development
Mesp1 and Mesp2 are expressed only in the early ingressed group of mesoderm and not in the paraxial and axial mesoderm. No paraxial mesoderm was generated in the dKO embryo, which resulted in a complete lack of somites. To clarify whether such mesodermal defects observed in the Mesp1,p2-dKO embryo are of a cellautonomous or non-cell-autonomous consequence, chimera analysis was conducted (see Figure 4 for the method). A chimeric embryo was generated by aggregating 8-cell embryos derived from wild-type and intercrossed heterozygous dKO mice (see Figure 4A for details). Thus, dKO/wild chimeric embryos should be composed of b-gal-negative wild-type cells and b-gal-positive Mesp12/2, Mesp22/2 cells. The results were extremely interesting. Chimera embryos containing Mesp12/2, Mesp22/2 cells developed normally but the heart was composed of only wild-type cells and lacked Mesp12/2, Mesp22/2 cells (Figure 4B). Cross-sections of these embryos confirmed the almost complete absence of Mesp12/2, Mesp22/2 cells in most of the heart tissue (Figure 4C). Myocardium and endocardium in the right and left ventricles and outflow tract, and the epicardium consisted of wild-type cells. However, b-gal-positive Mesp12/2, Mesp22/2 cells constituted a part of the mesenchymal cells in the outflow tract. The cells might be derived from the cardiac neural crest (Kirby and Waldo 1990). Interestingly, the paraxial mesoderm was composed of both types of cells, indicating that Mesp12/2, Mesp22/2 cells can contribute to somitogenesis. It is worth noting that the defect of Mesp12/2, Mesp22/2 cells in the chimera is closely correlated with the expression of Mesp1 and Mesp2. These genes are expressed in the early ingressed groups of mesodermal cells destined for the extraem-
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bryonic mesoderm and cranial-cardiac mesoderm, but not in the axial mesoderm, or early paraxial mesoderm, endoderm or ectoderm. Therefore, the inability of Mesp12/2, Mesp22/2 cells to generate the paraxial mesoderm in dKO embryos is not cell-autonomous but rather a consequence of earlier events, such as defective gastrulation. Similarly, it is reasonable that the b-gal-positive Mesp12/2, Mesp22/2 cells contributed to the neural tube, notochord, gut and the lateral plate mesoderm, except for the cardiac mesoderm. However, Mesp12/2, Mesp22/2 cells did contribute to the extraembryonic mesoderm and embryonic endothelial cells lining several vessels, such as the dorsal aorta and intersomitic vessels. Therefore, only the inability to contribute to the cardiac mesoderm and epicardium reveals a cell-autonomous defect. Figure 5 provides a summary of the lineage of Mesp1expressing cells of an early gastrulating embryo (6.5–7.0 dpc). • Discussion Mesodermal cells generate the heart tube. The embryonic mesoderm can be di-
vided into five regions; the chorda (axial) mesoderm, somitic dorsal (paraxial) mesoderm, intermediate mesoderm, lateral plate mesoderm, and head mesenchyme (Gilbert 2000). It is generally thought that the heart is derived from the lateral plate mesoderm. The results of our experiments and fate mapping studies (Tam et al. 2000) showed that the heart precursors in mouse are clearly localized as the cranial-cardiac mesoderm in the lateral plate mesoderm. Mesp1 and Mesp2 are co-expressed in the early mesoderm and function cooperatively. Our lineage study using Mesp1-cre mice revealed a primary mesoderm population expressing Mesp1 which included almost all endothelial precursors in both the extraembryonic and embryonic vascular systems. In the embryonic region, two populations of cardiac precursor cells, myocardium and endocardium, were also included in the cells which expressed Mesp1. Therefore, we propose that the cardiovascular system, including the myocardium and endocardium, emerges from the Mesp1-expressing cells, and Mesp1 is the earliest molecular marker of the cardiovascular system, although it
Figure 5. Summary of the lineage of Mesp1-expressing cells of an early gastrulating embryo (6.5–7.0 dpc). Because Mesp1 expression is down-regulated after 7.5 dpc (Figure 1), lineage analyses (Figure 2) demonstrated that only a small group of mesodermal cells was the source for almost all of the extraembryonic and embryonic vascular systems. In addition, Mesp1expressing mesoderm contains the precursors for myocardium and endocardium.
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is still unclear where the myocardium and endocardium diverge from the other cells. Various anomalies of heart tube formation and looping were observed in Mesp1-null mice. Delayed migration of the cardiac precursor cells must be a cause of the various degrees of cardia bifida in Mesp1-null mice. Several gene knockout mice have been shown to generate cardia bifida, which include fibronectin (George et al. 1993), Gata4 (Kuo et al. 1997, Molkentin et al. 1997, Narita et al. 1997), furin (Constam and Robertson 2000, Roebroek et al. 1998), Hrs (Kodama and Soriano 1999) and Smad5 (Chang et al. 1999) knockout mice. Cardia bifida found in the above-mentioned knockout mice is caused by a defect of ventral folding due to the failure of migration and/or proliferation of cells involved in the ventrolateral morphogenesis, which is a similar situation to that observed in Mesp1-null embryo. In addition, a responsible gene that causes cardia bifida in zebrafish mutant miles apart (mil) has recently been identified (Kupperman et al. 2000). The gene Mil, coding a receptor that binds lysosphingolipids, sphingosine-1phospate, has a role in generating an environment permissive for heart precursor cells to migrate to the midline. However, no direct molecular relationship between Mesp1 and these genes is known. Because Mesp1, Mesp2 double KO embryo lacks most of mesodermal layer, the genes implicated in the heart tube formation, such as Nkx2.5, Gata4 are not expressed in double KO embryos. The lack of these genes might be one of the causes leading to the migration failure, but Mesp genes might be required to regulate downstream target genes that are essential for mesoderm proliferation rather than migration, but this needs to be determined. Serum response factor (SRF) is known as a critical factor required for proper mesoderm formation. SRF-deficient embryos have a severe gastrulation defect and do not form any mesodermal cells (Arsenian et al. 1998). This phenotype resembles that of Mesp1, Mesp2 double knockout mouse. However the early expression of SRF is ubiquitous and not restricted in the mesoderm (Croissant et al. 1996, Li et al. 1997). The expression becomes restricted in the developing heart at a later embryonic stage and continues during heart morphogenesis in which Mesp1 expression was no longer observed. Therefore, TCM Vol. 10, No. 8, 2000
the SRF expression overlaps with that of Mesp1 only in some of the cells and during a certain period, which indicates that no direct relationship exists between them. Our studies revealed that the heart field is generated by migration of the cells which expressed Mesp1 and also suggested that the embryonic primary blood vessels are generated by the cells which expressed Mesp1. Endothelial precursor cells (angioblasts) form the first major blood vessels in the embryo, including the dorsal aorta and the endocardium. This process is called vasculogenesis (Risau et al. 1988). The ability of endothelial cells to migrate is critical for the formation of the primary vessels of the embryo. As for the mechanism of dorsal aorta development, VEGF is thought to be a crucial factor, since the homozygous mutant embryo shows a complete failure of dorsal aorta development (Carmeliet et al. 1996, Ferrara et al. 1996). Although the origin of the mouse embryonic angioblast required for development of the dorsal aorta remains unclear, vascular precursor cells have been shown to be derived from the lateral plate mesoderm in Xenopus embryo (Cleaver and Krieg 1998). In chick embryo, transplantation experiments showed that there are two endothelial lineages in the formation of the aorta; one is derived from the paraxial mesoderm (i.e., somite) and the other from the splanchnopleural mesoderm (Pardanaud et al. 1996). Because the expression of Mesp1 is transient and the Mesp1-expressing cells contribute little to the axial and paraxial mesoderm during gastrulation, our results suggest that the precursor cells of the dorsal aorta have already been determined by expression of the Mesp1 during gastrulation, and the precursor cells might belong to the cranial-cardiac mesoderm or, possibly, another distinct population in the lateral plate mesoderm. • Conclusion The series of experiments described here involving cell lineage, loss of function and chimeric analyses revealed a differential requirement of Mesp1 and Mesp2 for different cell lineages. For the extraembryonic mesoderm, lack of Mesp1 and Mesp2 did not result in a severe defect in either mesodermal formation or subsequent cell differentiation, suggest-
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ing that these genes may not be required for extraembryonic mesoderm development or, alternatively, other molecules may rescue the deficiency of Mesp1 and Mesp2. In contrast, Mesp1 and Mesp2 are essentially important for embryonic mesoderm formation. Mesp1 (and maybe Mesp2)-expressing cells are fated to be the precursor of the endothelium and the cardiovascular system. However, these genes are required cell-autonomously only for the precursor of the myocardium and endocardium of the ventricles since Mesp12/2, Mesp22/2 cells contributed to the formation of vascular endothelium. The actual function of Mesp1 and Mesp2 and the target gene(s), however, is not known. Without Mesp1 and Mesp2, mesoderm may not be specified as a cardiac mesodermal precursor. Alternatively, mesoderm may lack migratory activity for localization at the proper position to be incorporated into precardiac cells. These points remain to be investigated. In addition, we do not know how Mesp1 and Mesp2 work cooperatively. They might work as a heterodimer although, generally, cell type-specific bHLH factor dimelizes with a ubiquitously expressed bHLH factor such as E12 or E47. We do not know whether any human syndromes link to Mesp genes and more detailed analysis is necessary. As we could mark the cells which expressed Mesp1 with either LacZ or GFP (if we used the proper reporter line), it is possible to isolate these cells with a cell sorter and investigate their ability to generate the heart tube in vitro. In addition, we now have an excellent tool for delivering any gene to the cardiovascular cell lineage, which would be useful for investigation of the mechanism of cell fate determination and cell differentiation.
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Cleaver O, Krieg PA: 1998. VEGF mediates angioblast migration during development of the dorsal aorta in Xenopus. Development 125:3905–3914. Constam DB, Robertson EJ: 2000. Tissue-specific requirements for the proprotein convertase Furin/SPC1 during embryonic turning and heart looping. Development 127:245–254. Croissant JD, Kim JH, Eichele G, et al: 1996. Avian serum response factor expression restricted primarily to muscle cell lineages is required for alpha-actin gene transcription. Dev Biol 177:250–264. Eisenberg LM, Markwald RR: 1995. Molecular regulation of atrioventricular valvuloseptal morphogenesis. Circ Res 77:1–6. Ferrara N, Carver-Moore K, Chen H, et al.: 1996. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380:439–442. Fishman MC, Chien KR: 1997. Fashioning the vertebrate heart: earliest embryonic decisions. Development 124:2099–2117. George EL, Georges-Labouesse EN, PatelKing RS, et al.: 1993. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development 119:1079–1091. Gilbert S: 2000. Developmental Biology, 6th ed., Massachusetts, Sinauer Associates, Inc. Heikinheimo M, Scandrett JM, Wilson DB: 1994. Localization of transcription factor GATA-4 to regions of the mouse embryo involved in cardiac development. Dev Biol 164:361–373. Kirby ML, Waldo KL: 1990. Role of neurla crest in congenital heart disease. Circulation 82(2):332–340. Kitajima S, Takagi A, Inoue T, Saga Y: 2000. Mesp1 and Mesp2 are essential for the development of cardiac mesoderm. Development 127:3215–3226. Kodama M, Soriano P: 1999. Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis. Genes Dev 13:1475–1485. Kuo CT, Morrisey EE, Anandappa R, et al.: 1997. GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev 11:1048–1060. Kupperman E, An S, Osborne N, et al.: 2000. A sphingosine-1-phospate receptor regulates cell migration during vertebrate heart development. Nature 406: 192–195. Lawson KA, Pedersen RA: 1992. Clonal analysis of cell fate during gastrulation and early neurulation in the mouse. In postimplantation development in the mouse. Ciba Found Symp 165:3–26. Li L, Liu Z, Mercer B, et al.: 1997. Evidence for serum response factor-mediated regulatory networks governing SM22alpha transcription in smooth, skeletal, and cardiac muscle cells. Dev Biol 187:311–321.
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Lints TJ, Parsons LM, Hartley L, et al.: 1993. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119:419–431. Molkentin JD, Firulli AB, Black BL, et al.: 1996. MEF2B is a potent transactivator expressed in early myogenic lineages. Mol Cell Biol 16:3814–3824. Molkentin JD, Lin Q, Duncan SA, Olson EN: 1997. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev 11:1061–1072. Narita N, Bielinska M, Wilson DB: 1997. Wild-type endoderm abrogates the ventral developmental defects associated with GATA-4 deficiency in the mouse. Dev Biol 189:270–274. Olson EN, Srivastava D: 1996. Molecular pathways controlling heart development. Science 272:671–676. Pardanaud L, Luton D, Prigent M, et al.: 1996. Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis. Development 122:1363–1371. Risau W, Sariola H, Zerwes HG, et al.: 1988. Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development 102:471–478. Roebroek AJM, Umans L, Pauli IGL, et al.: 1998. Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin. Development 125:4863– 4876. Saga Y, Hata N, Kobayashi S, et al.: 1996. Mesp1: A novel basic helix-loop-helix protein expressed in the nascent mesodermal cells during mouse gastrulation. Development 122:2769–2778. Saga Y, Hata N, Koseki H, Taketo MM: 1997. Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev 11:1827–1839. Saga Y, Miyagawa-Tomita S, Takagi A, et al.: 1999. Mesp1 is expressed in the heart precursor cells and required for the formation of a single heart tube. Development 126:3437–3447. Sakai K, Miyazaki J: 1997. A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. Biochem Biophys Res Commun 237:318–324. Srivastava D, Cserjesi P, Olson EN: 1995. A subclass of bHLH proteins required for cardiac morphogenesis. Science 270:1995–1999. Tam PP, Goldman D, Camus A, Schoenwolf GC: 2000. Early events of somitogenesis in higher vertebrates: allocation of precursor cells during gastrulation and the organization of a meristic pattern in the paraxial mesoderm. Curr Top Dev Biol 47:1–32. Trainor PA, Zhou SX, Parameswaran M, et al.: 1999. Application of lacZ transgenic
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cific marking of the neural crest cell lineage in mice. Dev Biol 212:191–203.
Yamaguchi TP, Dumont DJ, Conlon RA, et al.: 1993. flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors. Development 118:489–498.
Zinyk DL, Mercer EH, Harris E, et al.: 1998. Fate mapping of the mouse midbrain-hindbrain constriction using a site-specific recombination system. Curr Biol 8:665–668.
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Studying Vascular Development in the Zebrafish Andreas M. Vogel and Brant M. Weinstein*
The zebrafish, a genetically accessible vertebrate with an externally developing, optically clear embryo, is ideally suited for in vivo functional dissection of the embryonic development of the circulatory system. Here, we review the advantages of the zebrafish as a model system for studying vascular development, and describe genetic and experimental tools, methods and resources that have been developed to exploit these advantages. We also discuss briefly how some of these tools and methods can be brought to bear on problems of relevance to human health. (Trends Cardiovasc Med 2000;10:352–360). © 2001, Elsevier Science Inc.
Determining the molecular basis for inherited or acquired cardiovascular defects is an important but still largely unmet priority in the fight against congenital heart malformations, atherosclerosis, and other acquired heart diseases. Furthermore, fundamental understanding of adult neoangiogenic processes and their critical role in both tumor progression and revascularization after injury and ischemia is still lacking. This understanding is critical because antiangiogenic therapies show enormous promise as a means of com-
Andreas M. Vogel and Brant M. Weinstein are from the Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, Maryland, USA. * Address correspondence to: Brant M. Weinstein, Laboratory of Molecular Genetics, NICHD, NIH, Building 6B, Room 309, 6 Center Drive, Bethesda, MD 20892. Tel.: 11 (301) 435-8264; fax: 11 (301) 435-6001. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
bating cancer by “starving” tumors of their blood supply. Dramatic tumor remissions have been observed in laboratory animals given antiangiogenic agents such as endostatin, with few or none of the side effects seen with chemotherapy. Proangiogenic therapies also show great promise for treating conditions such as limb and cardiac ischemia. Efforts to develop both pro- and anti-angiogenic therapies, as well as targeted treatments for atherosclerosis, depend on a detailed understanding of the genetic mechanisms of blood vessel formation and disease and on the identification of new molecular targets for these therapies. A great deal of progress has been made in recent years in identifying some of the major players. These include genes that regulate vascular proliferation and stability such as vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), and the angiopoietins (Ang-1 and Ang-2) and their receptor, Tie-2 (reviewed in TCM Vol. 10, No. 8, 2000
Mesp1 Expression Is the Earliest Sign of Cardiovascular Development Yumiko Saga,* Satoshi Kitajima, and Sachiko Miyagawa-Tomita
Understanding the molecular mechanism leading to formation of the heart and vasculature during embryogenesis is critically important because malformation of the cardiovascular system is the most frequently occurring type of birth defect. While the hearts of all vertebrates are derived from bilateral paired fields of primary mesodermal cells that are specified to the cardiac lineage during gastrulation, the mechanism for lineage restriction, and the origin of the myocardium and endocardium have not been defined. Recently, we found that a transcription factor, Mesp1, is expressed in almost all precursors of the cardiovascular system and plays an essential role in cardiac morphogenesis. Mesp1 may play a key role in the early specification for cardiac precursor cells. (Trends Cardiovasc Med 2000;10:345–352). © 2001, Elsevier Science Inc.
Wieland F, Harter C: 1999. Mechanism of vesicle formation: insights from the COP system. Curr Opion Cell Biol 11:440–446. Wiggins D, Gibbons GF: 1996. Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid. Biochem J 320:673–679. Williams KJ, Brocia RW, Fisher EA: 1990. The unstirred water layer as a site of control of apolipoprotein B secretion. J Biol Chem 265:16,741–16,744. Wu X, Zhou M, Huang L-S, et al.: 1996. Demonstration of a physical interaction between microsomal triglyceride transfer protein and apolipoprotein B during the assembly of apoB-containing lipoproteins. J Biol Chem 271:10,277–10,281. Xie Z, Wise H: 1997. Cyclic AMP-independent activation of neutrophil like HL-60 cells by prostaglandin E2. Cell Signal 7:531–537. Yeung SJ, Chen SH, Chan L: 1996. Ubiquitinproteasome pathway mediates intracellular degradation of apolipoprotein B. Biochemistry 35:13,843–13,848. Zhou M, Fisher EA, Ginsberg HN: 1998. Regulated co-translational ubiquitination of apolipoprotein B 100. J Biol Chem 273: 24,649–24,653. Zhou M, Wu X, Huang LS, Ginsberg HN: 1995. Apoprotein B 100, an inefficiently translocated secretory protein, is bound to the cytosolic chaperone, heat shock protein 70. J Biol Chem 270:25,220–25,224. PII S1050-1738(01)00071-8
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The critical phase of embryonic development is known as gastrulation. The immediate outcome of gastrulation is the formation of the definitive germ layers and the organization of a general body plan, which serves as a blueprint of the embryonic body. The developmental fate of cells in the epiblast has been analyzed within the last decade with the use of a transgenic lacZ marker, fluorescent dyes, chick-quail transplantation chimeras, or intracellular labels. The resulting fate map is essentially the same for the chick as for the mouse and urodeles, and indicates that, despite geometrical differences,
Yumiko Saga and Satoshi Kitajima are at Cellular & Molecular Toxicology Division, National Institute of Health Science, 1-18-1, Kamiyohga, Setagaya-ku, Tokyo 158, Japan. Sachiko Miyagawa-Tomita is at Department of Pediatric Cardiology, The Heart Institute of Japan, Tokyo Women’s Medical University, 8-1, Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. * Address correspondence to: Dr. Yumiko Saga, Cellular & Molecular Toxicology Division, National Institute of Health Sciences, 1-18-1, Kamiyohga, Setagaya-ku, Tokyo 158, Japan. Tel.: 81-3-3700-9652; fax: 81-3-37009647; e-mail address: [email protected]. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
topological fate relationships appear to be conserved among the vertebrates (Lawson and Pedersen 1992, Tam et al. 2000). The heart is the first functional organ formed during organogenesis. Cells destined to become cardiac mesoderm are fated, induced, and specified prior to and during gastrulation. The cardiac mesoderm cells invaginate through the primitive streak, and migrate and spread with the cranial mesoderm. Subsequently, the bilaterally symmetric cardiac precursors migrate and converge at the midline of the embryo to form the cardiac crescent, which then forms a linear, single heart tube. The tube formed by an outer myocardium and an inner endocardium undergoes rightward looping. The looped heart tube then undergoes septation to generate the mature, four-chambered cardiac structure. Many molecules are associated with cardiogenesis after formation of the crescent-shaped cardiac primordium. Nkx2.5 (Lints et al. 1993), Flt1 (Yamaguchi et al. 1993), Mef2B/C (Molkentin et al. 1996), Gata4 (Heikinheimo et al. 1994, Kuo et al. 1997), and dHand/ eHand genes (Srivastava et al. 1995) are expressed in the crescent-shaped cardiac primordium, myocardium and/or endocardium, respectively. Furthermore, many
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Figure 1. Mesp1 expression and the phenotype of the Mesp1-null embryo. (A) Mesp1 expression starts in the newly formed mesodermal cells at the onset of gastrulation (6.5 dpc). The expression continues until 7.5 dpc and is then quickly down-regulated. The axial and paraxial mesoderm does not express Mesp1. This early expression of Mesp1 completely disappeared before heart tube formation. Lateral view. A, anterior; P, posterior; Hf, head fold. (B) Cardiac malformation observed in Mesp1-null embryos at 9.5 dpc. Instead of a single heart tube generated in the wildtype, various types of malformations were observed in the mutants (from a single to two separated tubes). Each tube is sometimes separated into two small chambers. Frontal view. (C) Expression of Mesp1-LacZ indicating normal migration of the cranial-cardiac mesoderm in a heterozygous embryo at 9.5 dpc, but delay of migration in the homozygous embryo. Lateral view. Em, embryonic region; Ex, extraembryonic region.
signaling molecules are involved in the later cardiac morphogenesis (Fishman and Chien 1997, Olson and Srivastava 1996). Prior to the crescent-shaped cardiac primordium formation, however, little is known about the molecular signals that guide the cardiac mesoderm. In this article, we summarize a series of molecular and genetic analyses on the Mesp genes, Mesp1 and Mesp2, which are implicated in the early specification of cardiac precursor cells. • Mesp1 Is Expressed in the Early Mesoderm and Involved in Cardiac Morphogenesis Mesp1, belonging to the bHLH transcription factor family, is expressed in the early mesoderm at the onset of gastrulation (Figure 1 and Saga et al. 1996). The expression of the transcript is restricted to a part of mesoderm ingressed through the primitive streak of the early stage embryo (6.5–7.0 dpc) and quickly downregulated after 7.5 dpc and no expression has been observed in the paraxial or axial mesoderm. Only the slight expression observed at a later stage embryo (8.5 dpc in Figure 1) might represent a part of the cranial-cardiac mesoderm. The indication of involvement of Mesp1 in cardiac morphogenesis comes from a gene knockout study. Homozygous Mesp1 null mice exhibit various anomalies of heart tube formation and looping (Figure 1B). The heart tubes range from two completely sepa-
rated tubes to a single tube. The separated tubes show various degrees of cardia bifida, from full to partial bifurcations (Saga et al. 1999). This anomaly is most likely to be caused by a failure of ventral fusion of the pericardial mesoderm. However, these hearts show well-formed myocardium and endocardium. Therefore, cellular differentiation accompanied by heart tube formation occurs normally even in Mesp1-null embryos. Because Mesp1 is expressed in the nascent mesoderm at the onset of gastrulation, it is assumed that the abnormality is due to a precursor cell defect. To monitor the cell lineage, a knockin mouse (Mesp1LacZ) which has the b-galactosidase gene under the control of the Mesp1 promoter was analyzed (Saga et al. 1999). As the bgal activity is retained for a longer period than the mRNA signal, the cell movement can be traced from the onset of gastrulation to a later stage. The most anterior mesodermal cells expressing Mesp1-LacZ migrated to generate the future cardiac field and this migration was markedly delayed in the Mesp1-null embryo (Figure 1C), indicating that the cardiac anomaly is caused by the delay in mesodermal migration. However, since the b-gal activity was down-regulated before heart tube formation, it was not clear whether Mesp1 was expressed in the cardiac precursors or in other cells supporting cardiac morphogenesis. To define the cell lineage of Mesp1-expressing mesodermal cells from the beginning of the ex-
pression to the final destination, the knockin mouse (Mesp1-cre) which has Cre recombinase under the control of the Mesp1 promoter-enhancer was generated (Saga et al. 1999). • Mesp1-Expressing Cell Lineage Cell type-specific genomic alternations mediated by the Cre/loxP system constitute a powerful approach to the study of cell lineages (Trainor et al. 1999, Yamauchi et al. 1999, Zinyk et al. 1998). On crossing heterozygous Mesp1-Cre and reporter mice CAG-CAT-Z, one quarter of the progeny should consist of double transgenics (Figure 2A). Only cells that express Cre from the Mesp1 allele should undergo recombination between the loxP sites of the reporter construct, excising the CAT gene and permitting LacZ expression. As the Cre-mediated excision is cell-heritable, the marked cells and all their progeny should express LacZ at later stages, even after Cre is no longer expressed. Thus, LacZ staining in the double-transgenic embryo should reveal the progeny of all cells that express Cre transiently during development under the control of Mesp1 promoter and enhancer. Intercrossed embryos examined at 7.5 dpc revealed that the extraembryonic mesoderm and cranial-cardiac embryonic mesoderm in the lateral plate mesoderm was positive for LacZ staining (Figure 2B), while the paraxial and axial mesoderm were negative, indicat-
Figure 2. Lineage analysis of Mesp1-expressing cells. (A) Schematic presentation of the strategy for lineage analysis. Mesp1-cre which has the Cre recombinase gene under the Mesp1 promoter, is mated with a reporter mouse, CAG-CAT-Z, which has an inactive LacZ gene in the absence of Cre-mediated recombination (Sakai and Miyazaki 1997). Once Mesp1 is expressed, Cre recombinase eliminates the CAT gene which leads to activation of the LacZ gene. Because CAG promoter is ubiquitously active, the cells which expressed Mesp1 are expected to express LacZ continuously. (B) LacZ staining pattern of the 7.5 dpc intercrossed embryo, which exhibits strong staining in the extraembryonic mesoderm and progenitors of the cranial-cardiac mesoderm (CCM) (lateral view on left panel). Posterior view (right panel) shows no expression in paraxial (PM) and axial mesoderm (AM). A anterior; P, posterior; Ex, extraembryonic region. (C) LacZ staining of the 9.5 dpc intercrossed embryo, which exhibits strong staining in the heart (H). AL, anterior limb; M, mandibular arch; Otv, otic vesicle; OV, optic vesicle. (D) Cross-section of C (heart region), showing strong staining in both myocardium and endocardium including the cono-truncal and atrioventricular cushions. LacZ staining is seen in the endocardium, myocardium, and mesenchymal cells of the cushion at the atrioventricular canal, and the epicardium as shown in the upper right corner. Some cranial vessel primordia also show the LacZ staining. IC, inferior atrioventricular endocardial cushion; OFT, outflow tract; PRV, primitive right ventricle; SC, superior atrioventricular endocardial cushion. (E) Cross-section of C (caudal region) showing staining in the endothelial cells of the dorsal aorta (DA). (F) A section of the extraembryonic yolk sac showing staining in the endothelial cells (ET) but not in the endodermal cells (ED).
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Figure 2
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Figure 3. Comparison of gastrulating embryos (7.5 dpc) of wild-type and dKO. Photographs taken from the lateral view and schematic drawings are shown. In the wildtype, early ingressed groups of mesodermal, extraembryonic and cranial-cardiac cells have migrated out from the primitive streak. The paraxial mesoderm has also migrated anteriorly and the lateral mesoderm follows. Axial mesoderm has emerged from the most anterior part of the node and is elongated along the midline. In contrast, in the dKO embryo, no clear mesodermal layer was generated in the embryonic region although the primitive streak was formed and extraembryonic mesoderm was placed relatively normally. Axial mesoderm was initially generated normally but no further extension was observed in the dKO embryo.
Figure 4. Chimera analysis. (A) Schematic presentation of the strategy for chimera analysis. The double KO mouse line was genetically marked by mating with the Rosa26 mouse which shows ubiquitous b-gal activity in all cells. A dKO (2/2) 8-cell stage embryo obtained by intercross of the dKO mouse was aggregated with an 8-cell stage wild-type embryo and the blastocyst thus formed was transferred to the foster mother. The embryo was recovered at 9.5 dpc and stained for detection of b-gal activity. Only dKO (2/2) cells show b-gal activity and wild-type cells should not have any b-gal activity. (B) A chimera embryo which was generated by aggregating a dKO and a wild-type embryo. Note that the heart is composed of only wild-type white cells and lacks the LacZ-positive dKO cells. Lateral view. (C) A transverse section of B, confirming that heart tissues are exclusively composed of wild-type cells. The epicardium also consists of wild-type cells. PLV, primitive left ventricle; PRV, primitive right ventricle; OFT, outflow tract.
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ing that Mesp1-expressing cells contribute to the extraembryonic mesoderm and the cranial-cardiac mesoderm. Later embryos (9.5 dpc) were examined to further trace the lineage of cells expressing Mesp1. We previously reported that only the myocardium in the heart was derived from Mesp1-expressing cells (Saga et al. 1999). More detailed and careful analyses, however, revealed the b-gal activity not only in the heart tube but also in the vascular system, that is, the dorsal aorta, intersomitic vessels, and cranial vessels (Figure 2C–F). A cross-section showed the LacZ staining in the endocardium as well (Figure 2D), indicating that both cell types (myocardium and endocardium) were derived from Mesp1expressing cells. At the atrioventricular canal and outflow regions, the b-gal activity is seen in the endocardium, myocardium, and mesenchymal cells forming cardiac cushions. Furthermore, the epicardium and proepicardium show the LacZ staining. The strong LacZ staining indicates that the endothelial cells in the yolk sac are also derived from Mesp1-expressing cells (Figure 2F). These observations indicate that Mesp1 is activated at first in the extraembryonic mesoderm and then in the common precursor cells of myocardium, endocardium, and the vascular system. Therefore, Mesp1 may represent the earliest molecular marker for these cell lineages. Thereafter, flk-1 (an early molecular marker of endothelial precursor cells) (Yamaguchi et al. 1993) and Nkx-2.5 (an early molecular marker of cardiac progenitor cells) (Lints et al. 1993) transcripts become detectable in the 7.0 dpc and 7.5 dpc mouse embryo, respectively. In view of the endocardial cell lineage, in chick embryo, one hypothesis is that the endocardial cells originate from the two different precursors, that is, cardiac mesoderm and non-cardiac mesoderm (Eisenberg and Markwald 1995). We propose that the pre-myocardial and pre-endocardial cells have a common precursor (i.e., the Mesp1-expressing mesoderm). It is noted that no somitic mesoderm was stained, although intersomitic vessels were clearly stained. Although we are not sure of the origin of the intersomitic vessels, it is not likely to be the paraxial mesoderm. We previously showed that endogenous Mesp1 was expressed in the presomitic mesoderm just before segmentation (Saga et al. 1996). However, Cre is not expressed in the presomitic
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mesoderm from this Mesp1-cre allele since no b-gal activity was detected in any progenitor of the paraxial mesoderm. The reason is not clear but it could be due to interference by a pgk-neo cassette which was included in the targeting vector. This was fortunate for us because it enabled us to distinguish the cranial-cardiac mesoderm from the paraxial mesoderm.
(Figure 3). The embryos died at 9.5 dpc and exhibited only a head-like structure. They lacked any caudal structures behind the anterior head region, such as a posterior head region, heart or somites. These observations indicate that Mesp1 and Mesp2 function cooperatively during gastrulation and are essential for development of the embryonic mesoderm.
• Mesp1, Mesp2 Double Knockout Mouse
• Chimera Analysis Reveals Cell-autonomous Function of Mesp1 and Mesp2 in Cardiac Morphogenesis
The abnormal heart morphogenesis in Mesp1 (2/2) embryos resulting in cardia bifida is caused by delayed migration of the cardiac precursor cells. However, the initial suppression of cell migration is rescued at the later stages and the Mesp1 (2/2) embryo eventually generates an abnormal heart tube. The involvement of Mesp2, another gene of the same family, is indicated because of the similar expression pattern. An early expression study did not reveal Mesp2 expression at the early stage embryo, and Mesp2 knockout mice did not show any defect in cardiac morphogenesis (Saga et al. 1997). However, a prolonged in situ reaction revealed Mesp2 expression at 6.5–7.0 dpc in a pattern very similar to that of Mesp1. Furthermore, Mesp2 expression was retained longer in Mesp1 (2/2) embryos than in wild-type embryos. Accordingly, Mesp1- and Mesp2-doubleknockout mouse (Mesp1,p2-dKO) was generated to clarify the function of Mesp2 during gastrulation and to understand the cooperative functions of the two transcription factors (Kitajima et al. 2000). Mesp1,p2-dKO embryos exhibited abnormal gastrulation, because no clear embryonic mesodermal cell layer was observed between the endoderm and ectoderm in the embryonic region. The dense cell accumulation in the primitive streak indicated the initiation of gastrulation and the ingression of ectodermal cells into the primitive streak. However, the mesodermal cells appeared unable to depart from the primitive streak. On the other hand, extraembryonic mesoderm was generated and differentiated to generate blood and endothelial cells. Detailed analysis, using several mesodermal molecular markers, revealed that Mesp1,p2-dKO embryos lacked embryonic non-axial mesodermal cell layers, although the axial mesoderm initially generated also showed a defect during later development
Mesp1 and Mesp2 are expressed only in the early ingressed group of mesoderm and not in the paraxial and axial mesoderm. No paraxial mesoderm was generated in the dKO embryo, which resulted in a complete lack of somites. To clarify whether such mesodermal defects observed in the Mesp1,p2-dKO embryo are of a cellautonomous or non-cell-autonomous consequence, chimera analysis was conducted (see Figure 4 for the method). A chimeric embryo was generated by aggregating 8-cell embryos derived from wild-type and intercrossed heterozygous dKO mice (see Figure 4A for details). Thus, dKO/wild chimeric embryos should be composed of b-gal-negative wild-type cells and b-gal-positive Mesp12/2, Mesp22/2 cells. The results were extremely interesting. Chimera embryos containing Mesp12/2, Mesp22/2 cells developed normally but the heart was composed of only wild-type cells and lacked Mesp12/2, Mesp22/2 cells (Figure 4B). Cross-sections of these embryos confirmed the almost complete absence of Mesp12/2, Mesp22/2 cells in most of the heart tissue (Figure 4C). Myocardium and endocardium in the right and left ventricles and outflow tract, and the epicardium consisted of wild-type cells. However, b-gal-positive Mesp12/2, Mesp22/2 cells constituted a part of the mesenchymal cells in the outflow tract. The cells might be derived from the cardiac neural crest (Kirby and Waldo 1990). Interestingly, the paraxial mesoderm was composed of both types of cells, indicating that Mesp12/2, Mesp22/2 cells can contribute to somitogenesis. It is worth noting that the defect of Mesp12/2, Mesp22/2 cells in the chimera is closely correlated with the expression of Mesp1 and Mesp2. These genes are expressed in the early ingressed groups of mesodermal cells destined for the extraem-
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bryonic mesoderm and cranial-cardiac mesoderm, but not in the axial mesoderm, or early paraxial mesoderm, endoderm or ectoderm. Therefore, the inability of Mesp12/2, Mesp22/2 cells to generate the paraxial mesoderm in dKO embryos is not cell-autonomous but rather a consequence of earlier events, such as defective gastrulation. Similarly, it is reasonable that the b-gal-positive Mesp12/2, Mesp22/2 cells contributed to the neural tube, notochord, gut and the lateral plate mesoderm, except for the cardiac mesoderm. However, Mesp12/2, Mesp22/2 cells did contribute to the extraembryonic mesoderm and embryonic endothelial cells lining several vessels, such as the dorsal aorta and intersomitic vessels. Therefore, only the inability to contribute to the cardiac mesoderm and epicardium reveals a cell-autonomous defect. Figure 5 provides a summary of the lineage of Mesp1expressing cells of an early gastrulating embryo (6.5–7.0 dpc). • Discussion Mesodermal cells generate the heart tube. The embryonic mesoderm can be di-
vided into five regions; the chorda (axial) mesoderm, somitic dorsal (paraxial) mesoderm, intermediate mesoderm, lateral plate mesoderm, and head mesenchyme (Gilbert 2000). It is generally thought that the heart is derived from the lateral plate mesoderm. The results of our experiments and fate mapping studies (Tam et al. 2000) showed that the heart precursors in mouse are clearly localized as the cranial-cardiac mesoderm in the lateral plate mesoderm. Mesp1 and Mesp2 are co-expressed in the early mesoderm and function cooperatively. Our lineage study using Mesp1-cre mice revealed a primary mesoderm population expressing Mesp1 which included almost all endothelial precursors in both the extraembryonic and embryonic vascular systems. In the embryonic region, two populations of cardiac precursor cells, myocardium and endocardium, were also included in the cells which expressed Mesp1. Therefore, we propose that the cardiovascular system, including the myocardium and endocardium, emerges from the Mesp1-expressing cells, and Mesp1 is the earliest molecular marker of the cardiovascular system, although it
Figure 5. Summary of the lineage of Mesp1-expressing cells of an early gastrulating embryo (6.5–7.0 dpc). Because Mesp1 expression is down-regulated after 7.5 dpc (Figure 1), lineage analyses (Figure 2) demonstrated that only a small group of mesodermal cells was the source for almost all of the extraembryonic and embryonic vascular systems. In addition, Mesp1expressing mesoderm contains the precursors for myocardium and endocardium.
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is still unclear where the myocardium and endocardium diverge from the other cells. Various anomalies of heart tube formation and looping were observed in Mesp1-null mice. Delayed migration of the cardiac precursor cells must be a cause of the various degrees of cardia bifida in Mesp1-null mice. Several gene knockout mice have been shown to generate cardia bifida, which include fibronectin (George et al. 1993), Gata4 (Kuo et al. 1997, Molkentin et al. 1997, Narita et al. 1997), furin (Constam and Robertson 2000, Roebroek et al. 1998), Hrs (Kodama and Soriano 1999) and Smad5 (Chang et al. 1999) knockout mice. Cardia bifida found in the above-mentioned knockout mice is caused by a defect of ventral folding due to the failure of migration and/or proliferation of cells involved in the ventrolateral morphogenesis, which is a similar situation to that observed in Mesp1-null embryo. In addition, a responsible gene that causes cardia bifida in zebrafish mutant miles apart (mil) has recently been identified (Kupperman et al. 2000). The gene Mil, coding a receptor that binds lysosphingolipids, sphingosine-1phospate, has a role in generating an environment permissive for heart precursor cells to migrate to the midline. However, no direct molecular relationship between Mesp1 and these genes is known. Because Mesp1, Mesp2 double KO embryo lacks most of mesodermal layer, the genes implicated in the heart tube formation, such as Nkx2.5, Gata4 are not expressed in double KO embryos. The lack of these genes might be one of the causes leading to the migration failure, but Mesp genes might be required to regulate downstream target genes that are essential for mesoderm proliferation rather than migration, but this needs to be determined. Serum response factor (SRF) is known as a critical factor required for proper mesoderm formation. SRF-deficient embryos have a severe gastrulation defect and do not form any mesodermal cells (Arsenian et al. 1998). This phenotype resembles that of Mesp1, Mesp2 double knockout mouse. However the early expression of SRF is ubiquitous and not restricted in the mesoderm (Croissant et al. 1996, Li et al. 1997). The expression becomes restricted in the developing heart at a later embryonic stage and continues during heart morphogenesis in which Mesp1 expression was no longer observed. Therefore, TCM Vol. 10, No. 8, 2000
the SRF expression overlaps with that of Mesp1 only in some of the cells and during a certain period, which indicates that no direct relationship exists between them. Our studies revealed that the heart field is generated by migration of the cells which expressed Mesp1 and also suggested that the embryonic primary blood vessels are generated by the cells which expressed Mesp1. Endothelial precursor cells (angioblasts) form the first major blood vessels in the embryo, including the dorsal aorta and the endocardium. This process is called vasculogenesis (Risau et al. 1988). The ability of endothelial cells to migrate is critical for the formation of the primary vessels of the embryo. As for the mechanism of dorsal aorta development, VEGF is thought to be a crucial factor, since the homozygous mutant embryo shows a complete failure of dorsal aorta development (Carmeliet et al. 1996, Ferrara et al. 1996). Although the origin of the mouse embryonic angioblast required for development of the dorsal aorta remains unclear, vascular precursor cells have been shown to be derived from the lateral plate mesoderm in Xenopus embryo (Cleaver and Krieg 1998). In chick embryo, transplantation experiments showed that there are two endothelial lineages in the formation of the aorta; one is derived from the paraxial mesoderm (i.e., somite) and the other from the splanchnopleural mesoderm (Pardanaud et al. 1996). Because the expression of Mesp1 is transient and the Mesp1-expressing cells contribute little to the axial and paraxial mesoderm during gastrulation, our results suggest that the precursor cells of the dorsal aorta have already been determined by expression of the Mesp1 during gastrulation, and the precursor cells might belong to the cranial-cardiac mesoderm or, possibly, another distinct population in the lateral plate mesoderm. • Conclusion The series of experiments described here involving cell lineage, loss of function and chimeric analyses revealed a differential requirement of Mesp1 and Mesp2 for different cell lineages. For the extraembryonic mesoderm, lack of Mesp1 and Mesp2 did not result in a severe defect in either mesodermal formation or subsequent cell differentiation, suggest-
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ing that these genes may not be required for extraembryonic mesoderm development or, alternatively, other molecules may rescue the deficiency of Mesp1 and Mesp2. In contrast, Mesp1 and Mesp2 are essentially important for embryonic mesoderm formation. Mesp1 (and maybe Mesp2)-expressing cells are fated to be the precursor of the endothelium and the cardiovascular system. However, these genes are required cell-autonomously only for the precursor of the myocardium and endocardium of the ventricles since Mesp12/2, Mesp22/2 cells contributed to the formation of vascular endothelium. The actual function of Mesp1 and Mesp2 and the target gene(s), however, is not known. Without Mesp1 and Mesp2, mesoderm may not be specified as a cardiac mesodermal precursor. Alternatively, mesoderm may lack migratory activity for localization at the proper position to be incorporated into precardiac cells. These points remain to be investigated. In addition, we do not know how Mesp1 and Mesp2 work cooperatively. They might work as a heterodimer although, generally, cell type-specific bHLH factor dimelizes with a ubiquitously expressed bHLH factor such as E12 or E47. We do not know whether any human syndromes link to Mesp genes and more detailed analysis is necessary. As we could mark the cells which expressed Mesp1 with either LacZ or GFP (if we used the proper reporter line), it is possible to isolate these cells with a cell sorter and investigate their ability to generate the heart tube in vitro. In addition, we now have an excellent tool for delivering any gene to the cardiovascular cell lineage, which would be useful for investigation of the mechanism of cell fate determination and cell differentiation.
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PII S1050-1738(01)00069-X
TCM
Studying Vascular Development in the Zebrafish Andreas M. Vogel and Brant M. Weinstein*
The zebrafish, a genetically accessible vertebrate with an externally developing, optically clear embryo, is ideally suited for in vivo functional dissection of the embryonic development of the circulatory system. Here, we review the advantages of the zebrafish as a model system for studying vascular development, and describe genetic and experimental tools, methods and resources that have been developed to exploit these advantages. We also discuss briefly how some of these tools and methods can be brought to bear on problems of relevance to human health. (Trends Cardiovasc Med 2000;10:352–360). © 2001, Elsevier Science Inc.
Determining the molecular basis for inherited or acquired cardiovascular defects is an important but still largely unmet priority in the fight against congenital heart malformations, atherosclerosis, and other acquired heart diseases. Furthermore, fundamental understanding of adult neoangiogenic processes and their critical role in both tumor progression and revascularization after injury and ischemia is still lacking. This understanding is critical because antiangiogenic therapies show enormous promise as a means of com-
Andreas M. Vogel and Brant M. Weinstein are from the Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, Maryland, USA. * Address correspondence to: Brant M. Weinstein, Laboratory of Molecular Genetics, NICHD, NIH, Building 6B, Room 309, 6 Center Drive, Bethesda, MD 20892. Tel.: 11 (301) 435-8264; fax: 11 (301) 435-6001. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
bating cancer by “starving” tumors of their blood supply. Dramatic tumor remissions have been observed in laboratory animals given antiangiogenic agents such as endostatin, with few or none of the side effects seen with chemotherapy. Proangiogenic therapies also show great promise for treating conditions such as limb and cardiac ischemia. Efforts to develop both pro- and anti-angiogenic therapies, as well as targeted treatments for atherosclerosis, depend on a detailed understanding of the genetic mechanisms of blood vessel formation and disease and on the identification of new molecular targets for these therapies. A great deal of progress has been made in recent years in identifying some of the major players. These include genes that regulate vascular proliferation and stability such as vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), and the angiopoietins (Ang-1 and Ang-2) and their receptor, Tie-2 (reviewed in TCM Vol. 10, No. 8, 2000