Workflow diagram.

Embryos were either uninjected or injected with etsrp RNA at the one cell stage, then raised until the late gastrulation stages when the flk1-gfp transgenic reporter is induced ectopically. Equal paired groups of embryos were pooled for mRNA extraction, library construction, and solexa mRNA-sequencing. Samples were pre-validated prior to sequencing by RT-PCR with fli1A, the 32UTR of etsrp, as well as scl, which were all preferentially induced as expected by etsrp overexpression (e) relative to uninjected controls (u).

Verification of RNA-seq data.

Wild type and etsrp injected, etsrp oe, embryos were collected at late stages of gastrulation and processed by WISH. Ectopic induction was detected as random positively labeled cells in embryos injected with etsrp RNA at the one cell stage. Endogenous expression was observed in the presumptive forerunner cells (arrows) of wild-type uninjected embryos in (C) she and (E) spa17. Endogenous expression was also observed in (K) tmem119b (L) myl9 (AD2) sh3bp4 (AN2) hmha1 (AT2) aff3 and (AW2) acsbg2. Ectopic induction is clearly detected in 49 of the 50 genes examined, but not (AT2) aff3, which has a relatively high level of endogenous expression at this stage. Wild type uninjected embryos are positioned on the left column with their etsrp oe counterparts on the right for each gene, anterior is facing left where possible. Scale bar: 250 μm.

Genes with expression in vascular endothelial cells.

The following genes are restricted to the vasculature and are expressed in the cranial vasculature, dorsal aorta, caudal hemtopoietic tail region, posterior cardinal vein, and intersegmental vessels: (E) cd93; (F) gpr183; (S) tmem88a; (AF2) hmha1, while the following are expressed in the same structures but exclude the posterior cardinal vein: (A) lyg2 is also expressed in the posterior yolk extension; (AC2) tagap; (AK2) si:ch211-250g4.3. These genes are expressed in the cranial vasculature, dorsal aorta, caudal hematopoietic tail region, posterior cardinal vein and other tissues as noted: (B) cdc42gap1, is also expressed in the hypochord, hatching gland, and sparsely in spinal cord; (D) klhl4 is also expressed in the heart, intersegmental vessels, and epiphysis; (G) xirp2a is also expressed in the heart, somite boundaries and pronephric duct; (N) rgl2, is also expressed in the intersegmental vessels, forebrain and primitive erythorcytes; (AA2) myof is also expressed in the fin fold, and tailbud; (AM2) arhgap27 is also expressed in the intersegmental vessels, otic vesicles, pronephric duct, and hatching gland. The following genes are expressed in the cranial vasculature, dorsal aorta, caudal hematopoietic tail region, and intersegmental vessels as well as (C) spa17, the olfactory bulb, epiphysis, otoliths, pronephric duct, and caudal notocord; (H) rassf4 was not detected in the intersegmental vessels but is observed in the forebrain, floor plate of neural tube, and spinal cord neurons; (I) tmem119b is also expressed in spinal cord, and tailbud; (AJ2) aff3 is also expressed in the forebrain, spinal cord, and caudal notocord. (AD2) fhl3 is expressed in the cranial vasculature, posterior cardinal vein, caudal vein, fin fold, lateral line primordium, and lightly in caudal somites. (AL2) acsbg2 is expressed in the posterior cardinal vein, caudal vein, spinal cord neurons, optic stalk, forebrain, and midbrain-hindbrain boundary. The following genes have basal levels of ubiquitous expression with prominent labeling of the cranial vasculature, dorsal aorta, caudal hematopoietic tail region and other structures and tissues where indicated: (J) myl9 is also expressed in the floor plate; (K) cntn4, is also expressed in the intersegmental vessels, olfactory bulb, and spinal cord neurons; (L) myo1f is also expressed in primitive myeloid cells; (M) iclp2, also has slight expression in anterior intersegmental vessels; (Q) dgki; (R) ifit5-like; (U) samd10 is also expressed in the caudal somites, and neurons of the anterior spinal cord; (V) cald1 is also expressed in the intersegmental vessels and pronephric duct; (X) zgc:171494 is also expressed in the floor plate, and pronephric duct; (Y) ankdd1a is also expressed in the spinal cord neurons; (Z) sh3bp4 is also expressed in the posterior cardinal vein, and intersegmental vessels; (AB2) grtp1b is also expressed in the posterior cardinal vein; (AE2) irf9; (AG2) ankrd58; (AH2) ccdc135 is also expressed in the posterior cardinal vein, pronephric duct, posterior neural tube, and slightly in the hatching gland; (AI2) tmem151a is also expressed in the posterior cardinal vein, spinal cord neurons and intersegmental vessels (see Figure S1AI2). Basal ubiquitous expression with darker staining of the dorsal aorta and caudal hematopoietic tail region is noted in: (O) capn8 which includes expression in the forebrain, hatching gland, and lateral line primordium; (P) mhc1uea; (T) rasa4 is also expressed in the yolk extension; (W) sept5b is also expressed in the forebrain and somites. Embryos are positioned laterally with the anterior facing left. Abbreviations: cht, caudal hematopoietic tail region; cn, caudal notochord; cs, caudal somites; cv, cranial vasculature; da, dorsal aorta; e, epiphysis; ff, fin fold; fp, floor plate; h, heart; hg, hatching gland; isv, intersegmental vessels; llp, lateral line primordium; mhb, midbrain hindbrain boundary; o, otoliths; ob, olfactory bulb; os, optic stalk; ov, otic vesicle; pd, pronephric duct; pe, primitive erythroid cells; pm, primitive myeloid cells; py, posterior yolk extension; sb, somite boundaries; sc, spinal cord; scn, spinal cord neurons; ss, somites; tb, tailbud; ye, yolk extension. Scale bar: 250 μm.

Analysis of gene expression in etsrp morphants.

Flk1-gfp embryos were injected at the one cell stage with a mixture of translation blocking morpholinos for etsrp and analyzed by WISH at the 24hpf stage. Marked reduction is apparent in all genes examined in the axial vasculature, which includes the dorsal aorta and posterior cardinal vein, and is marked with a downward facing arrow in all images. The primitive myeloid cells stained in (L) myo1f wild-type controls are absent in their etsrp morphant counterparts. The staining in the axial trunk region of rgl2 in etsrp morphants marks primitive erythrocytes that are trapped due to lack of circulation. The expression of non-vascular structures is not affected otherwise in etsrp morphants. Embryos are positioned laterally with the anterior facing left. Abbreviations: cht, caudal hematopoietic tail region; cv, cranial vasculature; da, dorsal aorta. Scale bar: 250 μm.

Higher magnification of Figure 4. The axial trunk vasculature of embryos displayed in figure 4 were imaged at higher magnification to highlight the changes observed. Wild-type embryos are on the left half of each column with their etsrp morphant counterparts on the right for each gene. Embryos were positioned with anterior facing left.