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1 Supplementary Figure 1. Formation of the AA5x. a, Camera lucida drawing of embryo at 48 hours post fertilization (hpf, modified from Kimmel et al. Dev Dyn : ). b, Confocal microangiogram of wild type embryo at 60 hpf (image modified from Isogai et al, Dev Biol 230: ). a, b, Lateral views, anterior to the left, dorsal is up. Red box denotes region of aortic arches shown in c. c, Schematic drawing showing lateral and dorsal views of aortic arches (AA) 4, 5, and 6 at 46 hpf and 60 hpf. AA5x aortic arch connector vessel, VA ventral aorta. Arrows indicate direction of blood flow. d, Still images from 2-photon time-lapse of a wild type Tg(kdrl:egfp) la116 subjected to microangiography; endothelial cells are green, flow is red; see Supplementary Movie 1. Developmental time point (hours post fertilization; h - hours) is indicated for each frame. Arrows show lateral dorsal aortae (LDA). Arrowheads denote sprouting AA5x. White box in the right top figure indicates region magnified at 53.75h. e, Schematic drawings summarizing results shown in Figure 1 that demonstrate AA5x formation is dependent on both flow and Vegf signaling. Left panel denotes location of laser ablation following microsurgery to block flow from entering aortic arches 5 and 6 (see Fig. 1ac). 1

2 Supplementary Figure 2. Laser-assisted microsurgery to sever aortic arches 5 and 6. a. Right side of 46 hpf Tg(kdrl:egfp) la116 embryo before (top) and after (bottom) high power confocal illumination to ablate the connection between aortic arches (AA) 5 and 6 and the ventral aorta; lateral view, dorsal is up, anterior to the right. Top left, confocal micrograph of right side of embryo before ablation; white box indicates region imaged at right. Top right, magnified image of aortic arches targeted for ablation (indicated by red box). Bottom left, confocal micrograph of right side of embryo immediately after ablation; white box indicates region imaged at right. Bottom right, magnified image of aortic arches targeted for ablation (indicated by red box). Loss of the GFP signal in both AA5 and AA6 ventral connection is indicated by red box. b. Microangiogram of 72 hpf Tg(kdrl:egfp) la116 embryo 24 hours post-ablation. Endothelial cells visualized in green, circulation in red. Confocal image in the control left non-ablated site (left panel, anterior to the left) showed normal perfusion of all the aortic arches (left figure, white box) and overall normal vascular morphology. Right side on which the connection between AA5 and 6 to the ventral aorta was severed, showed absence of flow specifically in AA5 and 6 but normal perfusion of the rest of AAs (right panel, white box; magnified image in white box is shown in Figure 1b, anterior to the right), as well as other cranial vasculature. Lateral views, dorsal is up. The embryo in these images is the same as that shown in Figure 1a-c. 2

3 doi: /nature08889 Supplementary Figure 3. Overall vascular morphology and circulatory function in embryos lacking flow or Vegf signaling. Confocal microangiograms of Tg(kdrl:egfp)la116 embryos at 65 hpf (lateral view, anterior to the left, dorsal is up) showing endothelial cells (green) and circulation of QDots (red). Top, camera lucida drawing of zebrafish embryo at similar developmental stage. Black box denotes region of embryos shown in the head columns, red box indicates images shown in trunk column. Experimental manipulations are indicated on the left of each panel set. Treatment with DMSO ( control ; 0.1%), Tricaine (0.66mg/ml), BDM (15mM) and SU5416 (2.5 µm) was performed between 46 to 65 hours post fertilization. The vegfa MO was injected at 3 ng per embryo. White arrowheads indicate the normal formation and perfusion (when applicable) of the aortic arches 5 and 6 in each experimental condition. Perfusion (red fluorescence) is not apparent in BDM or Tricaine treated embryos due to the complete loss of circulation and inability to perform microangiography on these embryos. 3

4 Supplementary Figure 4. kdrl, vegfa, and klf2a expression in developing aortic arches. All images show embryos at approximately 50 hpf subjected to whole mount in situ hybridization with a digoxigenin-labeled antisense riboprobe against the indicated transcript. Yolk sacs were manually removed for ventral views. a, expression of kdrl in endothelial cells of the aortic arches (aa), AA5x and lateral dorsal aortae (LDA). Lateral and dorsal views are from different embryos. b, Ventral view of vegfa expression in the aortic arches (bracketed) and glomerulus (arrow). c, Higher magnification ventral view of vegfa expression in the glomerulus; position of the first somite is indicated. d, Schematic drawing depicting location of vegfa expression in relation to the AA5x vessel and 1st somite; dorsal view, anterior is up. e, Higher magnification lateral views of vegfa and fli1a expression in aortic arches 5 and 6. fli1a is expressed in both endothelial cells (outlined white arrow) and at lower levels in non-endothelial cells (white arrowhead) in the aortic arches. fli1a does not appear to be expressed in columnar-shaped cells within the arches (indicated by red arrowhead), while vegfa expression is apparent in these cells. f, Ventral view of klf2a expression in a developing AA5x sprout (black arrow). Brackets marked with asterisks denote lumens of the lateral dorsal aortae; a blood cell within the lumen one of the branches is apparent (white arrowhead). The bracket labeled with DA indicates the lumen of the dorsal aorta near the branchpoint to the lateral aortae. All images, except for b were captured using differential interference contrast (DIC) microscopy. 4

5 doi: /nature08889 Supplementary Figure 5. Notch signaling is not required for AA5x development. a, Ventral views of embryos subjected to whole mount in situ hybridization using an antisense riboprobe against cdh5 or dll4, anterior is up. Expression of cdh5, but not dll4, is clearly evident in the aortic arch blood vessels (bracket) at 60 hpf. b, Confocal micrographs of Tg(fli1ep:dsredex)um13;(tp1bglob:egfp)um14 double transgenic embryos at 50 hpf. Green fluorescence is indicative of Notch activation, while red fluorescence marks all endothelial cells. Left panel, GFP is robustly expressed in the eye and neuronal cell types within the brain, while no expression is seen within the aortic arch blood vessels (aa5, aa6). Lateral view, anterior is to the right, dorsal is up. Right panel, GFP expression was not apparent in the lateral dorsal aortae (LDA) or developing AA5x (right panel, white arrowheads). Dorsal view, anterior is up. c, Confocal microangiogram of a Tg(tp1bglob:egfp)um14 embryo subjected to angiography with QDots (pseudocolored in blue in the right panel) at 48 hpf showing robust GFP expression, indicative of Notch activation, within the endothelial cells lining the dorsal aorta (DA) and segmental arteries (indicated by white arrows), but not in the posterior cardinal vein (PCV) or segmental veins. d, Confocal micrographs of Tg(fli1a:negfp)y7 embryos in which endothelial cell nuclei are labeled with EGFP; embryos are at 60 hpf. Embryos were injected with 10 ng of scrambled or dll4 MO. Left panels, ventral view, image of lateral dorsal aortae in region of the connection to AA5x. Numbers indicate cell nuclei in the connected AA5x. We did not observe a difference in endothelial cell number between control and dll4deficient embryos. Right panels, lateral view, images show endothelial nuclei in segmental arteries at 60 hpf. As we have previously reported, we observed increased numbers of cells within the dorsal longitudinal anastomotic vessel (arrowheads) in dll4-deficient embryos. 5

6 Supplementary Figure 6. Expression analyses in the absence of flow or klf2a. a, Results of quantitative RT- PCR showing expression of previously identified flow- or klf2-responsive genes, as well as mir-126 and vegfa at 65 hpf. Embryos were injected with tnnt2 MO (2 ng) or splice-blocking klf2a MO (2.5 ng). Relative expression is comparison of beta-actin normalized expression in tnnt2- or klf2a-deficient embryos compared to sibling control embryos injected with scrambled MO at the same stage of development. Errors bar indicate standard deviation (n = 3 separate clutches of pooled embryos). PCR primers used for qpcr can be found in Methods. Only mir-126 expression was consistently reduced in the absence of both flow and klf2a. b, Whole mount in situ hybridization of zebrafish embryos fixed at 65hpf following 12 hours of treatment with Tricaine (0.66 mg/ml). Lateral views of head region (anterior to the left, dorsal is up) showing expression of klf2a, mir- 126, or cdh5; black boxes indicate location of aortic arch blood vessels. Loss of flow causes reduction in klf2a and mir-126 expression specifically in the aortic arches, while cdh5 expression is unchanged. Compare to control embryos in Fig. 2a and 3a. c, Whole mount in situ hybridization of zebrafish embryos fixed at 65 hpf following injection with control MO (11ng, left column), 12h Tricaine treatment (middle column), or injection with ATG klf2a MO (11 ng, right column). Top two rows show ventral views (anterior to the top) of embryos hybridized with vegfa or cdh5 riboprobes, respectively. Brackets indicate position of the aortic arches and arrows denote location of the glomerulus. Bottom row shows kdrl expression in the head region (lateral view, anterior to the left, dorsal is up); boxed regions denotes aortic arches. Together, the experiments in panels b and c failed to reveal any major changes in the expression of vegfa, kdrl, or cdh5 following loss of flow or klf2a. 6

7 Supplementary Figure 7. Description and characterization of a splice-blocking klf2a Morpholino. a, Zebrafish klf2a intron-exon structure. Location of Morpholino at the exon 3 splice acceptor site is indicated (spl MO), as is location of forward (F) and reverse (R) primers used for RT-PCR and quantitative RT-PCR (qpcr) in b. b, RT-PCR analysis using primers to amplify a fragment spanning the exon 2-exon 3 boundary in klf2a coding sequence using RNA isolated from embryos at 48 hpf. The expected fragment size for a properly spliced klf2a transcript is 293 bp, while inclusion of the second intron would yield a 399 bp fragment. An unspliced fragment is observed with increasing amounts of spl MO from 0.5 ng to 2.5 ng per embryo. qpcr analysis was performed using similarly positioned primers and demonstrated reduced levels of klf2a transcript with increasing MO doses. c, Embryos injected with scrambled or klf2a spl MO at 60 hpf showing normal overall morphology; lateral views, anterior to the left. d, Confocal micrangiograms of embryos injected with 2.5 ng klf2a splice Morpholino (left panels; 72 hpf) or 11 ng klf2a ATG Morpholino (right panels; 65 hpf). Top panels show grossly normal patterning of head vascular and perfusion of aortic arches 5 and 6 (arrowheads), bottom panels show normal patterning of the trunk vascular system. For comparison, see Supplementary Figure 3. e, Confocal microangiograms of Tg(kdrl:egfp) la116 at 72 hpf following injection with 2.5 ng scrambled or klf2a spl MO; dorsal view, anterior is up; arrows indicate normal position of the AA5x. f, Graph depicting penetrance of AA5x defects in klf2a MO-injected embryos. Error bars are standard deviation (n = 3 clutches of embryos). See Supplemental Table 3 for raw data. 7

8 Supplementary Figure 8. Effects of mir-126 deficiency on zebrafish development. a, Whole mount in situ hybridization at indicated time points using a digoxigenin-labeled locked nucleic acid probe against mir-126. Expression is apparent in the dorsal aorta (arrow) at 18 somite stage (ss) and 24 hpf. By 65 hpf mir-126 expression is at higher levels in the aortic arch blood vessels compared to other anatomical locations. Positions of aortic arches 4-6 are indicated at 65 hpf. Lateral views, anterior to the left, dorsal is up. b, Northern analysis showing mature mir-126 and let-7a expression in 65 hpf embryos injected with 11 ng control, 2 ng tnnt2, or 11 ng klf2a MOs. mir-126 expression is reduced in embryos lacking flow or klf2a, while let-7 is not affected. c, Northern analysis from embryos injected with 20 ng of control or mir-126 Morpholino. Probed with LNA against mir-126 (left) or let-7a (right). d, Transmitted light images, lateral views, anterior to the left, dorsal is up. Left panels, embryos at 26 hpf. Right panels, embryos at 60 hpf. Bottom right panel shows focal hemorrhage near posterior aortic arches (arrow) in embryo deficient for mir-126. e, Confocal microangiograms of Tg(kdrl:egfp) la116 embryos at 60 hpf subjected to angiography with rhodamine-conjugated dextran; lateral views, dorsal is up, anterior to the left. Top panels, embryos injected with control Morpholino. Bottom panels, embryos injected with mir-126 Morpholino, white arrows denote ectopic branches that emanate from segmental arteries. These are not apparent in control Morpholino injected embryos. 8

9 Supplementary Figure 9. Genetic interaction between klf2a and mir-126. a, Light and fluorescent images from embryos injected with 400 pg mrna encoding a monomeric red fluorescent protein (mcherry), or mrna encoding Mcherry and Klf2a separated by an in-frame viral 2A peptide sequence (mcherry-2a-klf2a). Expression of Mcherry (indicated by red fluorescence) from both mrnas is apparent at 48 hpf. b, qrt-pcr of klf2a expression using RNA isolated from injected embryos at 48 hpf. These results demonstrate that injected mrna persists until 48 hpf. c, Whole mount in situ hybridization using a DIG-labeled LNA probe against mir- 126 on embryos injected with mcherry or mcherry-2a-klf2a mrna at 48 hpf. Ventral views, anterior is up. Expression is apparent in aortic arch blood vessels of control embryos injected with mrna encoding Mcherry (left panel), but is reduced in tnnt2-deficient embryos that lack flow (middle panel). Injection of mrna encoding Mcherry-2A-Klf2a can rescue expression of mir-126 in tnnt2-deficient embryos (right panel); the morphology of aortic arch vessels in this case is abnormal due to extensive pericardial edema in the absence of flow. d, Northern analysis of mir-126 and let-7 in embryos injected with increasing amounts of mir-126 Morpholino to demonstrate extent of partial knockdown used in low-dose Morpholino injections in Fig. 3i, j. e, Penetrance of defects in AA5x formation in MO-injected embryos at 65 hpf; Error bars denote standard deviation, n = 3 clutches for high dose injections, n = 4 for low-dose injections; see also Supplementary Table 3. 9

10 Supplementary Figure 10. Validation of a transgenic mirna expression construct in zebrafish embryos. a, Diagram of the ptol-fli1ep:mir-126/mcherry construct (see Supplementary Methods). This cassette consists of the non-coding first exon of zebrafish ef1a, as well as the accompanying splice donor (SD) and splice acceptor (SA) sites and the first intron. Within the intron, we placed the pri-mir-126 sequence. This construct would be expected to generate a pre-mrna containing the pri-mir-126 sequence and mcherry coding sequence. Subsequent splicing and processing should yield separate mature mir-126 and mcherry mrnas. b, To validate the mir-126/mcherry cassette, we generated a bactin2:mir-126/mcherry construct to drive high level ubiquitous expression in the zerbafish embryo. Left, image shows a zebrafish embryo at 24 hpf following co-injection of ptol-bactin2:mir-126/mcherry and mrna encoding Tol2 transposase. Lateral view, anterior to the left, dorsal is up. Red fluorescence was detected throughout the embryo indicating mcherry expression. Right, northern blot analysis of similarly injected embryos probed with DIG-labeled mir-126 LNA showed increased expression of mature mir-126 mature sequence compared to control embryos injected with bactin2:mcherry. c, whole mount in situ hybridization of zebrafish embryos at 65 hpf with LNA mir-126 probe; brackets indicate aortic arches. Left, expression of mir-126 in aortic arches of control embryos (bracket; 11ng control MO, left figure). Middle, reduced mir-126 expression in aortic arches in embryos injected with 11 ng klf2a MO. Right, mosaic mir-126 expression in embryos co-injected with the klf2a MO, ptol2-fliepmir126/mcherry and mrna encoding Tol2 transposase (arrowheads). 10

11 Supplementary Figure 11. mirna sensors in zebrafish embryos. a, Left, constructs used for whole embryo mirna sensor assays. In this case, the 3 UTR of interest was cloned downstream of the enhanced green fluorescent protein (egfp) coding sequence. A control construct was generated with the SV40 polya signal downstream of the monomeric red fluroscent protein, mcherry. Both plasmids were used to synthesize mrna, which was subequently co-injected along with mir-126 duplex into 1-cell zebrafish embryos. Right, injected embryos observed under epifluorescent illumination at 24 hours post fertilization. In each case, mcherry expression is evident. In the presence of mir-126, the spred1 3 UTR leads to repression of Egfp expression, while the pik3r2 does not. b, An endothelial cell autonomous mirna sensor construct. The plasmid, flanked by Tol2 direct repeat elements, was injected into 1-cell zebrafish embryos with mrna encoding the Tol2 transposase. The control egfp-sv40-polya cassette is driven by expression of an enhancer element in the fli1ep cassette and the basal E1a promoter. Endothelial expression of the mcherry-spred1 3 UTR is driven by the same enhancer element in conjunction with a fli1a promoter element in the fli1ep fragment; the basal E1a promoter and fli1ep promoter elements are in opposite orientations. c, Quantification of fluorescence in endothelial cell autonomous sensor assay. Red/green ratio indicates comparison of relative voxel intensities derived from 3-dimensional reconstructions of confocal micrographs of embryos co-injected with the endothelial sensor construct, Tol2 transposase mrna, and the indicated Morpholino (see Methods). Red refers to red fluorescence from the mcherry-3 spred1 UTR, while green is fluorescence from control egfp expression. An increase in Red/green ratio is indicative of increased expression from the mcherry- 3 spred1 UTR cassette. Error bars represent standard deviation; for control and klf2a, n = 6; for tnnt2 MO, n = 5, for mir-126 MO, n = 4. 11

12 doi: /nature08889 Supplementary Figure 12. Flow, klf2a, and mir-126 are required for xenograft angiogenesis in zebrafish. a, Confocal microangiogram of blood vessels surrounding a Ras-transformed NIH3T3 xenograft in the yolk sac of Tg(kdrl:egfp)la116 zebrafish embryos at 72 hpf. Endothelial cells in green, blood flow in red. White arrow denotes position of common cardinal vein, arrowheads denote tip cells sprouting around the xenograft and H labels the heart. b, mir-126 expression as assayed by whole mount in situ hybridization using a DIG-labeled LNA probe. Same xenograft and labeling as that shown in a. c, klf2a expression in endothelial cells (black arrowheads) within a xenograft (denoted by asterisk); this embryo is not the same as that shown in a. d-h, Confocal microangiograms of xenografts in the yolk sacs of Tg(kdrl:egfp)la116 zebrafish embryos at 72 hpf; each treatment is indicated with the respective panel. Xenografted cells are red; blood flow is pseudocolored blue, endothelial cells are green. White arrow denotes common cardinal vein in all panels. i, Quantification of ratio of vessel volume to tumor volume in embryos subjected to indicated treatment; error bars are standard deviation, n=3 xenograft-bearing embryos for each indicated manipulation. 12

13 Supplementary Table 1. Effects of flow and Vegf signaling of AA5x formation. Failure to connect AA5/6 to LDA total embryos DMSO treatment exp exp Unilateral microsurgery exp exp BDM treatment exp exp Tricaine treatment exp exp vegfa MO 3 ng exp exp exp SU5416 treatment exp exp kdrl y17 mutant exp exp gata1 MO exp Proportion with defect AA5x formation assayed at 65 hours post fertilization. 1. In cases where embryos displayed normal AAx formation, they had reformed the connection between aortic arches 5 and 6 to the ventral aorta. 2. All observed embryos failed to exhibit red blood cell formation. 13

14 Supplementary Table 2. Quantification of in situ hybridization scoring at 65 hpf. mir-126 LNA probe total loss of embryos expression Proportion with defect klf2a riboprobe total embryos loss of expression control exp exp exp tnnt2 MO 1 exp exp exp Tricaine-treated 1 exp exp exp BDM-treated 1 exp exp exp klf2a MO exp exp Proportion with defect 1. All embryos injected with tnnt2 MO, treated with Tricaine, or treated with BDM did not exhibit a heartbeat. 14

15 Supplementary Table 3. AAx phenotypes in Morpholino injected embryos. total embryos Failure to connect AA5/6 to LDA mir-126 MO 20 ng exp exp exp exp klf2a MO 11 ng exp exp exp klf2a spl MO 2.5 ng exp exp exp klf2a spl MO 1.5 ng exp exp exp klf2a spl MO 0.5 ng exp exp exp ctrl MO 2.5 ng exp exp exp ctrl MO 20 ng exp exp exp mir-126 MO 7 ng exp exp exp exp klf2a MO 2 ng exp exp exp exp Proportion with defect 15

16 mir-126 MO 7 ng + klf2a MO 2 ng exp exp exp exp fliep:mir126/mcherry + klf2a MO 11ng exp exp exp exp fliep:mcherry + klf2a MO 11ng exp spred1 mrna exp exp exp spredmo+mir126mo exp exp

17 Supplementary Movie Legends. Below are brief descriptions for Supplementary Movies. Please note that while the sequence of events that we observe is consistent with Anderson et al. Dev Biol 318, (2008), our developmental staging is somewhat later than previously reported. This is likely due to some delay in handling and mounting embryos prior to time lapse, or slight differences in incubation temperature (Beth Roman, personal communication). For time-lapse movies, number (n) indicates number of total movies in each case. Movies 1, 2, 5-8, and 11 were obtained by 2- photon microscopy; images captured every 15 minutes and presented at 30 frames per second (fps). Movies 3, 4, 9, and 10 were captured by video using light microscopy. Supplementary Movie 1. Wild type Tg(kdrl:egfp) la116 embryo injected with QDots. Dorsal view of lateral dorsal aortae (LDA) and formation of connecting aortic arch vessel. Anterior is up. Movie spans from 46 hpf to 60 hpf at 15 minute intervals. Circle and square denote growing left and right AAx, respectively. Green is endothelial cells, red fluorescence is Qdots in circulation. n = 3 Supplementary Movie 2. Wild type Tg(kdrl:egfp) la116 embryo injected with QDots. Dorsal view of right lateral dorsal aortae (LDA) and formation of connecting aortic arch vessel; the tip of the fluid-filled sprouting right AAx LDA is denoted by an asterisk. Anterior is up. Movie spans from 50 hpf to 57 hpf, just prior to fusion of the AAx with the LDA. Green is endothelial cells, red fluorescence is Qdots in circulation. Supplementary Movie 3. Video of aortic arch circulation in a wild type embryo at 57 hpf. Lateral view, anterior is to the left, dorsal is up. Circulation of red blood cells is apparent in aortic arch 4 (aa4), while blood cells appear to be caught in aortic arches 5 and 6 (aa5, aa6). Supplementary Movie 4. Video of aortic arch circulation in a wild type embryo at 65 hpf. Lateral view, anterior is to the left, dorsal is up. Circulation of red blood cells is apparent in aortic arches (aa) 4, 5, and 6. Supplementary Movie 5. Wild type Tg(kdrl:egfp) la116 embryo treated with BDM beginning at 46 hpf. Dorsal view of lateral dorsal aortae (LDA). Anterior is up. Movie spans from 46 hpf to 60 hpf. Asterisks denote region of LDA that normally connects to the AAx. n = 3 Supplementary Movie 6. Wild type Tg(kdrl:egfp) la116 embryo treated with Tricaine beginning at 46 hpf. Dorsal view of lateral dorsal aortae (LDA). Anterior is up. Movie spans from 46 hpf to 60 hpf. Asterisks denote region of LDA that normally connects to the AAx. n = 1 Supplementary Movie 7. Wild type Tg(fli1a:negfp) y7 embryo. Lateral view of left aortic arches and initial sprouting of AA5x (growing tip denoted by red line). Primordial head sinus (PHS) is indicated by a blue line. Movies spans from 42 hpf to 50 hpf. n = 3. Supplementary Movie 8. Wild type Tg(fli1a:negfp) y7 embryo treated with Tricaine. Lateral view of left aortic arches and initial sprouting of AA5x (tip denoted by red line). Movies spans from 42 hpf to 50 hpf. n = 2. 17

18 Supplementary Movie 9. Video of aortic arch circulation in an embryo injected with 11 ng klf2a Morpholino at 65 hpf. Lateral view, anterior is to the left, dorsal is up. Circulation appears normal through aortic arch 4 (aa4) but is blcoked in aortic arches (aa) 4 and 5. n =2. Supplementary Movie 10. Video of aortic arch circulation at 60 hpf in an embryo injected with 20 ng of mir-126 Morpholino hpf. Lateral view, anterior is to the left, dorsal is up. Similar to wild type embryos before full lumenization of the AAx, circulation of red blood cells is apparent in aortic arch 4 (aa4), while circulation in aortic arches 5 and 6 (aa5, aa6) is blocked, leading to pulsatile flow. Supplementary Movie 11. Tg(kdrl:egfp) la116 embryo injected with 20 ng of mir-126 Morpholino hpf and imaged by 2-photon microscopy. Dorsal view of lateral dorsal aortae (LDA). Anterior is up. Movie spans from 46 hpf to 57 hpf. Asterisks denote region of LDA that normally connects to the AAx. n = 3 18