So far, no detailed data are available about the expression and function of ATIPs during development. Therefore, we examined in this study the expression of ATIPs during mouse development. To determine the histological location of Mtus1 expression during mouse development, beta-galactosidase stainings of a gene-trapped Mtus1 mouse line (Zuern et al., 2012) were performed (Figs. 1-5). To further verify these Mtus1 promoter activity data, we also examined the Mtus1 mRNA expression by whole-mount in situ hybridization experiments (Figs. 6 and 7) and the corresponding ATIP protein expression by immunohistochemistry (Fig. 8) in defined stages of mouse development. In addition, to further distinguish in more detail between the three different murine ATIP isoforms, we determined the mRNA expression pattern in various tissues of embryonic stages E7.5 to E14.5 by RT-PCRs.
Cardiovascular ATIP Expression
During mouse development, a very prominent Mtus1 expression was detected in the entire cardiovascular system. Heart formation in mice is initiated by cardiac progenitor cells migrating to the ventral midline of the embryo at E7.5. At this early stage, Mtus1 promoter activity is not yet detectable in the FHF (first heart field) cells of the cardiac crescent (Fig. 1A,C), indicating that ATIP is not playing a role during these early steps of cardiac differentiation. As Mtus1 is also not expressed in the SHF (second heart field), ATIPs seem to be irrelevant for the determination of FHF or SHF. The initial expression of Mtus1 in the cardiovascular system was visible when the first functional cardiomyocytes form the primitive heart tube at E8.0 (Fig. 1D). Here, Mtus1 promoter activity was seen in the endocardial tissue as well as in the myocardial tissue of the primitive heart tube (Fig. 1G). Moreover, Mtus1 was highly expressed in the visceral yolk sac, where the blood islands have fused together to form the extra-embryonic vasculature (Fig. 1D–G), pointing to an additional role not only in embryonic but also in extra-embryonic blood vessel formation.
At E8.5, when the S-shaped cardiac looping begins, Mtus1 is strongly expressed in the entire heart, including endocardial and myocardial tissue of the atrial and ventricular heart chambers, bulbus cordis, and sinus venosus (Figs. 1F,I,J, 6A). At this stage, the embryonic and extra-embryonic circulation assembles, and Mtus1 promoter activity is clearly visible in the first forming embryonic blood vessels, like the dorsal aorta and the vitelline artery (Fig. 1J–L), and in the extra-embryonic circulation of the visceral yolk sac (Fig. 1E,F).
The strong Mtus1 expression in the heart remains in E9.5, where it was globally found in the heart (Figs. 2A–C, 6B,C). Notably, in the common atrial heart chamber, Mtus1 was expressed in the entire myocardial wall (Figs. 2D,E,G, 7A,C), mainly in the myocardial trabeculation of the ventricular heart chambers (Figs. 2D–G, 7A–C), whereas in the outflow tract, including the truncus arteriosus and the aortic sac, Mtus1 expression was focused to the endothelium (Figs. 2D–G, 7B,C). In addition to a remaining strong extra-embryonic vascular expression in the yolk sac, Mtus1 promoter activity was detected in the expanding embryonic vascular system, visualized in the branchial arch arteries (Fig. 2G,K), dorsal aorta in the head region and primary head veins (Fig. 2I), dorsal aorta in the truncus region with branching somitic arteries (Fig. 2A,G, J–L) and vitelline and umbilical blood vessels (Fig. 2L).
Figure 2. Mtus1 promoter activity at E9.5. Whole mount X-Gal stained Mtus1+/− embryos. A: Lateral right view. B: Lateral left view. C: Higher magnification of cardiac region of B. D–L: Sections through X-Gal stained embryos demonstrating detailed Mtus1 promoter activity in cardiovascular tissues and in the eye. Eye (1), heart (2), first branchial arch artery (3), second branchial arch artery (4), dorsal aorta (5), somitic arteries (6), notochordal plate (7), ventricular heart chamber (8), atrial heart chamber (9), myocardium of bulbus cordis (10), endocardium of bulbus cordis (11), endocardium of truncus arteriosus (12), myocardium of truncus arteriosus (13), bulbo-ventricular canal (14), trabeculation of myocardial wall of common ventricular heart chamber (15), aortic sac (16), perioptic vascular plexus (17), outer layer of optic cup/future pigment layer of retina (18), inner layer of optic cup/future neural layer of retina (19), dorsal aorta in head region (20), primary head vein (21), vitelline artery (22), umbilical vein (23).
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At E10.5, when the four heart chambers are already aligned as in the mature heart, prominent Mtus1 expression persists in the heart (Figs. 3A,C, 6D, 7D,E, 8A), with high levels in the atrial (Figs. 3G,H, 7E) and ventricular heart chambers (Figs. 3G–I, 7D,E, 8A), and the endothelium of the outflow tract (Figs. 3H,I, 7D), and with lower levels in the valves (Figs. 3G,I, 7E, 8B). At this stage, further differentiation of the outflow tract occurs with the first evidence of aortico-pulmonary spiral septum formation. This is seen by an increase of mesenchyme cells between the outer myoepithelial layer and the inner layer of endocardial cells. Expression of Mtus1 in the vascular system of this stage is detected in the paired dorsal aorta (Figs. 3G,H,N, 6C, 7G), somitic arteries (Figs. 3A,H, 6C, 7G), branchial arch arteries (Fig. 3K), perioptic vascular plexus (Fig. 3M), the broad forming network of cerebral blood vessels (Fig. 3M) and the vitelline vein (Figs. 3N, 8G). Regarding extra-embryonic vasculature, Mtus1 promoter activity remained high in the yolk sac (Figs. 3D,F, 6I), and appeared strongly in the umbilical blood vessels (Fig. 7H) and in the fetal vascular labyrinth part of the primitive placenta (Fig. 3E,O).
Figure 3. Mtus1 promoter activity at E10.5. Whole mount X-Gal stained Mtus1+/− embryos. A: Lateral left view. B: Dorsal view. C: Ventral view. D: Whole embryo in intact yolk sac with placenta. E: Fetal view of placenta. F: Isolated yolk sac and amnion. G: Transverse cardiac section. H,I: Sagittal cardiac sections. J: Transverse head section. K: Sagittal head section. L: Sagittal eye section. M: Sagittal brain section. N: Transverse lower truncus section. O: Sagittal placenta section. Midbrain (1), eye (2), heart (3), somitic arteries (4), forelimb bud (5), umbilical cord (6), hindlimb bud (7), tail (8), olfactory pit (9), placenta (10), yolk sac with embryo (11), maternal decidua part of primitive placenta (12), fetal labyrinth part of primitive placenta (13), yolk sac (14), amnion (15), right part of atrial heart chamber (16), left part of atrial heart chamber (17), bulbus cordis (18), ventricular heart chamber (19), dorsal aorta (20), notochord (21), somitic artery (22), hindgut diverticulum (23), aortic sac (24), myocardium of truncus arteriosus (25), endocardium of truncus arteriosus (26), endocardium of aortic sac (27), endocardial cushion of atrio-ventricular canal (28), myocardium of bulbus cordis (29), endocardium of bulbus cordis (30), outer pigment layer of retina (31), inner neural layer of retina (32), perioptic vascular plexus (33), first branchial arch artery (34), network of cerebral blood vessels (35), neuroepithelium of neural tube (36), vitelline vein (37).
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Analogous cardiovascular Mtus1 expression was found at E11.5 (Figs. 4, 8B,C) and E12.5 (Fig. 5). Mtus1 remained excessively expressed in the atria, the myocardial ventricular wall, and in the outflow tract of the heart (Figs. 4B,D–F, 5E,F, 8B,C), and in the vascular embryonic circulation system, as demonstrated in the dorsal aorta (Fig. 4D–F,L), somitic arteries (Fig. 4L,M), expanding branching network of cerebral blood vessels (Figs. 4A,D, I–K, 5O), hyaloid plexus of vessels within the hyaloid cavity (Figs. 4G,H, 5H), and the hepatic sinusoids (Fig. 5P). Once again, Mtus1 expression was observed in extra-embryonic vascular tissue, e.g., in blood vessels and the endodermal component of the yolk sac (Figs. 4C,O, 5D,T, 6H,I, 7J–L, 8N), umbilical blood vessels (Figs. 4L, 5C,S), and the fetal vascular labyrinth part of placenta (Figs. 4O, 5C, 6H, 8M).
Figure 4. Mtus1 promoter activity at E11.5. Whole mount X-Gal stained Mtus1+/− embryos. A: Lateral left view. B: Isolated heart and lung. C: Placenta with yolk sac and amnion. D, E: Sagittal heart sections. F: Transverse heart section. G: Transverse eye section. H: Sagittal eye section: I: Transverse head section. J, K: Sagittal head sections. L: Sagittal truncus section. M: Transverse tail section. N: Sagittal limb bud section. O: Transverse placenta section. Network of cerebral blood vessels (1), eye (2), forelimb bud (3), hindlimb bud (4), heart (5), lung (6), placenta (7), yolk sac (8), amnion (9), myocardial wall of heart ventricle (10), truncus arteriosus (11), aortic sac (12), atrial heart chamber (13), dorsal aorta (14), endocardial cushion of atrio-ventricular canal (15), first branchial arch artery (16), aorta (17), outer pigment layer of retina (18), inner neural layer of retina (19), primitive hyaloid plexus of vessels within hyaloid cavity (20), cerebral artery (21), umbilical blood vessel (22), intersomitic arteries (23), dorsal root ganglia (24), neural tube blood vessels (25), maternal decidua part of placenta (26), fetal labyrinth part of placenta (27).
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Figure 5. Mtus1 promoter activity at E12.5. Whole mount X-Gal stained Mtus1+/− embryos and isolated organs. A: Lateral left view. B: Isolated forelimb bud. C: Embryonic side of placenta. D: Visceral yolk sac with vascular network. E: Isolated heart and lung. F: Sagittal isolated heart section. G: Sagittal isolated lung section. H: Transverse eye section. I, J: Transverse brain sections. K: Transverse palate section. L: Transverse isolated truncus section. M: Transverse tail section. N: Sagittal truncus section. O: Sagittal head section. P: Transverse section of isolated liver. Q: Sagittal isolated forelimb bud section. R: Transverse isolated hindlimb bud section. S: Transverse umbilical cord section. T: Sagittal yolk sac section. Umbilical cord (1), chorionic plate (2), heart (3), lung (4), right atrium (5), left atrium (6), atrio-ventricular cushion (7), myocardial wall of future left ventricle (8), bulbus cordis (9), segmental bronchi (10), homogenous lung parenchyma (11), outer pigment layer of retina (12), inner neural layer of retina (13), primitive hyaloid plexus of vessels within hyaloids cavity (14), mesencephalic vesicle (15), neuroepithelium (16), choroid plexus within fourth ventricle (17), Jacobson's organ (18), olfactory epithelium (19), mantle layer of neural tube (20), dorsal root ganglia (21), network of cerebral blood vessels (22), cerebral nerves (23), hepatic sinusoids (24), pre-cartilage of digital bones (25), pre-cartilage of femur (26), pre-cartilage of tibia (27), pre-cartilage of metatarsal bone (28), umbilical arteries (29), umbilical vein (30), endodermal yolk sac component (31), mesodermal yolk sac component (32).
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Figure 6. Whole mount in situ hybridization of Mtus1 (A–I) and Agtr2 (J–O) mRNA expression in wild-type embryos. Mtus1 mRNA expression at E8.5 ventral view (A), E9.0 lateral left view (B), E9.5 lateral left view (C) E10.25 lateral right view (D), E10.75 lateral right view (E), E11.5 lateral right view (F), E12.5 lateral right view (G), E11.5 placenta with yolk sac fetal view (H), and E10.5 yolk sac (I). Agtr2 mRNA expression at E9.0 lateral left view (J), E10.25 lateral left view (K), E10.75 dorsal view (L), E12.5 lateral right view (M), E11.5 placenta with yolk sac fetal view (N), and E10.5 yolk sac (O). Heart (1), yolk sac (2), eye (3), somitic arteries (4), dorsal aorta (5), forelimb bud (6), tail tip (7), hindlimb bud (8), whiskers (9), ear (10), fetal labyrinth part of placenta (11), maternal decidua part of placenta (12). The very strong staining in the bigger cavities of the brain, tail, and ear placodes in some of these embryos (B, C, D, E, K, and L) turned out to be just trapped probe but not specific staining.
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Figure 7. Mtus1 mRNA expression pattern demonstrated on sections of Mtus1 whole mount in situ hybridization analyses of wildtype embryos. A–C: Heart sections at E9.5. D, E: Heart sections at E10.5. F: Eye section at E10.5. G: Sagittal truncus section at E10.5. H: Sagittal umbilical cord section at E10.5. I: Forelimb bud section at E10.5. J: Transverse yolk sac section at E12.5. K: Sagittal yolk sac section at E12.5. L: Higher magnification of yolk sac section at E12.5. Wall of common atrial heart chamber (1), myocardial wall of common ventricular heart chamber (2), endocardium of common ventricular heart chamber (3), endocardium of aortic sac (4), myocardium of truncus arteriosus (5), endocardium of truncus arteriosus (6), myocardium of bulbus cordis (7), endocardium of bulbus cordis (8), left and right first branchial arch artery (9), bulbo-ventricular canal (10), endocardial cushion of atrio-ventricular canal (11), inner neural layer of retina (12), outer pigment layer of retina (13), somitic arteries (14), umbilical blood vessel (15), dorsal aorta (16), forelimb bud (17), yolk sac blood vessels (18), endodermal yolk sac component (19), mesodermal yolk sac component (20), blood islands within yolk sac (21).
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Figure 8. ATIP protein expression demonstrated by immunhistochemistry analyses of cryosections of wildtype embryos. A: Heart section at E10.5. B, C: Heart sections at E11.5. D: Eye section at E9.5. E: Eye section at E10.5. F: Eye section at E11.5. G: Truncus section at E10.5. H: Forelimb bud section at E10.5. I: Forelimb bud section at E11.5. J, K: Sagittal truncus sections at E12.5. L: Sagittal nose section at E12.5. M: Horizontal placenta section at E11.5. N: Yolk sac section at E11.5. O: Sole secondary antibody (2.ab) staining of yolk sac section at E11.5 as exemplary negative control. Endocardium of common ventricular heart chamber (1), myocardium of common ventricular heart chamber (2), bulbus cordis (3), endocardial cushion of atrio-ventricular canal (4), myocardial wall of common atrial heart chamber (5), outer pigment layer of retina (6), inner neural layer of retina (7), primitive hyaloid plexus of vessels within hyaloid cavity (8), wall of vitelline vein (9), forelimb bud (10), dorsal root ganglia (11), neural tube (12), olfactory epithelium (13), fetal labyrinth part of placenta (14), maternal decidua part of placenta (15), yolk sac (16). Scale bars = 50 μm.
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The RT-PCR expression pattern of the 3 different known ATIP isoforms revealed that in the whole cardiovascular system, examined in isolated samples of heart, umbilical cord vessels, yolk sac, and placenta, isoform 1 and isoform 4 were both expressed from E8.5 to E14.5 (Fig. 9).
Figure 9. RNA expression pattern of different Mtus1 isoforms and Agtr2 at indicated developmental stages and tissues by RT–PCRs. Gapdh expression confirmed equal cDNA loading, RT- PCR excludes DNA contaminations. In E10.5–E13.5 brain equates to brain tissue without eyes. U.cord, umbilical cord; M, marker.
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Summing up these data, ATIP1 and ATIP4 are very strongly and well-defined as expressed in the extra-embryonic and embryonic cardiovascular system during mouse development.
ATIP Expression During Eye Development
The development of the eyes is initiated by paired outgrowings of the brain, which come in contact with the surface ectoderm to induce the formation of the lens placodes. The earliest Mtus1 expression during these processes was found at E9.5 when the optic vesicle has formed (Figs. 2A,B, 6B,C). Here, Mtus1 is expressed in the outer layer of the optic cup, forming the future pigment layer of the retina, as well as in the inner layer of the optic cup, forming the future neural layer of the retina (Figs. 2G,H, 8D). At E10.5, when the lens vesicle is forming, Mtus1 remained expressed in the inner and outer layer of the optic cup, with intensified expression at the margins (Figs. 3A,B,J–L, 7F, 8E). After lens formation at E11.5, Mtus1 expression in the eyes was mainly located in the outer layer of the optic cup, (Figs. 4A,G,H, 8F). Furthermore, the blood vessels supplying the developing eyes are positive for Mtus1 expression, indicated by the Mtus1 signal in the primitive hyaloid plexus of the hyaloid cavity (Figs. 4G,H, 8F), and the perioptic vascular plexus (Figs. 2H, 3J).
The apportionment of Mtus1 isoform expression by RT-PCRs revealed a clear expression of isoform 3 in combination with isoform 4 and faintly with isoform 1 (Fig. 9). As the eye is partly developing from brain, the so far described restricted expression of isoform 3 to the brain fits to the expression in the eye. The expression of isoform 1 and isoform 4 in the eye most likely reflects the expression of ATIP isoforms in the blood vessels of the eyes.
ATIP Expression in Limbs
During limb bud formation, Mtus1 expression was well-defined when detected at E10.5 and E11.5 in the middle-distal part of the forelimb and hindlimb buds (Figs. 3A–,N, 4A,N, 6D–F, 7I, 8H,I). Additionally, Mtus1 was expressed in the ectoderm of the forelimb and hindlimb bud (Figs. 4N, 7I, 8H,I). At E12.5, Mtus1 expression was localized in the ectoderm and in the pre-cartilage of future bones, like digital bones, metatarsal bones, tibia, or femur (Figs. 5A,B,Q,R, 6G).
RT-PCR results demonstrated mRNA expression of Mtus1 isoforms 1 and 4 in the limbs during development (Fig. 9). This indicates that ATIPs might play a role during the complex regulation of limb bud development, in concert with other well-known important factors like FGFs, Shh, dHand, Hoxd, and Tbx (reviewed in Towers and Tickle, 2009a; Towers and Tickle, 2009b; Butterfield et al., 2010).
ATIP Co-Expression With AT2
ATIPs are described as proteins functionally interacting with the angiotensin 2 Type II receptor. To determine where ATIPs are co-expressed with AT2, we performed Agtr2 whole mount in situ hybridization analyses in different embryonic stages (Fig. 6, bottom panel) and RT-PCRs of embryonic samples (Fig. 9). During cardiovascular development, we detected at least a faint Agtr2 expression in heart and umbilical cord in all examined stages from E8.5 to E14.5, indicating that ATIP could interact with AT2 in heart and blood vessels. Also during eye and limb development, there was a common Mtus1 and Agtr2 expression detectable (Figs. 6B–G,J–M, 9). During eye development, both Mtus1 (Figs. 2A,B, 3A, 4A, 6B–G) and Agtr2 (Fig. 6J–M) expression patterns seem to overlap partially, whereas the prominent Agtr2 expression in the limbs (Fig. 6L,M), in particular the central Agtr2 expression in the limb bud at E10.5 (Fig. 6L), is not similar to the Mtus1 expression (Figs. 3A–C,N, 6E, 8H), which is detected at the more distal part of the limb bud. In placenta, brain, and liver at E11.5–14.5, we could not detect any Agtr2 expression even if there was an expression of Mtus1 (Figs. 6 and 9). This supports the notion that during developmental processes and in adult organs, ATIPs may act in different signaling pathways, which are dependent and independent of AT2 signaling.
Molecular Interactions of ATIP
Mouse ATIP isoform 4 interacts with the C-terminal part of the AT2, while ATIP isoform 3 and isoform1 fail to interact (Nouet et al., 2004; Wruck et al., 2005). A C-terminal deletion mutant of ATIP isoform 4 is still able to interact with AT2, indicating that the N-terminal part of ATIP isoform 4, which is different in the other 2 ATIP isoforms, is relevant and sufficient for this interaction (Wruck et al., 2005). Accordingly, only ATIP isoform 4 is obviously able to exert a direct interaction with the AT2.
As we detected Mtus1 isoform 4 ubiquitiously in all examined RT-PCR samples and, additionally, we found Mtus1 expressed in the entire vascular system, we assume that Mtus1 isoform 4 is mainly expressed in blood vessels. ATIP isoform 4 is shown to interact with AT2; however, Agtr2 is not expressed ubiquitously. Therefore, ATIP isoform 4 most likely acts in the vascular system not exclusively in an AT2-dependent manner.
The high and exclusive expression of Mtus1 isoform 3 in fetal brain suggests a main function during neural development or memory processes. Due to the missing interaction of ATIP isoform 3 with AT2, its putative physiological functions are, in contrast to the mostly abundant Agtr2 expression in the cerebellum, most likely AT2 independent (Wruck et al., 2005).
During development, we detected Mtus1 isoform 1 mostly co-expressed with isoform 4, except for brain, liver, and gonads, where isoform 1 was not detectable. The coiled-coil C-terminal regions of ATIPs suggest possible homo- as well as hetero-dimerization (Nouet et al., 2004), hence a concomitant function is conceivable.
Physiological Functions of ATIP
Mice overexpressing ATIP4 (Fujita et al., 2009) show a significant reduction in neointima formation, together with reduced vascular cell proliferation, oxidative stress, and inflammation but without effects on blood pressure. However, it is not clear whether the protective effect of ATIP1 on vascular injury is AT2-dependent. ATIP4 has been shown to contribute to diverse intracellular cascades. In rat fetal neurons, ATIP4 and AT2 constitutively interact at the plasma membrane in a multimeric complex also comprising tyrosine phosphatase SHP-1 (Li et al., 2007). Upon AT2 stimulation, ATIP4 and SHP-1 dissociate from the receptor and translocate together to the methanesulfonate sensitive 2 (MMS2) ubiquitin ligase variant involved in neuronal differentiation.
Since Mtus1 is abundantly expressed during cardiovascular development, we would expect severe cardiac malformations and early lethality in Mtus1 knockout mice. However, this is not the case (Zuern et al., 2012), suggesting that other factors are compensating for the loss of ATIP. One predestinated candidate for this is the Mtus2 gene product CAZIP (also designated TIP150 or KIAA0774 in chicken). The amino acid sequences of ATIP and CAZIP are conserved (approximately 35% identity in humans), showing the highest homology in their C-termini, the part of the proteins that is also present in all described ATIP isoforms (Rodrigues-Ferreira and Nahmias, 2010), and the expression profile during cardiac mouse development matches perfectly to that of Mtus1 (Du Puy et al., 2009).