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Keywords:

  • ATIP (Angiotensin receptor type 2 interacting protein);
  • ATBPs (Angiotensin receptor type 2 binding proteins);
  • Mtus1 (Mitochondrial tumor suppressor 1 gene);
  • AT2 (Angiotensin type 2 receptor);
  • Agtr2 (Angiotensin type 2 receptor gene);
  • cardiovascular development;
  • LacZ;
  • gene trap

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental Procedures
  6. Acknowledgments
  7. References

Background: ATIPs (Angiotensin receptor type 2 [AT2] interacting proteins) are described as AT2 interacting protein variants, whereas their expression and functions during development are not known yet. Results: Here, we provide a detailed expression pattern of ATIP variants during mouse development by visualizing Mtus1 promotor activity, Mtus1 RNA, and subsequent ATIP protein expression. ATIPs are strongly expressed in the developing cardiovascular system, including the vascular plexus of the yolk sac and the fetal vascular part of the placenta. Moreover, ATIP is expressed spatially distinct during eye and limb bud development, and in later stages in lung and nervous system. The three murine ATIP isoforms are expressed in a distinct manner, whereupon isoform 1 and 4 are correlated to cardiovascular, lung, and limb bud development and isoform 3 is restricted to brain and eye development. Interestingly, Mtus1 expression is not necessarily correlated to Agtr2 expression, suggesting novel but yet unknown functions for ATIP, independent of AT2 signaling. Conclusions: ATIPs seem to be mainly involved in the developmental regulation of the cardiovascular system and may act in different AT2-dependent and -independent manners. Hence, these results deliver valuable information to further elucidate the different functions of ATIPs in the processes of mammalian development. Developmental Dynamics 243:699–711, 2014. © 2013 Wiley Periodicals, Inc.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental Procedures
  6. Acknowledgments
  7. References

ATIP, initially identified as MTUS1 (Mitochondrial Tumor Suppressor 1) (Seibold et al., 2003), was later shown to be expressed in a variety of splice variants (reviewed in (Rodrigues-Ferreira and Nahmias, 2010), whereof certain variants interact with the AT2, hence named AT2 Interacting Protein (ATIP) (Nouet et al., 2004; Di Benedetto et al., 2006) or AT2 Binding Protein (ATBP) (Wruck et al., 2005). For reasons of clarity, we use the ATIP nomenclature throughout this study and all data correspond to the mouse ATIP isoforms (Table 1). In humans, the Mtus1 gene is coding for five different transcript variants, whereas the homologous mouse Mtus1 gene lacks exons 3 and 4 and is, therefore, only coding for three isoforms, ATIP1, ATIP3, and ATIP4, respectively. All three isoforms harbour the same C-terminal conserved coiled-coil region, but differ in their N-terminal parts. In man, Mtus1 is regulated by four different gene promoters, whereas little is known for the murine system (reviewed in Rodrigues-Ferreira and Nahmias, 2010).

Table 1. Comparative Nomenclature of ATIP Isoforms In Mouse, Human, Rat, and Zebrafish (AA)
MouseHumanRatZebrafish
 ATIP3a (1,270)  
ATIP1 = ATBP135 (1210)ATIP3b (1,216)ATIP3 (1,210)ICIS (1,338)
ATIP3 = ATBP60 (520)ATIP4 (517)ATIP4 (520)
ATIP4 = ATBP50 (440)ATIP1 (436)ATIP1 (440)

By using the C-terminal part of the AT2 as bait in yeast two-hybrid systems, ATIP was identified as new AT2 interacting protein, resulting in the newer ATIP nomenclature (Nouet et al., 2004; Wruck et al., 2005). Human ATIP1 (Table1: nomenclature of corresponding orthologues) interacts with AT2 and mediates inhibition of growth factor-induced ERK2 activation and cell proliferation (Nouet et al., 2004). Mouse ATIP4, the orthologue of human ATIP1 (Table1), was shown to be involved in the transport of AT2 from the Golgi compartment to the plasma membrane and to mediate the inhibitory effects on MAP kinases and anti-proliferative effects of the AT2 (Wruck et al., 2005). Since human ATIP1 trans-inactivates receptor tyrosine kinases, it was considered as an early component of growth inhibitory signaling cascades (Nouet et al., 2004). Poly (ADP-ribose) polymerase-1 (PARP-1) activates transcription of the Mtus1 gene and concurrently represses Agtr2 gene transcription (Reinemund et al., 2009). Mice ubiquitously over-expressing mouse ATIP isoform 4 showed attenuated superoxide anion production, activation of cell proliferative signaling cascades, and elevated expression of tumor necrosis factor α (Fujita et al., 2009). After femoral artery cuff placement, neointima formation was reduced in these mice, indicating a role in vascular remodeling (Fujita et al., 2009). Taken together, these data suggest a dual mechanism of AT2 activity regulation, in which ATIP mediates intracellular translocation of AT2 to defined areas of the cell membrane, but also through direct interaction of ATIP with the AT2.

During development, the cardiovascular system is the first functional system, which is essential for a proper supply of oxygen and nutrition for the growing embryo. Initially, cardiac progenitor cells are positioned in the cardiac crescent at the ventral midline of the embryo, and then form the primitive heart tube. At this stage, the heart also connects to the arterial and venous circulation system, where blood circulates. Within the following looping process, the cardiac chambers are formed and the final septation and formation of valves facilitates the controlled blood flow. As disturbances during cardiac development lead to severe heart defects in 1% of newborns and 10% of stillbirths, it is of major interest to understand in detail the spatial and temporal fine-tuning of these processes.

The Renin-Angiotensin System (RAS) plays an important role in cardiovascular physiology. In this system, the AT2 seems to counteract the pro-inflammatory, pro-hypertrophic, and pro-fibrotic actions of the AT1 receptor and it is therefore of major interest to define the regulatory mechanisms, which inhibit the activated RAS. Amongst other AT2 interacting proteins, e.g., ErbB3 (Knowle et al., 2000), PLZF (Senbonmatsu et al., 2003), CNK1 (Fritz and Radziwill, 2005), or TIMP-3 (Kang et al., 2008), functional ATIP interaction with AT2 is regarded to be essential for the AT2-mediated growth inhibition (Reinemund et al. 2009). The AT2 receptor was found to be expressed in a variety of tissues of developing mammalian embryos, including mice, rats, and humans (Millan et al., 1989; Zemel et al., 1989, 1990). Especially in the mammalian heart, AT2 expression decreases with age, thus its functions in the adult heart remained widely discussed (Timmermans et al., 1992; Bottari et al., 1993; de Gasparo et al., 2000). Nowadays, mainly based on studies using genetically modified mice, it is becoming more and more evident that the AT2 antagonizes some effects of the AT1. Thereby, the majority of available data support the hypothesis of pro-hypertrophic but anti-fibrotic actions of the AT2 in pressure-overload or Angiotensin-infusion models in adult mice (reviewed in Widdop et al., 2003), and it is re-expressed in adult hearts in the event of myocardial infarction (Nio et al., 1995). However, the function and regulation of AT2 during heart development remains unclear, since AT2-deficient mice are born healthy and display an almost normal phenotype (Hein et al., 1995; Ichiki et al., 1995).

Gene targeting or gene trapping constructs that comprise the lacZ gene encoding beta-galactosidase allow to knock out gene function on the one hand and monitor endogenous promoter activity on the other hand (Austin et al., 2004). Thereby, it provides a very sensitive and cell-specific method to illustrate the endogenous promoter activity, whereas the X-Gal intensity is considered as proportional to the beta-galactosidase expression level. In this study, we describe the spacial and temporal promoter activity pattern of the Mtus1 gene during embryonic mouse development from day E7.5 to E12.5 by using a gene-trapped Lac-Z reporter that was placed under control of the endogenous Mtus1 gene promoter (Zuern et al., 2012), and we confirmed these data by investigating mRNA and protein expression profiles.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental Procedures
  6. Acknowledgments
  7. References

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.

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Figure 1. Mtus1 promoter activity at E7.5 to E8.5. A–F: Whole mount X-Gal stained Mtus1+/− embryos. A: E7.0; B: E7.25; C: E7.5; D: E8.0; E: E8.5 dorsal view; F: E8.5 ventral view. G: Transverse section through cardiac region of embryo demonstrated in D. H–L: Transverse sections of embryo visualized in F. Node/notochordal plate (1), visceral yolk sac with vascular plexus and blood islands (2), primitive heart tube covered with amnion (3), primitive heart tube (4), endocardial tissue of the primitive heart tube (5), myocardial tissue forming the outer wall of the primitive heart tube (6), dorsal aorta (7), outflow tract (8), myocardial trabeculation of common ventricular heart chamber (9), bulbus cordis of primitive heart (10), left horn of sinus venosus (11), right horn of sinus venosus (12), vitelline artery (13), hindgut diverticulum (14). EE, extraembryonic.

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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).

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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).

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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).

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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).

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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 in the Node and Notochordal Plate

Besides the prominent expression of Mtus1 in the whole cardiovascular system from E8.0 onwards, the earliest although faint Mtus1 promoter activity was detected in the node (Fig. 1A–C) and the emerging notochordal plate (Figs. 1E–H,J–L, 2B,L) at E7.0–E7.5. We suppose that in the node both isoform 1 and isoform 4 are expressed. In the early stage of E7.5, the only Mtus1 promoter activity was seen in the node (Fig. 1C) and in the RT-PCRs of whole embryo RNA these two isoforms were detectable (Fig. 9). However, we couldn't observe this signal in our Mtus1 in situ hybridization experiments, assuming that the RNA detection level might be too low here.

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 Expression in Lung and Neural Tissues

Starting at E12.5, Mtus1 promoter activity was additionally detected in the homogenous lung parenchyma all around the bronchii (Fig. 5E,G) and neural tissues, like neural-epithelium of the brain (Fig. 5I), plexus choroideus of the fourth ventricle (Fig. 5J), Jacobson's organ and olfactory epithelium (Figs. 5K, 8L), neural tube (Figs. 5L–N, 8K), dorsal root ganglia (Figs. 5L–N, 8J), and cerebral nerves (Fig. 5O). In the lung, isoform 1 and isoform 4 were found, whereas in brain isoform 3 and 4 were detected by RT-PCR.

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).

ATIP in Cancer

Down-regulation or copy number variations in the candidate tumor suppressor gene Mtus1 were described to exert their suppressing function in pancreatic cancer (Seibold et al., 2003), hepatocellular carcinoma (Di Benedetto et al., 2006), prostate cancer (Louis et al., 2010), breast cancer (Frank et al., 2007; Tchatchou and Burwinkel, 2008; Rodrigues-Ferreira et al., 2009), and colorectal cancer (Bacolod and Barany, 2010; Zuern et al., 2010; Melcher et al., 2011). It has been shown that ectopic expression of Mtus1 gene products inhibit the proliferation of eukaryotic cells including tumor cell lines (Seibold et al., 2003; Nouet et al., 2004). This supports the notion that ATIP might be a relevant tumor suppressor in different cancer types and a potential future target of cancer therapies. However, most studies did not distinguish between the different ATIP isoforms. As tumor growth, vascularization, and metastasis exhibit parallels with developmental processes, it will be helpful to further elucidate the functional roles of the different ATIP isoforms during development.

Conclusion

Taken together, Mtus1 is strongly expressed during extra-embryonic and embryonic cardiovascular mouse development. Moreover, Mtus1 expression was found during eye and limb development, and in later stages during lung and neural development. Isoform 3 seems to be restricted to neural tissues whereas during development isoforms 1 and 4 are present in combination in all other investigated tissues. With respect to their co-expression with Agtr2, the appearance of ATIPs suggests AT2-dependent and -independent functions of ATIPs during development. These new findings about the Mtus1 expression and distribution of the 3 different isoforms during mouse development now provide a starting point for future investigations regarding cardiovascular developmental processes and the different functions of ATIP isoforms.

Experimental Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental Procedures
  6. Acknowledgments
  7. References

Mice

Mtus1 reporter mice were generated by a gene trap approach as previously described (Bundschu et al., 2005; Bundschu et al., 2006; Ullrich and Schuh, 2009; Zuern et al., 2012). Briefly, this Mtus1 mouse line harbors a beta-geo fusion construct between exons 9 and 10 of the Mtus1 locus. Therefore, the endogenous Mtus1 promoter activity can be monitored specifically on a single cell level by X-Gal staining. To minimize possible inbred effects, this mouse strain was kept on a mixed 129Ola × C57Bl/6 genetic background. Offspring were genotyped as previously described (Zuern et al., 2012). To gain heterozygote offspring, Mtus1−/− males were mated with wild-type females overnight in a controlled artificial light regime with 12 hr light:12 hr dark and plug-controlled the following morning. Embryos were dissected at stages E7.5–E14.5.

Beta-Galactosidase Embryo Assay

Pregnant females were sacrificed by cervical dislocation and embryos were dissected in ice-cold PBS/0.1% Tween 20 (PBST) at the appropriate stage. Embryos were fixed for 20 min rocking at 4°C in 0.2% glutaraldehyde/2 mM MgCl2/0.02% Igepal CA-630 in PBST. Fixed embryos were washed twice for 10 min in PBST, and stained overnight at 37°C in the dark with X-Gal staining solution (5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-2-D-galactopyranoside [X-Gal] in PBST). After 3 washes with PBST, embryos were post-fixed overnight at 4°C in 4% paraformaldehyde and washed again in PBST. For vibratome sections, embryos were embedded in gelatine mix solution (0.5% gelatine, 27% BSA, and 1.8% saccharose in PBS) at 4°C overnight. Blocks were prepared with gelatine mix solution and 2.5% glutaraldehyde. Sections (50 µm) were cut with a 1500-Vibratome section system and cover-slipped with 75% glycerol.

Whole Mount In Situ Hybridization (WMISH)

Pregnant females were sacrificed at the appropriate stage by cervical dislocation and embryos were dissected in ice-cold PBS/ 0.1% Tween20 (PBST) by removing the extra-embryonic membranes. Embryos were fixed in 4% paraformaldehyde/PBST overnight at 4°C and washed twice for 10 min in PBST. Embryos older than E10.5 were bleached in 6% hydrogen peroxide/PBST for 1 hr and washed twice with PBST for 10 min. Proteinase K treatment was performed with 10 μg/ml proteinase K/PBST for 7–12 min, depending on the embryonic stage. Proteinase K reaction was terminated with 2 mg/ml glycine in PBST for 15 min and embryos were washed twice with PBST. After re-fixation with 0.2% glutaraldehyde/4% PFA/PBST for 1 hr at 4°C, embryos were washed twice with PBST for 10 min. Then, embryos were incubated with pre-hybridization solution (50% formamide, 5× SSC pH 4.5, 1% SDS, 50 μg/ml heparin, 50 μg/ml yeast tRNA) first for 10 min at room temperature and then with pre-warmed pre-hybridization solution for 1 hr at 70°C. Hybridization was performed overnight at 70°C, by adding 5 μl of appropriate Mtus1 or Agtr2 probe, respectively. To generate the templates for the specific RNA probes, PCR reactions with cDNA of E9.5 wt embryos and the following primers were used: For Mtus1: ATIP-forw (T3, 830): 5′-aat taa ccc tca cta aag ggc acc tcc tgt ctg agc ggg-3′; and ATIP-rev (T7, 830): 5′-taa tac gac tca cta tag gca agg gac tcc tgc agc gcg-3′, producing an 830 bp PCR product, which is specifically recognizing all Mtus1 isoforms in the c-terminal part. For Agtr2: AT2R-forw (T3, 860): 5′-aat taa ccc tca cta aag gac cct ccc tct ctg ggc aac c-3′ and AT2R-rev (T7, 860): 5′-taa tac gac tca cta tag gtg tgg gcc tcc aaa cca atg gc-3′, producing an 860-bp PCR product, which specifically recognizes Agtr2. The antisense RNA probes were labeled with digoxigenin UTP, using DIG-RNA labeling mix (Roche, Mannheim, Germany) with T7 RNA polymerase according to the manufacturer's instructions. Prior to use, the RNA probes were purified with Microspin G50 columns (GE Healthcare, Germany).

After hybridization overnight, embryos were washed once with pre-warmed pre-hybridization buffer for 10 min, three times with pre-warmed Solution 1 (50% formamide, 5× SSC, pH 4.5, 1% SDS) for 30 min at 70°C, three times with pre-warmed Solution 2 (50% formamide, 2× SSC, pH 4.5) for 30 min at 65°C, and three times with TBST (140 mM NaCl, 2.7 mM KCl, 25 mM Tris-HCl, pH 7.5, 1% Tween20) for 10 min at RT. Then, embryos were blocked with 10% heat-inactivated sheep serum and 2% blocking reagent (Boehringer, Mannheim, Germany) in TBST for 90 min at RT. Blocking solution was replaced with pre-absorbed Anti-digoxigenin antibody coupled to alkaline phosphatase (AP; 1:8,000, Roche) in TBST/1% sheep serum/2% Boehringer blocking reagent, and incubated overnight at 4°C. The third day, embryos were washed three times for 10 min, five times for 1 hr, and overnight at 4°C with TBST. The next day, embryos were washed three times for 10 min at RT with NTMT (100 mM NaCl, 100 nM Tris-HCl, pH 9.5, 50 mM MgCl2, 0.1% Tween20) containing 2 mM Tetramisole hydrochloride (SIGMA-ALDRICH, Germany). To visualize the probe binding, embryos were incubated with 1 ml NTMT (+ Tetramisole hydrochloride) + 4 μl BCIP (5-bromo-4-chloro-3-indolyl-phosphate; Roche; 50 mg/ml) + 4 μl NBT (4-Nitro blue tetrazolium chloride; Roche; 100 mg/ml), as chromogenic substrate for the anti-DIG-AP antibody. Color development was determined by washing with PBST. Vibratome-sections were performed as described above for the X-Gal stained embryos.

Immunohistochemistry

Mouse embryos were fixed in 4% PFA/PBS for 4 hr at 4°C and overnight in 20% DMSO/80% Methanol at −20°C. Embryos were washed for 30 min at RT in 100 mM NaCl/100 mM Tris/HCl, pH 7.4, and overnight at RT in 15% saccharose/15% gelatin from cold water fish skin. After another equilibration overnight at RT in 15% saccharose/25% gelatine from cold water fish skin, embryos were embedded in 15% saccharose/20% gelatine from cold water fish skin on dry ice at −80°C. Frozen tissue blocks were fixed with TissueTek (Sakura, Staufen, Germany) on section blocks and cryosections (10 μm) were cut with microtome blades (Jung Frigocut 2800N; Leica, Germany). Cryosections were transferred to Superfrost Ultra plus glass slides (Thermo Scientific, Germany) and stored at −80°C. For immunostaining, cryosections were washed for 2 min in acetone to remove the surrounding gelatine embedding medium, washed two times for 10 min in PBST, permeabilized with 0.1% TritionX-100/PBS for 20 min, and blocked with 10% horse serum/PBS for 1 hr to reduce non-specific bindings. Sections were incubated overnight at 4°C with ATIP antibody (1:75; Zuern et al. 2010). The next day, sections were washed three times in PBS/0.1% Tween20, followed by an incubation with secondary antibody, donkey anti-rabbit Cy3 (1:1,200; Dianova, Hamburg, Germany) for 1–2 hr. Stained sections were washed 3 times with PBS/0.1% Tween20 and mounted in IS mounting medium DAPI (Dianova, Hamburg, Germany). Sole secondary antibody staining was performed in parallel as negative control (example shown in Fig. 8O).

Image Acquisition

Digital images of whole mount X-Gal and WMISH embryos were taken with an Olympus (Hamburg, Germany) SZX12 binocular equipped with an Olympus DP50 camera and Studio Lite1.0 software. Digital images of sections were taken with an Olympus BX60 microscope equipped with an Olympus DP72 camera and Analysis Start 5.1 software. Fluorescence images were taken with a Keyence BIOZERO microscope.

RT-PCR

Mouse tissues were quickly prepared on ice and snap frozen in liquid Nitrogen. In cases of small tissue amounts, samples of up to 5 embryos were pooled. Total RNA was extracted with peqGOLD RNAPure™ Kit (Peqlab, Erlangen, Germany). RNA concentrations were estimated photometrically at 260 nm and 200 μg/ml total RNA of each sample was used for cDNA synthesis with SuperScript II™ reverse transcriptase (Invitrogen, Germany). Semi-quantitative RT-PCRs were performed with Phire™ Hot Start II DNA Polymerase (Thermo Scientific). DNA contaminations were excluded by RT- (Gapdh) PCR. Equal cDNA loading amounts were verified by Gapdh expression.

The following specific primer pairs were used:

GAPDH(forw): 5′-ACC ACA GTC CAT GCC ATC AC-3′, GAPDH(rev): 5′-TCC ACC ACC CTG TTG CTG TA-3′; mATIPiso1(forw): 5′-CGG CGG AGC CAT TTG CAC AG-3′, mATIPiso1(rev): 5′-GCT CCG CGA GTC TGG CTT GG-3′; mATIPiso3(forw): 5′-ACT GGC ACT GCT TGT CGT GGG-3′, mATIPiso3(rev): 5′-CAA GTG CGA GTG AGC TCT GGG T-3′; mATIPiso4(forw): 5′-CAC GTC CGC CTA ACC GCC AA-3′, mATIPiso4(rev): 5′-GCT CTC CCC GGA GGC TGA CA-3′. To recognize all 3 murine ATIP isoforms, the following ATIPiso-all primers were used: mATIPiso-all(forw): 5′-TGT CAG CCT CCG GGG AGA GC-3′, mATIPiso-all(rev): 5′-GTG GTC TCA TGG GCG GCG TT-3′. To detect the murine Angiotensin type 2 receptor the following primers were used: mAT2R(forw): 5′-ACC CTC CCT CTC TGG GCA ACC-3′, mAT2R(rev): 5′-TGT GGG CCT CCA AAC CAA TGG C-3′.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results and Discussion
  5. Experimental Procedures
  6. Acknowledgments
  7. References

We thank Baygenomics, San Francisco, CA (http://baygenomics.ucsf.edu), for providing the murine ATIP ES cell line and T. Fischer for providing materials. Margarete von Wrangell scholarship to K.B. K.B. is indebted to the Baden-Württemberg foundation for the financial support of this research project by the Eliteprogramme for Postdocs. K.S. was supported by grants from the German Bundesministerium für Bildung und Forschung (BMBF01 EO1004; Start-Up B.8).

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  4. Results and Discussion
  5. Experimental Procedures
  6. Acknowledgments
  7. References
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