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Developmental Origins of Increased Nuchal Translucency Burger, N.B.
2016
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citation for published version (APA) Burger, N. B. (2016). Developmental Origins of Increased Nuchal Translucency.
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Download date: 10. Oct. 2021 Chapter 4
Increased nuchal translucency origins from abnormal lymphatic development and is independent of the presence of a cardiac defect
N.B. Burger M.N. Bekker E. Kok C.J.M. de Groot J.F. Martin W. Shou P.J. Scambler Y. Lee V.M. Christoffels M.C. Haak
Prenatal Diagnosis 2015 Dec;35(13):1278-86 62 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 63
Introduction Abstract Increased nuchal translucency (NT) is associated with aneuploidy1-3, cardiac defects4, skeletal Objective To assess whether cardiac failure, due to cardiac defects, and abnormal dysplasias, other structural abnormalities and genetic syndromes5. Yet, an increased NT in jugular lymphatic development are involved in nuchal edema (NE) – the morphological euploid fetuses is not pathological per se, as most euploid fetuses with nuchal thickening are equivalent of increased nuchal translucency – in various euploid mutant mouse models. born healthy6. A complete pathophysiological explanation of increased NT in relation to the heterogeneous Methods Mouse embryos with lymphatic abnormalities and NE (Ccbe1-/-), with group of associated anomalies remains to be determined. Cardiac failure7 and abnormal cardiac defects and NE (Fkbp12-/-, Tbx1-/-, Chd7fl/fl;Mesp1Cre, Jarid2-/-NE+) and with cardiac lymphatic development8-11 are the two main theories to explain increased NT. The theory of malformations without NE (Tbx2-/-, Pitx2-/-, Fgf10-/-, Jarid2-/-NE-) were examined. Embryos were cardiac failure has been based on the finding of abnormal blood flow velocity waveforms in the analyzed from embryonic day 11.5 to 15.5. Markers for lymphatic vessels, endothelium, ductus venosus7,12, the strong relationship between cardiac defects and increased NT13-17 and the smooth muscle cells and nerves were used to study the nuchal region. Haematoxylin- fact that the risk of a cardiac defect strongly increases with enlargement in NT15. The established Azophloxine staining was performed to examine cardiac morphology. association between cardiac defects and increased NT, however, does not prove a cause-effect 4 relation. In fact, a causal relation between cardiac failure – due to a cardiac defect – and increased Results Mouse embryos with lymphatic abnormalities and NE (Ccbe1-/-) showed no NT is unproven. Despite the lack of supportive evidence, cardiac failure is frequently referred to formation of the jugular lymphatic sac but normal cardiac morphology. In mouse as explanation for increased NT in fundamental and clinical research. embryos with cardiac defects and NE (Fkbp12-/-, Tbx1-/-, Chd7fl/fl;Mesp1Cre, Jarid2-/-NE+) The theory of abnormal lymphatic development has been based on the observation that enlarged jugular lymphatic sacs or large nuchal cavities within the NE were found. In aneuploid fetuses with increased NT morphologically show nuchal mesenchymal edema4, mouse embryos with a cardiac malformation without NE (Tbx2-/-, Pitx2-/-, Fgf10-/-, Jarid2-/-NE-) accompanied by bilaterally distended jugular lymphatic sacs (JLSs)8;9. The JLSs are the first part normal jugular lymphatic sacs were observed. of the lymphatic system to develop and function as a primitive drainage site9. This implies that disturbed lymphatic development is a likely explanation for increased NT in aneuploid fetuses. Conclusions Nuchal edema consistently coincides with abnormal jugular lymphatic Whether abnormal jugular lymphatic development is also involved in nuchal edema formation development in euploid mouse embryos, independent of cardiac anatomy. Nuchal in euploid fetuses is unknown. edema is unlikely to be caused by temporary cardiac failure solely due to a cardiac defect. We hypothesize that in euploid fetuses, increased NT results from abnormal jugular lymphatic development and is not causally related to cardiac failure induced by cardiac defects. This study aims to investigate the nuchal region, the jugular lymphatic system and cardiac morphology in three different groups of euploid mutant mouse models; mouse embryos with (i) abnormal lymphatic development and nuchal edema, (ii) cardiac defects with nuchal edema and (iii) cardiac defects without nuchal edema. By performing a morphological examination of both jugular lymphatic development and cardiac anatomy in these three different groups, we aim to examine whether cardiac failure due to a cardiac defect induces nuchal edema. Furthermore, we attempt to gain insight into the relationship between increased NT, lymphatic anomalies and cardiac anatomy (see Figure 1).
64 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 65
Figure 1. Relationship between increased NT, cardiac failure due to cardiac defects and abnormal jugular lymphatic region in mouse models with cardiac defects and nuchal edema and littermate wild-type embryos. development We examined (i) Tbx1-/- embryos17, showing abnormal development of the cardiac outflow tract, ventricular septal defects and aortic arch anomalies18, (ii) Jarid2-/-NE+ embryos, displaying non- compaction of the ventricular wall, double outlet right ventricle and ventricular septal defects19, (iii) Fkbp12-/- embryos, showing myocardial non-compaction, ventricular septal defects, hypertrophic trabeculae and a thinner left ventricular wall20 and (iv) Chd7fl/fl;Mesp1Cre embryos21, demonstrating a variety of pharyngeal arch artery defects21 and ventricular septal defects22. In the third group we examined cardiac morphology, jugular lymphatic development and the nuchal region in mouse models with cardiac anomalies without nuchal edema and littermate wild-type embryos. We studied (i) Tbx2-/- embryos23, showing enlarged and dilated ventricles, small endocardial cushions and outflow tract septation defects, such as double outlet right ventricle24, (ii) Fgf10-/- embryos, displaying abnormal direction of the ventricular apex and absent pulmonary arteries and veins25, (iii) Pitx2-/- embryos, showing left-right asymmetry, incomplete 4 closure of the ventral body wall, severe defects in atrioventricular valve and septal formation, disturbed sinuatrial morphogenesis, ventricular-arterial malalignment, hypoplastic right ventricle and arrested rotation of the embryonic heart26-28 and (iv) Jarid2-/-NE- embryos, showing non- compaction of the ventricular wall, double outlet right ventricle and ventricular septal defects19. Methods Table 1. Number of mouse embryos examined per embryonic day
Embryos Mouse embryos Embryonic day Number of mouse embryos Three different groups of mouse embryos were analyzed from embryonic day (E) 11.5 to 15.5 Mouse embryos with a lymphatic defect +/+ to gain insight into the relationship between increased NT, lymphatic anomalies and cardiac Ccbe1 control 15.5 4 Ccbe1-/- 15.5 4 defects. During this period lymphatic development starts and cardiovascular development has Mouse embryos with a cardiac defect with nuchal edema almost completed. These stages also coincide with the presence of nuchal edema and correlate Chd7fl/fl;Mesp1Cre+/+ control 15.5 2 with the timing of the visibility of nuchal translucency in human fetuses. Chd7fl/fl;Mesp1Cre-/- 15.5 2 +/+ Various knockout and one knockdown mouse models were analyzed and compared to wild- Fkbp12 control 11.5-13.5 5 Fkbp12-/- 11.5-13.5 5 type control embryos. In knockout and knockdown embryos a specific gene is completely Jarid2+/+ control 14.0 1 (knockout), or partially (knockdown) deleted, resulting in the induction of a cardiac or lymphatic Jarid2-/- NE+ 14.0-14.5 7 abnormality. Additionally, nuchal edema is present or absent in these embryos. In the human Tbx1+/+ control 14.5 2 -/- clinical situation, increased nuchal translucency is not related to a specific cardiac defect, but is Tbx1 14.5 3 Mouse embryos with a cardiac defect without nuchal edema associated with a spectrum of cardiac anomalies. Therefore, multiple different mutant mouse Fgf10+/+ control 13.5 2 models with lymphatic abnormalities or various cardiac defects with and without the presence Fgf10-/- 13.5 6 of nuchal edema were studied. Jarid2+/+ control 14.5 1 In the first group we analyzed mouse embryos with severe lymphatic developmental defects. Jarid2-/- NE- 14.5 2 Pitx2+/+ control 12.5-14.5 5 Ccbe1-/- embryos display a lack of lymphatic structures and show nuchal edema16. We investigated Pitx2-/- 12.5-14.5 11 -/- the heart, jugular lymphatic development and the nuchal region in Ccbe1 embryos and Tbx2+/+ control 12.5 3 littermate wild-type embryos. Tbx2-/- 12.5 3
In the second group we studied cardiac anatomy, jugular lymphatic development and the nuchal Overview of the number of mouse embryos examined per embryonic day. 66 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 67
A subset of Jarid2-/- embryos showed nuchal edema, whereas other Jarid2-/- embryos Figure 2. Phenotype, immunohistochemical analysis of the JLS and cardiac anatomy in mouse embryos with a lymphatic defect and nuchal edema demonstrated normal nuchal thickness. Jarid2-/- embryos with nuchal edema (Jarid2-/-NE+) and without nuchal edema (Jarid2-/-NE-) were consequently studied in two separate groups according Ccbe1+/+ control Ccbe1 -/- 8 to the presence of nuchal edema . E15.5 E15.5 The number of examined mouse embryos per mutant mouse model was dependent on availability. Because of limited availability we could not examine an equal number of all different mouse models. Guidelines for care and use of mice approved by the Department of Anatomy, Embryology & Physiology, Academic Medical Center, Amsterdam, the Netherlands, were followed. Mice were a b mated overnight and the day of the vaginal plug detection was established as E0.5. Embryos Lyve1 E15.5 Lyve1 E15.5 were isolated and fixed in 4% paraformaldehyde at 4°C overnight. Subsequently, embryos were ME CA dehydrated and the whole embryos were embedded in paraffin. Serial, transverse sections of JLS JV 7µm were made of the nuchal area and the heart. Every 5th section was mounted on a slide and CA JV 4 ventral ventral the slides were dried at 37°C for at least 24 hours. ↕ ↕ Details on histological and immunohistochemical stainings are provided in the Supplementary c dorsal d dorsal Material. Lyve1 E15.5 Lyve1 E15.5 CA JV JLS JV rESuLTS ventral ventral ↕ ↕ dorsal dorsal Normal cardiac morphology in mouse embryos with lymphatic defects and nuchal edema e f HA E15.5 General nuchal morphology HA E15.5 Ccbe1-/- embryos presented with nuchal edema, whereas the wild-type embryos showed normal nuchal thickness at E15.5 (see Figure 2).
ventral ventral Jugular lymphatic system ↕ ↕ g dorsal h dorsal In Ccbe1-/- embryos the JLSs were not observed, as described earlier16 (see Figure 2). In wild-type embryos the JLS were located directly lateral to the jugular vein and carotid artery. The JLS had a -/- normal size of a maximal diameter of approximately 2-3 times the size of the diameter of the jugular Absence of JLS in Ccbe1 embryo with nuchal edema and normal cardiac morphology. Phenotype of Ccbe1+/+ control (a) and Ccbe1-/- (b) embryos at E15.5. Note the nuchal edema in the Ccbe1-/- embryo (see vein9. Lymphatic endothelial cells (LECs) in the JLS stained similarly positive for Lyve1 and Pecam1 arrow). Transverse sections of the neck region in Ccbe1+/+ control embryo (c) and Ccbe1-/- embryo (d) stained for lymphatic marker Lyve1. The JLS is absent in the Ccbe1-/- embryo and was expected at the location of the dotted box. Magnification in the wild-type embryos. SMA-positive cells were not observed in the subendothelial LEC space in of sections of the boxed areas in (e-f) demonstrate the staining results of the lymphatic endothelium of the JLS using all examined embryos. Ncam1 positive nerve fibers were positioned close to the jugular vein and lymphatic marker Lyve1. Transverse sections of the heart in Ccbe1+/+ control embryo (g) and Ccbe1-/- embryo (h) stained with Haematoxylin and Azophloxine reveal normal cardiac anatomy. CA, carotid artery; JLS, jugular lymphatic sac; JV, -/- the area where the JLS was expected in Ccbe1 embryos. In the wild-type embryos Ncam1 positive jugular vein; Lyve1, Lymphatic vessel endothelial receptor 1; Scale bars (c-d) represent 160 μm, scale bars (e-f) represent nerve fibers were located in proximity to the JLS (see Supplementary Figure 1). 40 μm and scale bars (g-h) represent 160 μm.
Cardiac morphology Normal cardiac morphology was observed in all analyzed Ccbe1-/- and wild-type embryos at E15.5 (see Figure 2). Details of the examined mouse embryos are presented in Table 1. 68 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 69 ↕ ↕ ↕ JV dorsal dorsal ventral ventral
Abnormal jugular lymphatic development in mouse embryos with cardiac defects and dorsal ventral E14.5 E14.5 E14.5 E14.5 JV - nuchal edema - / / - - NC JLS
General nuchal morphology (h) embryos. Tbx1 Tbx1 -/- JLS Chd7fl/fl;Mesp1Cre, Fkbp12-/-, Jarid2-/-NE+ and Tbx1-/- embryos presented with nuchal edema at ME control (i), Chd7fl/ control x Lyve1 h p h Lyve1 Lyve1 E12.5-15.5 (see arrows in Figure 3). Nuchal edema was not observed in all wild-type embryos +/+ ↕ ↕ ↕ dorsal ventral dorsal JV
-/- ventral dorsal ventral E14.5 E14.5 E14.5 at E11.5-15.5 (see Figure 3 and Supplementary Figure 2). All Tbx1 embryos and three out of E14.5 seven Jarid2-/-NE+ embryos showed a large cavity in the nuchal mesenchyme in the posterior neck control control JV control (g) and Tbx1 control
region (see Figure 4). These large nuchal cavities extended from posterior to the antero-lateral JLS +/+ Tbx1+/+ Tbx1+/+
part of the neck. Lyve1, Pecam1 and SMA staining were absent in the lining of the nuchal cavities JLS Lyve1 g w o Lyve1 g Lyve1
(see Figure 4 and Supplementary Figure 3). The nuchal region of the other four out of seven (f), Tbx1 ↕ JV ↕ ↕ dorsal dorsal dorsal ventral ventral ventral E14.0 E14.5 E14.0
-/-NE+ fl/fl -/- E14.0 Jarid2 embryos, the Chd7 ;Mesp1Cre embryos and Fkbp12 embryos showed mesenchymal -/-NE+ JV ME NE+ NE+ - - JLS edema (see Figure 3). Wild-type embryos showed no nuchal cavities or mesenchymal edema / / - NC -
(see Figure 3 and Figure 4). JLS 4 Jarid2 Jarid2 ME f Lyve1 n f Lyve1 Lyve1 (p) embryos demonstrate an enlarged JLS in all mutant embryos. an enlarged (p) embryos demonstrate v control (e), Jarid2 (e), control -/- -/- JV ↕ Jugular lymphatic system ↕ ↕ dorsal dorsal dorsal ventral ventral ventral E14.0 E14.0 E14.0 The JLS had a normal size9 in all wild-type embryos at E11.5-15.5. In E11.5 Fkbp12-/- a normal size E14.0 control -/-NE+ control JV
of the JLS was observed. In the three E14.5 Jarid2 embryos presenting with large nuchal Jarid2 (d), JLS -/- fl/fl -/- -/- cavities a normal size of the JLS was found. The JLS in Chd7 ;Mesp1Cre, Fkbp12 , Tbx1 and the JLS Jarid2+/+ -/-NE+ Jarid2+/+ e Lyve1 Lyve1 e control (o) and Tbx1 (o) control Lyve1 m
four Jarid2 embryos without nuchal cavities was approximately five to ten times larger than u
-/- +/+ ↕ ↕ ↕ the JLS in the wild-type embryos at E12.5-15.5 (see Figure 3). In one out of one E13.5 Fkbp12 dorsal dorsal ventral dorsal ME ventral ventral E13.5 E13.5 E13.5 E13.5 JV
-/-NE+ JV - - / embryo and in one out of seven E14.5 Jarid2 embryos the JLS contained red blood cells. This / - - control (c), Fkbp12 (c), control (n), Tbx1 was not observed in the JLS or in the nuchal cavities in the other mutant and wild-type embryos. +/+ JLS -/-NE+ Fkbp12 Fkbp12 The JLSs were positioned lateral to the jugular vein and carotid artery in all examined mutant JLS ME t l Lyve1 d Lyve1 d and wild-type embryos (see Figure 3). The LECs in the JLS showed similar positive staining for Lyve1 JV ↕ ↕ ↕
-/- -/-NE+ dorsal ventral dorsal dorsal ventral ventral E13.5 E13.5 and Jarid2 embryos and wild-type embryos. Slightly positive E13.5
Lyve1 and Pecam1 in Fkbp12 E13.5 -/- fl/fl control
expression of Lyve1 was observed in the JLS in Tbx1 embryos and Chd7 ;Mesp1Cre embryos, control embryos. Lyve1, Lymphatic vessel endothelial hyaluronan receptor 1. Scale bars represents 80 μm. bars represents 1. Scale receptor endothelial hyaluronan vessel Lymphatic embryos. Lyve1, control (m), Jarid2 control -/- -/- JV whereas wild-type embryos showed apparent positive Lyve1 expression in the JLS. SMA-positive JLS JLS Fkbp12+/+
cells were not found in the cell layers surrounding the JLS in all examined mutant and wild-type Fkbp12+/+ s Lyve1 c Lyve1 c k Lyve1 (l), Jarid2 and Tbx1
embryos. Ncam1 positive nerve fibers were closely located to the JLS in all analyzed mutant and -/- ↕ ↕ ↕ ME -/-NE+ dorsal dorsal JV ventral dorsal ventral ventral E15.5 JV E15.5 E15.5 wild-type embryos (see Supplementary Figure 2). Details of the examined mouse embryos are E15.5 JLS enlisted in Table 1. Fkbp12 (a), Chd7fl/fl;Mesp1Cre(b), control +/+ JLS
Cardiac morphology (k), Fkbp12 control Chd7fl/fl;Mesp1Cre Chd7fl/fl;Mesp1Cre Lyve1 j Lyve1 b r b Lyve1
-/-NE+ +/+
Two out of six E14.0-14.5 Jarid2 embryos showed ventricular septal defects. In 1/6 E14.5 ↕ ↕ ↕ dorsal dorsal CA ventral dorsal ventral ventral +/+ +/+ E15.5 E15.5 E15.5 E15.5 JV Jarid2-/-NE+ embryos a transposition of the great arteries and an atrioventricular septal defect CA was observed. In 1/6 E14.5 Jarid2-/-NE+ embryos a transposition of the great arteries together JLS JV control with an atrial and ventricular septal defect was identified. Three out of six E14.0-14.5 Jarid2-/- control
NE+ fl/fl JLS i Chd7fl/fl;Mesp1Cre Chd7fl/fl;Mesp1Cre q Lyve1
embryos showed normal cardiac morphology. The two E15.5 Chd7 ;Mesp1Cre embryos a Lyve1 a Lyve1 fl;Mesp1Cre(j),Fkbp12 Phenotype and immunohistochemical analysis of the JLS in mouse embryos with a cardiac defect and nuchal edema analysis of the JLS in mouse embryos 3. Phenotypewith a cardiac and immunohistochemical Figure defects and nuchal edema. JLS in mouse embryos Enlarged with cardiac Phenotype of Chd7fl/fl;Mesp1Cre defects and nuchal edema in mouse embryos neck region with cardiac 4. Posterior Figure (NC) of nuchal cavities in Jarid2 Presence Magnifications of the boxed areas of sections in (q-x) show the staining results of the lymphatic endothelium of the JLS using lymphatic marker Lyve1. CA, carotid artery;carotid Lyve1. CA, results of the lymphatic endothelium JLS using marker JLS, jugular the staining (q-x) show of sections in areas of the boxed Magnifications 40 μm. represent bars (q-x) 160 μm and scale bars (i-p) represent 1; Scale receptor endothelial hyaluronan vessel Lymphatic Lyve1, jugular vein; lymphatic sac; JV, Note the nuchal edema in mutant embryos (see arrows). Transverse sections of the neck region stained for lymphatic marker Lyve1 in Chd7fl/fl;Mesp1Cre Lyve1 stained for lymphatic marker sections of the neck region Transverse Note the nuchal edema in mutant embryos (see arrows). 70 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 71
+/+ ↕ ↕ JV dorsal
showed an asymmetrical heart, abnormal formation of the apex, ventricular septal defects and ventral dorsal E12.5 ventral E12.5 E12.5 -/-
double inlet left ventricle. All five E11.5-13.5 Fkbp12 embryos showed non-compaction of the - / - (l), Pitx2 JV myocardium, ventricular septal defects, disturbed trabecules of the myocardium and failure of JLS Tbx2 -/NE+- the right ventricle to form. In all three E14.5 Tbx1-/- embryos a persistent truncus arteriosus was JLS h Lyve1 p x observed. In 2/3 E14.5 Tbx1-/- embryos a ventricular septal defect was found (data not shown). Lyve1 ↕ ↕ dorsal ventral dorsal E12.5 ventral E12.5 JV E12.5 Normal jugular lymphatic development in mouse embryos with cardiac defects without control JV control (k), Jarid2 control +/+ JLS the presence of nuchal edema JLS Tbx2+/+
General nuchal morphology of nuchal (h) embryos. Note the absence -/- g Lyve1 o w Lyve1 Nuchal edema was not observed in Fgf10-/-, Jarid2-/-NE-, Pitx2-/- and Tbx2-/- embryos or in wild-type (j), Jarid2 -/- JV ↕ ↕ dorsal ventral dorsal E14.5 E14.5 ventral embryos at E12.5-14.5 (see Figure 5). Nuchal cavities or mesenchymal edema were not observed E14.5 - / in all analyzed mutant and wild-type embryos at E12.5-14.5 (see Figure 5). - JV Pitx2 4 JLS JLS control (i), Fgf10 control Jugular lymphatic system (g) and Tbx2 control +/+ +/+ f Lyve1 n Lyve1 -/- -/-NE- -/- -/- v JV The JLS showed a similar size in Fgf10 , Jarid2 , Pitx2 and Tbx2 embryos compared to the ↕ ↕ dorsal ventral E14.5 dorsal E14.5 ventral E14.5 9 JV
normal size of the JLS in wild-type embryos at E12.5-14.5 (see Figure 5). The JLSs were located (f), Tbx2 JLS -/- lateral to the jugular vein and carotid artery in all investigated embryos. A similar, positive control expression of Lyve1 and Pecam1 was observed in LECs located in the JLS in all mutant and wild- JLS Pitx2+/+ Lyve1 e m type embryos. The subendothelial LEC layer stained negative for SMA in all analyzed mutant u Lyve1 ↕ control (e), Pitx2 (e), control ↕ dorsal and wild-type embryos. Ncam1 positive nerve fibers were observed adjacent to the JLS in all ventral dorsal ventral E14.5 E14.5 E14.5 E14.5 +/+ - JV
examined embryos (see Supplementary Figure 4). Details of the investigated mouse embryos JV NE - JLS / are presented in Table 1. - (d), Pitx2 (d), JLS Jarid2 -/-NE- d t l Lyve1 Cardiac morphology d Lyve1
-/-NE- ↕ ↕ dorsal dorsal ventral ventral JV E14.0 E14.0 One out of two E14.5 Jarid2 embryos showed non-compaction of the ventricular wall and E14.0 -
abnormal growth of the heart. Normal cardiac morphology was found in the other E14.5 Jarid2 JV control
/-NE- JLS embryo. Jarid2 (c), control (p) embryos show a similar size of the JLS in all mutant and wild-type embryos. Magnifications of the boxed areas of sections areas of the boxed of the JLS in all mutant and wild-type (p) embryos a similar size embryos. show Magnifications JLS +/+ -/- -/-
One out of three E12.5 Tbx2 embryos displayed a thin atrial wall, thin endocardial cushions Jarid2+/+ k s c Lyve1 and double outlet right ventricle. Two out of three E12.5 Tbx2-/- embryos showed enlarged Lyve1 JV ↕ ↕ (b), Jarid2 dorsal ventral dorsal ventral E13.5 E13.5 -/- endocardial cushions. E13.5
-/- - / JV -
Four out of six E13.5 Fgf10 embryos displayed an abnormal direction of the ventricular apex JLS
-/- JLS control (o) and Tbx1 (o) control and absent pulmonary arteries, 2/6 E13.5 Fgf10 embryos showed a normal cardiac morphology. Fgf10 +/+ In all eleven E12.5-14.5 Pitx2-/- embryos a defect of the ventral body-wall closure was observed, r j b Lyve1 Lyve1 resulting in externalization of the hearts. All eleven E12.5-14.5 Pitx2-/- embryos showed a (a), Fgf10 control +/+ ↕ ↕ (n), Tbx1 JV dorsal ventral dorsal ventral E13.5 E13.5 -/- disturbed myocardial trabeculation of the ventricles and non-septated atria. In 8/11 E12.5-14.5 E13.5 JV JLS Pitx2-/- embryos ventricular septal defects were detected. In 2/11 E14.5 Pitx2-/- embryos both a control JLS membranous and muscular ventricular septal defect was observed. In 1/11 E12.5 Pitx2-/- embryos JLS Fgf10+/+ i a the ventricular septum could not be examined due to the position of the heart. In all eleven Lyve1 Lyve1 q edema in all mutant embryos. Transverse sections of the neck region stained for lymphatic marker Lyve1 in Fgf10 Lyve1 stained for lymphatic marker sections of the neck region edema in all mutant embryos. Transverse control (m), Pitx2 control in (q-x) show the staining results of the lymphatic endothelium of the JLS using lymphatic marker Lyve1. CA, carotid artery; JLS, jugular lymphatic sac; JV, jugular vein; Lyve1, artery; Lyve1, vein; jugular carotid CA, Lyve1. JV, sac; lymphatic jugular JLS, marker lymphatic using JLS the of endothelium lymphatic the of results staining the show (q-x) in 40 μm. represent bars (q-x) 160 μm and scale bars (i-p) represent 1. Scale receptor endothelial hyaluronan vessel Lymphatic Phenotype and immunohistochemical analysis of the JLS in mouse embryos with a cardiac defect without nuchal edema analysis of the JLS in mouse embryos 5. Phenotypewith a cardiac and immunohistochemical Figure defects without nuchal edema. of JLS in mouse embryosNormal size with cardiac Phenotype of Fgf10 72 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 73
E12.5-14.5 Pitx2-/- embryos the aorta and pulmonary trunk arose from the middle of the ventricles The fourth phenotype reports on embryos with severe nuchal edema, a normal size of the JLS and were in parallel position. The aorta was located to the right of the pulmonary trunk. In 6/11 and extremely enlarged nuchal cavities. In a very small number of embryos this phenotype was E13.5-14.5 Pitx2-/- embryos a persistent truncus arteriosus was observed (data not shown). observed in this study. We cannot fully understand the mechanism of nuchal edema formation The cardiac defects of the mutant mouse models analyzed in this study have been described in in this subgroup. The presence of nuchal cavities is restricted solely to embryos with nuchal previous studies (see Methods section). Specific information regarding the general development edema, such as in human fetuses with Turner syndrome, trisomy 21 or trisomy 189,29,32. Nuchal of the mutant mouse models is presented in Supplementary Table 1. cavities are never observed in embryos with the absence of nuchal edema9;29. Nuchal cavities are most probably formed by an accumulation of fluid in the intercellular spaces of the connective tissue29,33. Accordingly, their presence is dependent on the amount of edema. The larger the Discussion amount of nuchal edema, the more likely that nuchal cavities develop and the larger the nuchal cavities will become. We hypothesize that this phenotype represent a small, heterogeneous Despite the worldwide use of NT measurement as part of prenatal screening for aneuploidy and group of patients with various rare conditions, such as skeletal dysplasias and genetic syndromes5. for detection of fetuses at increased risk for cardiac defects, the pathophysiology of increased A lymphatic or cardiac abnormality does not seem to be involved in the development of nuchal NT is insufficiently understood. For the first time, this study demonstrates that the majority of edema in this subgroup. Other mechanisms, such as an altered composition of the extracellular 4 euploid mouse models with nuchal edema show a coincidental abnormal development of the matrix34, hemodynamic disturbances (Burger et al. unpublished data), altered gene expression35 JLS. The mouse models with normal nuchal thickness demonstrate a normal size of the JLS, and various etiological factors may be involved. Importantly, as we performed a morphological irrespective of cardiac anatomy. examination of the JLS, we cannot draw any conclusions on the function of the lymphatic Based on our findings, we can classify four different phenotypic subgroups in the spectrum structures. Whether appropriate drainage through the JLS is impaired in this subgroup due to of nuchal thickness (see Table 2). The first subgroup describes normal nuchal thickness (i.e. no abnormal function of the JLS, remains unanswered. Future research should examine JLS function nuchal edema), a normal size of the JLS and absence of nuchal cavities. This phenotype is visible in embryos with normal and increased nuchal thickness. in all wild-type embryos and reflects the normal, physiological development of nuchal thickness and the jugular lymphatic system. Furthermore, it stresses the relationship between jugular Table 2. Various phenotypic subgroups in the spectrum of nuchal thickness lymphatic development and nuchal thickness; a normal size of the JLS consistently corresponds Nuchal edema Jugular lymphatic sac Nuchal cavities to the absence of nuchal edema. Phenotype 1; normal fetus absent normal size absent The second phenotype is visible in embryos with nuchal edema and coincidentally enlarged Phenotype 2; trisomy 21, trisomy 18 present increased size mostly absent JLS. The vast majority of the investigated embryos with nuchal edema show dilated JLSs without Phenotype 3; Turner Syndrome severe absent extremely large nuchal cavities. This phenotype is similar to that previously observed in trisomy 16 mouse Phenotype 4; rare genetic syndromes severe normal size extremely large embryos, a mouse model for human trisomy 21, and in trisomy 18 and trisomy 21 human fetuses Overview of the four different phenotypic subgroups in the spectrum of nuchal thickness. with nuchal edema8,11. This subgroup seems the most common phenotype in the spectrum of nuchal edema. The third phenotype involves embryos with severe nuchal edema, absence of JLSs and Cardiac failure is a broadly prevailed explanation for increased NT, however, it is not supported extremely large nuchal cavities. The Ccbe1-/- mouse model shows this phenotype. This entity is by evidence. Intracardiac flow velocities do not differ between fetuses with normal or increased analogue to human fetuses with Turner Syndrome, who show extremely enlarged NT, absence NT, irrespective of cardiac anatomy36,37. Abnormal ductus venosus flow velocity waveforms are of JLSs and enormous nuchal cavities containing a large amount of fluid29. Drainage of fluid also found in fetuses with normal NT, regardless of a cardiac defect38,39. Furthermore, ductal flow from the nuchal region is not possible, because of JLS agenesis. The JLS normally functions as velocity waveforms are not related to a specific type of cardiac defect in fetuses with increased a drainage site9. Subsequently, nuchal cavities develop within the nuchal edema, persist and NT40. If cardiac failure causes increased NT, specific types of cardiac defects that could result become extremely enlarged. On ultrasound examination the massive nuchal edema is referred in hemodynamic compromise – such as tricuspid valve insufficiency or stenosis – should be to as ‘cystic hygroma’30 and is associated with hydrops fetalis and fetal death in more than 95% overrepresented, which is not the case41. This is supported in this study, which uses mouse of cases31. models in which a variety of cardiac defects was found. This is analogue to the fact that any type 74 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 75
of cardiac defect, both mild and severe abnormalities, are associated with increased NT36,40,41. Academic Medical Center, Amsterdam, the Netherlands). We would also like to thank Robert Ventricular septal defects are the most common cardiac defect related to increased NT, yet Kelly for providing Fgf10 mutant model, Stefan Schulte-Merker for providing Ccbe1 mutant it is untenable that a ventricular septal defect causes cardiac failure in fetal life. Notably, the model and Antonio Baldini for providing the Tbx1 mutant model. majority of cardiac malformations do not evidently affect fetal cardiac function41;42. An exception to this might be the Fkbp12-/- embryos, who suffer from dilated cardiomyopathy that resembles morphological features of cardiac failure in humans43. Importantly, enlarged JLS were observed in Fkbp12-/- embryos, which indicates at least a role for abnormal jugular lymphatic development in the formation of nuchal edema in this mouse model. If cardiac failure causes increased NT, then ascites, cardiomegaly, peripheral edema and pericardial or pleural effusions would be expected. Fetuses with increased NT, however, solely show edema in the neck region. Nuchal edema normally resolves after 14 weeks of human gestation and is therefore a temporary phenomenon. Cardiac failure can not explain the regional and transient character of increased NT. A delay or disturbance in lymphatic development can clarify both the local and temporary 4 nature of increased NT44. The theory of cardiac failure as an explanation for increased NT, hence, seems obsolete and further research to elucidate the pathophysiology of increased NT should no longer focus on this theory. A recent review on genetic mechanisms in mouse embryos with nuchal edema identified genes involved in both embryonic lymphatic and cardiac abnormalities45. Given this partially shared genetic background of lymphatic and cardiac defects, the association between nuchal edema and jugular lymphatic abnormalities will consequently result in an increased risk for cardiac defects in fetuses with nuchal edema. A limitation of this study is that we could not relate the cardiac structural defects to dysfunction. We can therefore not provide direct evidence to contradict the theory of cardiac failure in the etiology of increased NT. Another limitation of this study is the limited number of mouse embryos examined, because for many models large numbers of mutant embryos were not available.
Conclusion
For the first time, this study shows that abnormal jugular lymphatic development consistently coincides with nuchal edema in euploid embryos, independent of cardiac anatomy. Whether abnormal jugular lymphatic development is causally related to nuchal edema needs to be addressed in further research. Given the broad spectrum of fetal anomalies and the variable extent of severity, it seems probable that there is not one single cause of increased NT.
Acknowledgements The authors wish to thank Corrie de Gier-de Vries for technical assistance and Kees de Jong for cardiac morphology analysis (both Department of Anatomy, Embryology & Physiology, 76 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 77
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43. Shou W, Aghdasi B, Armstrong DL, Guo Q, Bao S, Supplementary Material Charng MJ, Mathews LM, Schneider MD, Hamilton SL, Matzuk MM. Cardiac defects and altered ryanodine receptor function in mice lacking Histological staining FKBP12. Nature 1998;391:489-492. 44. Bekker MN, Twisk JW, Bartelings MM, Gittenberger- Sections of the embryonic heart were examined using Haematoxylin and Azophloxine staining. de Groot AC, van Vugt JM. Temporal relationship Slides were deparaffinized using a xylene to ethanol series, followed by rinsing in bidest water between increased nuchal translucency and enlarged jugular lymphatic sac. Obstet Gynecol for 5 minutes. The slides were incubated with filtered Mayer’s Haematoxylin for 6 minutes and 2006;108:846-853. rinsed in running tap water for 10 minutes. Slides were washed in bidest water for 2 minutes and 45. Burger NB, Bekker MN, de Groot CJ, Christoffels VM, Haak MC. Why increased nuchal translucency incubated with Azophloxine for 3 minutes. Next, the sections were differentiated in bidest water is associated with congenital heart disease; a for 1 minute, dehydrated to xylene and mounted using Entellan (Merck). systematic review on genetic mechanisms. Prenat Diagn 2015;35:517-28. Immunohistochemistry The nuchal region was examined from below the eye to the clavicle level. We used an antibody for lymphatic endothelium (Lyve1, rabbit polyclonal antibody clone 103-PABi50 (1:500, 4 Reliatech, Wolfenbüttel, Germany), for smooth muscle actin (SMA, mouse monoclonal antibody clone 1A4 (1:4000), Sigma-Aldrich, St Louis, USA), for endothelium (Platelet endothelial cell adhesion molecule-1 (Pecam1); goat polyclonal antibody clone SC-1506 (1:2000); Santa Cruz Biotechnology, Santa Cruz, USA) and for nerves (Ncam1 (Neural Cell Adhesion Molecule 1; rabbit polyclonal antibody clone AB5032 (1:1500), Chemicon, Temecula, USA). The slides were deparaffinated using a xylene to ethanol series. Then the slides were incubated in a solution of
0.3% H2O2 in PBS (phosphate buffered saline: 150 mM NaCl, 10 mM NaPi, pH 7.4)/50% ethanol for 30 minutes to block endogenous peroxidase activity. Subsequently, the slides were rinsed twice in PBS for 5 minutes. In case of Pecam1 the slides were placed in 200 ml 1% Antigen Unmasking solution (Vector Laboratories, Burlingame, USA) in a rack and cooked for 5 min at 1000 Watt in a high pressure cooker. Then the rack was cooled in bidistilled water and once the pressure cooker was depressurized, the rack was placed on ice for ± 20 minutes. Next, the slides were rinsed in PBS for 5 minutes. All slides (staining for Lyve1, SMA, Pecam1 and Ncam1) were blocked in Tris-sodium buffer (TNB; 1M Tris, 1.5M NaCl, adjust to pH 7.4 using HCl with 0.5% blocking reagent) for 30 minutes. Subsequently, all slides were incubated overnight with the specific primary antibody. On the following day, all slides were rinsed three times in TNT (0.1M Tris-HCl (pH 7.5), 0.15M NaCl, 0.05% Tween-20) for 5 minutes. Slides stained for Lyve1, SMA and Ncam1 were incubated with the second antibody (Bright Vision+ HRP anti-mouse or -rabbit depending on the first antibody) for 30 minutes. In case of Pecam1, sections were incubated in biotinylated Donkey-anti-goat IgG (H+L) (Jackson ImmunoResearch Laboratories, #705065147, 1:200) for 30 minutes. The slides were rinsed three times in TNT for 5 minutes, followed by incubation with Streptavidin- horseradish peroxidase (SA-HRP) (Dako, #P0397, 1:100). Then all slides (slides stained for Lyve1, SMA, Pecam1 and Ncam1) were rinsed three times in TNT for 5 minutes followed by ± 5 min incubation with 3-3’diaminobenzidin tetrahydrochloride (DAB; Dako kit) for visualization. The 80 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 81
reaction was stopped in bidistilled water. Finally, all slides were counterstained using Mayer’s- Supplementary Figure 1. Phenotype, immunohistochemical analysis of the JLS and cardiac anatomy in mouse embryos with a lymphatic defect and nuchal edema Hematoxylin for 1 minute, rinsed in running tap water for 10 minutes and dehydrated to xylene. The sections were mounted using Entellan (Merck) and the slides were analyzed by microscopy using Leica DFC 320.
Supplementary Table 1
Mouse model Abnormal development observed in Abnormal development reported in the examined mouse models literature Ccbe1-/- embryos Absence of jugular lymphatic sacs A lack of definitive lymphatic structures Chd7fl/fl;Mesp1Cre No other abnormalities besides cardiac Craniofacial defects, such as cleft palate embryos defects (Sperry et al. 2014) Fkbp12-/- embryos No other abnormalities besides cardiac 9% of Fkbp12-/- embryos show exencephaly defects (Shou et al. 1998, Maruyama et al. 2011, Chen et al. 2013) Jarid2-/- embryos No other abnormalities besides cardiac No other abnormalities besides cardiac 4 defects defects Tbx1-/- embryos Micrognathia and thymus agenesis Micrognathia, cleft palate, thymus and parathyreoid agenesis were reported (Jerome and Papaioannou 2001, Lindsay et al. 2001, Vitelli et al. 2002) Fgf10-/- embryos Skeletal malformations; a disturbed Skeletal malformations; a disturbed outgrowth of the limbs outgrowth of the limbs (Sekine et al. 1999, Marguerie et al. 2006) Pitx2-/- embryos Abnormal left-right asymmetry and ventral Left-right asymmetry, ventral body wall body wall defect defects, abnormal development of the maxillary and mandibular facial prominences and cleft palate, displaced eyes with irregular pupils were reported (Lu et al. 1999, Liu et al. 2001, Liu et al. 2002) Tbx2-/- embryos Duplication of the distal digit IV Duplication of the distal digit IV and eye abnormalities (Harrelson et al. 2004), reduced retinal volume (Behesti et al. 2009)
Phenotype of Ccbe1+/+ control (a) and Ccbe1-/- (b) embryos at E15.5. Note the nuchal edema in the Ccbe1-/- embryo (see arrow). Transverse sections of the neck region in Ccbe1+/+ control embryo (c) and Ccbe1-/- embryo (d) stained for Lyve1. The JLS is absent in the Ccbe1-/- embryo and was expected at the location of the dotted box. Magnification of consecutive sections of the boxed areas in (e-l) demonstrate the staining results of the lymphatic endothelium of the JLS with different antibodies. Transverse sections of the heart in Ccbe1+/+ control embryo (m) and Ccbe1-/- embryo (n) stained with Haematoxylin and Azophloxine reveal normal cardiac anatomy. CA, carotid artery; JLS, jugular lymphatic sac; JV, jugular vein; Lyve1, Lymphatic vessel endothelial receptor 1; Pecam1, Platelet endothelial cell adhesion molecule-1; SMA, Smooth muscle actin; Ncam1, Neural cell adhesion molecule. Scale bars (c-d) represent 80 μm, scale bars (e-l) represent 40 μm and scale bars (m-n) represent 160 μm. 82 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 83
+/+ (h) embryos. -/- control (g) and Tbx1 control +/+ control (i), Chd7fl/ fl;Mesp1Cre(j), Chd7fl/ (i), control Fkbp12 (f), Tbx1 +/+ -/-NE+
4 control (e), Jarid2 (e), control -/- (d), Jarid2 (d), -/- (p) embryos demonstrate an enlarged JLS in all mutant embryos. Magnifications of the JLS in all mutant embryos. Magnifications an enlarged (p) embryos demonstrate -/- control (c), Fkbp12 (c), control +/+ control (o) and Tbx1 (o) control +/+ (n), Tbx1 -/-NE+ control (a), Chd7fl/fl;Mesp1Cre(b), Fkbp12 (a), Chd7fl/fl;Mesp1Cre(b), control +/+ control (m), Jarid2 control -/- (l), Jarid2 -/- boxed areas of consecutive sections in (q-vv) show the staining results of the lymphatic endothelium of the JLS with different antibodies. CA, carotid artery;carotid CA, antibodies. of the lymphatic endothelium JLS with different the staining results show sections in (q-vv) JLS, jugular lymphatic of consecutive areas boxed Neural adhesion molecule-1; endothelial cell SMA, Smooth muscle actin; Ncam1, Platelet 1; Pecam1, receptor endothelial hyaluronan vessel Lymphatic Lyve1, jugular vein; sac; JV, 40 μm. represent bars (q-vv) 80 μm and scale bars (i-p) represent Scale adhesion molecule. cell control (k), Fkbp12 control Phenotype and immunohistochemical analysis of the JLS in mouse embryos with a cardiac defect and nuchal edema analysis of the JLS in mouse embryos Supplementary 2. Phenotypewith a cardiac and immunohistochemical Figure Phenotype of Chd7fl/fl;Mesp1Cre defects and nuchal edema in mouse embryos neck region with cardiac Supplementary 3. Posterior Figure 80 μm. adhesion molecule-1; endothelial cell bars represents SMA, smooth muscle actin. Scale Platelet 1; Pecam1, receptor endothelial hyaluronan vessel Lymphatic Lyve1, Note the nuchal edema in mutant embryos (see arrows). Transverse sections of the neck region stained for Lyve1 in Chd7fl/fl;Mesp1Cre in Lyve1 for stained region neck the of sections Transverse embryosmutant in edema nuchal the Note arrows). (see 84 Chapter 4 Increased nuchal translucency: originating from lymphatic or cardiac defects? 85
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(p) embryos show a similar size of the JLS in all mutant and wild-type embryos. Magnifications of the boxed areas of consecutive sections in sections consecutive of areas boxed the of wild-typeand mutant all in JLS the of embryos. Magnifications embryos (p) size similar a show is required for local cell movement intoatrioventricular -/- cushions. Development 2002;129:5081-91. Behesti H Papaioannou VE, Sowden JC. Loss of Tbx2 delays optic vesicle invagination leading to small optic control (a), Fgf10 control cups. Dev Bio 2009;15;333:360-72. +/+ control (o) and Tbx1 and (o) control +/+ (n), Tbx1 (n), /- Phenotype and immunohistochemical analysis of the JLS in mouse embryos with a cardiac defect without nuchal edema analysis of the JLS in mouse embryos Supplementary 4. Phenotypewith a cardiac and immunohistochemical Figure defects without nuchal edema. of JLS in mouse embryosNormal size with cardiac Phenotype of Fgf10 edema in all mutant embryos. Transverse sections of the neck region stained for Lyve1 in Fgf10 stained for Lyve1 sections of the neck region edema in all mutant embryos. Transverse (q-vv) show the staining results of the lymphatic endothelium of the JLS with different antibodies. CA, carotid artery; JLS, jugular lymphatic sac; JV, jugular vein; Lyve1, Lymphatic Lyve1, artery;carotid vein; jugular CA, antibodies. of the lymphatic endothelium JLS with different the staining results show (q-vv) JLS, jugular lymphatic sac; JV, bars (i-p) Scale adhesion molecule. cell Neural adhesion molecule-1; endothelial cell SMA, smooth muscle actin; Ncam1, Platelet 1; Pecam1, receptor endothelial hyaluronan vessel 40 μm. represent bars (q-vv) 80 μm and scale represent