Vascular anatomy of the developing medaka, Oryzias latipes: A complementary fish model for cardiovascular research on vertebrates



The zebrafish has become a very useful vertebrate model for cardiovascular research, but detailed morphogenetic studies have revealed that it differs from mammals in certain aspects of the primary circulatory system, in particular, the early vitelline circulation. We searched for another teleost species that might serve as a complementary model for the formation of these early primary vessels. Here (and online at, we present a detailed characterization of the vascular anatomy of the developing medaka embryo from the stage 24 (1 day 20 hr) through stage 30 (3 days 10 hr). Three-dimensional images using confocal microangiography show that the medaka, Oryzias latipes, follows the common embryonic circulatory pattern consisting of ventral aorta, aortic arches, dorsal aorta, transverse vessels, vitelline capillary plexus, and marginal veins. The medaka, thus, may serve as a valuable model system for genetic analysis of the primary vasculature of vertebrates. Developmental Dynamics 235:734–746, 2006. © 2006 Wiley-Liss, Inc.


Because of the small size and fecundity of adult zebrafish and the external development and optical clarity of its embryo, this species has provided great advantages for experimental, genetic, and morphological analysis of vertebrate vascular development (Weinstein et al.,1995; Zhong et al.,2000; Isogai et al.,2001; Lawson and Weinstein,2002; Torres-Vazquez et al.,2004). These features have made it possible to perform large-scale forward genetic screening to isolate embryonic and early larval lethal mutants specifically showing defects in their circulatory system (Stainier et al.,1996).


AA1 First aortic arch, Mandibular arch AA2 Second aortic arch, Hyoid arch AA3 Third aortic arch, First branchial arch AA4 Fourth aortic arch, Second branchial arch ACeV Anterior (rostral) cerebral vein ACV Anterior (rostral) cardinal vein AMA Anterior (rostral) mesenteric artery AMCtA Anterior (rostral) mesencephalic central artery BA Basilar artery BCA Basal communicating artery CA Caudal artery CaDI Caudal division of the primitive internal carotid artery CCV Common cardinal vein, duct of Cuvier (DC) CrDI Cranial division of the primitive internal carotid artery CV Caudal vein DA Dorsal aorta DAV Dorsal anastomotic vein DCV Dorsal ciliary vein DLAV Dorsal longitudinal anastomotic vessel H Heart HeV Hepatic vein HPVp Primary hepatic portal vein HyA Hyaloid artery HyV Hyaloid vein LDA Lateral dorsal aorta MCeV Middle cerebral vein MtA Metencephalic artery MV Marginal vein, Subintestinal vein NCA Nasal ciliary artery OA Optic artery OV Optic vein PCeV Posterior (caudal) cerebral vein PCS Posterior (caudal) communicating segment PCV Posterior (caudal) cardinal vein PHBC Primordial hindbrain channel PHS Primary head sinus PICA Primitive internal carotid artery PMBC Primordial midbrain channel PMCtA Posterior (caudal) mesencephalic central artery PMsA Primitive mesencephalic artery PS Pancreatic sinusoids Sep Primary intersegmental vessel Ses Secondary intersegmental vessel SIA Supraintestinal artery SIV Subintestinal vein SV Sinus venosus TV Transverse vessel, Vitelline artery UA Umbilical artery UV Umbilical vein VA Ventral aorta

Detailed embryonic studies on Amphioxus (Ura,1949), Lampetra (Yamada,1951), Trygon (Ura,1956), Neoceratodus (Saito,1984), Hynobious (Aoyama,1956), Caretta (Kawanishi,1956), Gallus (Ishida,1956), and Cricetus (Tada,1956) have revealed that the developmental process of the vascular system follows a reproducible stereotypic program in each species. From the comparative anatomical point of view, a common developmental pattern is highly conserved throughout the vertebrates during early development, although some species-specific variations do exist. In every vertebrate species, the primary circulatory system plays an important role in yolk absorption, and blood cells and blood vessels develop in blood islands on the yolk sac. The primary vascular plexus formed from blood islands on the yolk sac connects to the vascular system in the embryonic body via transverse vessels (the so-called vitelline arteries). This connection completes the primary circulation circuit, which consists of ventral aorta, aortic arches, dorsal aorta, transverse vessels, vitelline capillary plexus, and subintestinal vein (marginal vein), all generated by vasculogenesis (see the Discussion section and Fig. 7A,B). The posterior cardinal veins subsequently form along the pronephric ducts as the pronephric portal veins (Takaoka,1956; Inui,1960; Abe,1960; Miura,1960; Meier,1980; Hirakow and Hiruma,1981; Poole and Coffin,1988). Teleosts are an exception among the vertebrates (Swaen and Brachet,1899), because Oellacher's intermediate cell mass, where hematopoietic and vascular endothelial cell lineages appear, is located intraembryonically beneath the notochord within the trunk (Oellacher,1872,1873). This exceptional pattern has been confirmed in the zebrafish (Al-adhami and Kunz,1977; Isogai et al.,2001). Transverse vessels (vitelline arteries), vitelline capillary plexus, and marginal veins on the yolk sac are all absent. The common cardinal veins act as vitelline veins, with the posterior cardinal vein acting as the afferent vein into the common cardinal veins at the earliest stages.

Figure 7.

A–C: Primary vascular system of giant salamander Andrias japonicus (A; Ura,1944), human after Ura (B; unpublished data modified by Isogai), and zebrafish Danio rerio (C; Isogai et al.,2001). D: Medaka Oryzias latipes. This diagram is modified from that of Figure 1C for a better comparison. Arteries and veins, respectively, are colored light gray and black in A, red and blue in B, and black and gray in C and D.

Figure 1.

Circulation in the developing medaka at stage 24 and stage 25. A: Diagram of patent cranial vessels in the stage 24 embryo just before the initiation of circulation. Dorsolateral view. B: Diagram of cranial vessels at stage 25, after the initiation of circulation. Dorsolateral view. C: Diagram of the primary circulatory system at stage 25 (vessels on the right side are not shown). D: A Berlin-blue dye-injected specimen (boxed portion in C). Lateral view. E: Angiogram of the head at stage 25. Dorsal view. F: Diagram of E. Rostral is to the left in each panel. Arteries and veins are colored black and light gray, respectively. A list of the names corresponding to all vessels in this and subsequent figures is provided in the Abbreviations List. All the abbreviations with a prime superscript in this and subsequent figures represent the vessels on the right side. The names of anlages are noted as landmarks.

The zebrafish has proven itself a useful model system for studying human disease, especially for elucidating the molecular basis of congenital diseases. Genetic studies on zebrafish, particularly mutagenesis screenings, can be expected to play a significant role in assigning functions to genes (reviewed in Ward and Lieschke,2002; North and Zon,2003; reviewed in Ackermann and Paw,2003; reviewed in Keating,2004). Many mutants resembling human cardiovascular genetic diseases have been uncovered and characterized (Stainier et al.,1995; Weinstein et al.,1995; reviewed in Dodd et al.,2000; reviewed in Dooley and Zon,2000). However, as mentioned above, the zebrafish never forms transverse vessels, which are essential to form the celiac and superior and inferior mesenteric arteries of humans (Tada,1956; Arey,1966; Romer and Parsons,1977). Furthermore, the posterior cardinal vein, which is also essential to form the inferior vena cava of humans, develops in a different manner in the zebrafish (Isogai et al.,2001). The zebrafish, therefore, might not serve as an adequate genetic model for the analysis of defects in these fundamental vessels. However, Colle-Vandevelde demonstrated in 1963 that, in certain teleosts, blood cells formed not only in the intermediate cell mass but also on the yolk sac. In rainbow trout embryos, for example, vestigial transverse vessels form and the posterior cardinal veins develop as in higher vertebrates (Isogai and Horiguchi,1997). These data suggest that, in some teleosts, the common plan of vertebrate vascular development is conserved.

Like the zebrafish, the medaka, Oryzias latipes, is an excellent model for studies on early development of vertebrates (reviewed in Ishikawa,2000; reviewed in Wittbrodt et al.,2002; reviewed in Furutani-Seiki and Wittbrodt,2004; Shima and Mitani,2004). Recent work on this organism has also made a variety of genomic resources available, including expressed sequence tag (EST) libraries (Katogi et al.,2004; Kimura et al.,2004) and other resources (Sakaizumi et al.,1983; Hyodo-Taguchi and Egami,1985; Kubota et al.,1992; Wada et al.,1995). Notably, mutagenic screening in medaka has revealed unique phenotypes not previously detected in the zebrafish (Loosli et al.,2000,2004; reviewed in Ishikawa,2000; Elmasri et al.,2004; Furutani-Seiki et al.,2004; Iwanami et al.,2004; Tanaka et al.,2004; Watanabe et al.,2004). Some of these mutants, as well as spontaneous mutants, have been successfully cloned (Fukamachi et al.,2001; Kondo et al.,2001; Loosli et al.,2001; Sakamoto et al.,2004). During vascular development, the medaka also uses a pair of common cardinal veins (Ducts of Cuvier) for yolk absorption. However, unlike the zebrafish, the medaka also has an extra vitelline vein, the so-called vitello–caudal vein or median yolk vein (Iwamatsu,1994,2004), which might be homologous to the fundamental route consisting of transverse vessels, vitelline capillaries, and the subintestinal vein of other vertebrate embryos. Our preliminary data have also shown that the posterior cardinal vein of the medaka develops along the pronephric ducts in the same manner as in mammals (unpublished data, Isogai). However, a more complete description of the vascular anatomy of the developing medaka is needed to establish which vessels correspond to the common and essential vessels of all vertebrates.

Here, we present a detailed study on the vascular anatomy of the developing medaka embryo from the onset of the heart beat at stage 24 (1 day 20 hr) through stage 30 (3 days 10 hr) by using confocal microangiography (Weinstein et al.,1995) and dye microinjection (Ura,1944). Our results reveal that the medaka follows the common plan for the formation of the early vessels found in most other vertebrates, including humans, thus establishing it as a useful complementary model system for genetic and experimental analysis of these earliest vessels. In addition, the online version is available on the Medaka Web site (


Stage 24 (44 hr)

The heartbeat starts, but a patent vascular system is not established yet at this stage. Although the confocal microangiography is unavailable, the major embryonic arteries, ventral aorta (VA), mandibular (first aortic) arches (AA1), and lateral dorsal aortae (LDA) are detectable in dye-injected specimens (Fig. 1A). The designated names and abbreviations of all vessels are listed in the abbreviations list. The VA, which is subsequent to the heart, branches to form the right and left AA1s. The right and left LDAs from the AA1s run caudally to merge into a single medial dorsal aorta (DA) beneath the notochord. Rostrally extending primitive internal carotid arteries (PICA) from each AA1 are also visible and begin to divide into the cranial division (CrDI) and caudal division (CaDI). The common cardinal veins (CCV, equal to the duct of Cuvier [DC]), still without lumens, are observable stereomicroscopically on both sides.

Stage 25 (50 hr)

In the medaka, hemangioblasts cohere in the intermediate cell mass (ICM; Oellacher,1872,1873), which was designated inappropriately as the blood island (Iwamatsu,1994,2004), between the 7th and 15th somite levels beneath the notochord, and the embryonic erythrocytes come out from the ICM into circulation at this stage.

With the initiation of the blood flow, blood cells pass through the VA and AA1s and flow caudally to the LDAs and the single DA (Fig. 1B,C). Several transverse vessels (TV; so-called vitelline arteries) from the DA empty, by means of the left surface of the midgut and hindgut boundary, into a robust vessel, the marginal vein (MV), on the yolk sac (Fig. 1C,D). The MV, which lacks any accompanying vascular plexus on the surface of the yolk, drains into the sinus venosus (SV). This primary vascular system for yolk absorption represents the common pattern formed by vasculogenesis in the majority of vertebrates, except most teleosts, including the zebrafish (Ura,1943; Isogai et al.,2001). The zebrafish lacks transverse vessels and the marginal vein totally (see Fig. 7C in the Discussion section) and, therefore, uses a specialized venous route consisting of the caudal and posterior cardinal veins and common cardinal veins to absorb the yolk (Isogai et al.,2001).

In the head, the cranial circulatory route also comes on line at this stage (Fig. 1B,C,E,F). The CrDI extends rostrally along the medial side of the optic capsule and then curves caudally to the primordial midbrain channel (PMBC) and continues to the primordial hindbrain channel (PHBC) located lateral to the hindbrain and medial to the otic capsule. The CaDI goes deep inside the head, looping dorsally and caudally, and drains into the PMBC. At the caudal end of the otic capsule, the PHBC bends ventrolaterally to empty into the anterior cardinal vein (ACV), and then, into the CCV. As in the zebrafish, in the medaka the CCVs also play an important role in yolk absorption before their return to the SV. However, unlike zebrafish but like most other vertebrates, the medaka never forms apparent posterior cardinal veins (PCV) at this developmental stage.

Stage 26 (54 hr)

With the initiation of circulatory flow, confocal microangiography becomes a useful tool to detect the wiring patterns of the embryonic circulation three-dimensionally (see Fig. 1E). The DA extends caudally (the part caudal to the anal pore is designated as the caudal artery [CA]), and the CA comes to end as a blind vessel, where blood cells oscillate back and forth, in the tail consisting of six to seven pairs of somites (Fig. 2). The caudal vein (CV) forms along the ventral side of the tail gut as a return route from the CA. Cranial to the anus, the PCV followed by the CV extends rostrally between the right and left pronephric ducts to join the TV in the trunk and drains into the MV. At this stage, the TVs lose their connection with the DA, and so the yolk absorption is performed only by the venous blood. This venous route represents the typical vitelline circulatory system of teleosts (Ura,1943; Ballard,1964; Balinsky,1976; Portmann,1976).

Figure 2.

Circulation in the developing medaka at stage 26. Dorsolateral view. Rostral is to the left. In this and subsequent figures, the arteries and veins are colored black and light gray, respectively, and dark gray is applied for the arteries of the complicated overlapping vascular region.

In the head, the optic artery (OA) sprouts from the PICA along the optic stalk. The basal communicating artery (BCA) emerges as the connection of the equivalent branches from both CaDIs on the ventral wall of the brain (Fig. 2). The primitive mesencephalic artery (PMsA) comes out dorsally from the CrDI and drains into the PMBC. The nasal ciliary artery (NCA) from the CrDI sprouts dorsolaterally on the frontal surface of the eye capsule. The dorsal ciliary vein (DCV) is recognized as just a dorsolateral sprout from the PMBC. The middle cerebral veins (MCeV) extend dorsally up along the midbrain–hindbrain boundary from the PMBC–PHBC junction, and with the formation of the metencephalic artery (MtA), they will have blood stream by stage 27 (see Fig. 3A,C,D). It must be noted that the PCV begins to sprout caudally from the CCV along the pronephric tubules at this developmental stage (Fig. 2). Unlike most teleosts, the medaka conserves the common developmental manner of PCV formation by vertebrates.

Figure 3.

Circulation in the developing medaka at stage 27. A: Diagram of the embryonic circulatory system. Dorsolateral view. B: Diagram of vessels around the left eye. Ventrolateral view. C: Angiogram of the head. Dorsal view. D: Diagram of C.

Stage 27 (58 hr)

An odd connection appears between the paired LDAs in some embryos (asterisk in Fig. 3A). Primary intersegmental vessels (Sep) begin to sprout dorsally from the DA in the vertical myosepta of the trunk at this stage. As the tail, having 14 pairs of somites, extends, the CA and CV develop caudally, but the caudal tip remains still as a blind undifferentiated vessel. The PCV followed by the CV elongates cranially between the pronephric ducts to join the PCVs from the CCVs. At the first somite level, the anterior mesenteric artery (AMA) is recognized as just a ventral sprout from the DA. The subintestinal vein (SIV) sprouts from the MV and extends cranially beneath the midgut. The MV receives only venous blood from the CV via the PCV and TV.

The optic circulatory route has also been established at this stage; however, the increased number of melanocytes in the eyes makes observation of it difficult. The OA extending laterally along the optic stalk enters the optic fissure as the hyaloid artery (HyA) and forms a caudal hemi-loop in the inner surface of the optic capsule (Fig. 3B). The hyaloid vein (HyV), passing through the optic fissure, finally empties into the PMBC–PHBC junction via the optic vein (OV). The NCA runs dorsolaterally on the outer surface of the optic capsule to link to the DCV. As a consequence, blood from the CrDI flows through the NCA and DCV into the PMBC–PHBC junction.

The CaDIs extend caudally beyond the BCA junction as the posterior communicating segments (PCS) along both ventrolateral sides of the midbrain (Fig. 3C,D). At the midbrain–hindbrain boundary, the MtAs branching dorsally from the PCSs approach one another at the dorsal midline. The MtA connects with the MCeV to drain into the PMBC–PHBC junction. The posterior cerebral vein (PCeV) begins to sprout by this developmental stage (Fig. 3A,C,D). Like that in other vertebrates, the PHBC is the unique venous route in the cranial circulation to drain into the ACV and then into the CCV at this developmental stage. The CCV and cranial PCV remain at the same status as in the previous stage.

Stage 28 (64 hr)

In the trunk, several Seps from the DA extend dorsally up along the lateral wall of the notochord and then along the spinal cord (Fig. 4A). Dorsally beyond the spinal cord, they bifurcate craniocaudally to form a paired dorsal longitudinal anastomotic vessel (DLAVs, see Fig. 6A,E). The CA and CV end as an undifferentiated blind vessel in the tail having 20 pairs of somites. The PCV elongates cranially from the CV along the pronephric ducts to join the equivalent PCV sprouts from the CCVs and will make their connection at the next stage (see Fig. 5A, boxed portion). The PCVs from both CCV and CV form odd paths with the Seps (Fig. 4A: Sep+Ses). They represent the emergence of secondary intersegmental vessels (Ses) from the PCV in the process that forms the intersegmental artery and vein (Isogai et al.,2001,2003). Blood cell flow through these paths is confirmed at this stage.

Figure 4.

Circulation in the developing medaka at stage 28. A: Diagram of embryonic circulatory system. Dorsolateral view. B: Diagram of vessels around the left eye. Ventrolateral view. Rostral is to the left in each panel.

Figure 6.

Circulation in the developing medaka at stage 30. A: Diagram of embryonic circulatory system. Dorsolateral view. B: Angiogram of the head. Dorsal view. C: Diagram of B. D: Angiogram of the trunk. Dorsolateral view. E: Diagram of D. Rostral is to the left in each panel.

Figure 5.

Circulation in the developing medaka at stage 29. A: Diagram of embryonic circulatory system. Dorsolateral view. B: Diagram of vessels around the left eye. Ventrolateral view. C: The dissected digestive anlages of a Berlin-blue dye-injected specimen demonstrating the hepatic portal system at the boundary between fore- and mid-gut. Dorsolateral view. D: Diagram of C. Rostral is to the left in all panels. The names of major anlages are noted in D as landmarks.

With the development of the digestive system, the dorsal pancreatic anlage swells on the right side of the gut at the level of the third somite, while the liver anlage protrudes to the left at the level of the third and fourth somites (see Fig. 5C,D). The AMA takes its course, as the supraintestinal artery (SIA), right-caudally to the opening of the swim bladder on the right dorsal gut wall, and it then drains into the developing reticular sinusoids in the hepatic anlage via the ventral venous route beneath the foregut and midgut boundary (Figs. 4A, 5A,C, asterisk in D). On its way, a sprout branches out from the SIA to the dorsal pancreatic anlage in which the reticular pancreatic sinusoid (PS) develops at later stages (see Fig. 5C,D). The SIV extends cranially along the left ventral wall of the midgut and reaches the developing reticular hepatic sinusoids at this stage (Fig. 4A). Although blood cells seldom flow through this path, this route corresponds to the primary hepatic portal vein (HPVp; Ura,1949; Aoyama,1956). By stage 34, the proximal HPVp regresses, disconnecting the primary hepatic portal route from the reticular hepatic sinusoids. At the dorsal side of the fore- and mid-gut boundary, a dorsal anastomotic vein (DAV), anastomosing the HPVp and the ventral venous route to the hepatic sinusoid, appears (see Fig. 5C,D). The reticular hepatic sinusoids develop, and the hepatic vein (HeV) sprouts to connect to the left CCV at stages 28–30 (see Figs. 5A, 6A). With the formation of the HeV and the reduction in the HPVp after hatching, blood cells take their course through the SIV, the DAV, the ventral venous route beneath the fore- and mid-gut boundary, and then drain into the HeV. Thus, the hepatic portal vein along the bile duct into the reticular hepatic sinusoid is formed (our unpublished results). This change in the hepatic portal system is equivalent to the change that has been shown to happen in other vertebrates and is also the essential process in the formation of the hepatic portal circulation in humans (Ura,1949; Yamada,1951; Aoyama,1956; Ishida,1956; Kawanishi,1956; Tada,1956; Miki,1968; Saito,1984).

In the optic capsule, another hemi-loop appears connecting the HyA and HyV rostrally (Fig. 4B). The right and left PCSs extend caudomedially to merge into the single basilar artery (BA) leaving the arterial circle on the ventral surface of the midbrain. This circle is not homologous to the so-called circle of Willis, because the BCA connecting the PCSs might not correspond to the anterior communicating artery of circle of Willis that connects the anterior cerebral arteries in the human brain. The anterior cerebral vein (ACeV) sprouts from the rostral end of the PMBC into the boundary between the forebrain and the midbrain. The PCeV is still just a sprout. The hindbrain has no arterial supply at this stage, because the BA is just a blind vessel. So, all the blood supplied by the PCSs is directed to the MtAs and drained into the PHBCs by the MCeVs. From the caudomedial edge of the otic capsule, the PHBC goes ventrolaterally to drain into the ACV. The right primary head sinus (PHS) appears along the ventral side of the otic capsule, and the sprouts of the left PHS come on line by stage 29 (see Fig. 5A). The PHS is also called the “lateral head vein” in contrast to the “medial head vein (PHBC)”. The CCVs and MV begin to meander on the yolk surface for the efficient absorption of nutrients before their return to the SV.

Stage 29 (74 hr)

The second aortic arch (hyoid arch; AA2) has not been detected. The third aortic arch (AA3, and the fourth aortic arch, AA4, in few embryos) is formed but does not yet have a sufficient caliber to allow the flow of blood cells at this stage (Fig. 5A). The CA and CV have almost differentiated up to their end in the tail. In the trunk, both PCVs from the CCVs extend more caudally along the pronephric ducts as a pronephric portal vein, and at last, the right cranial PCV makes connection with the PCV from the CV by the end of this stage (Fig. 5A, boxed portion). However, the pronephric glomus will emerge at the later stage, and thus the pronephric portal system is not actually functional yet. Unlike the zebrafish, the medaka follows the developmental process conserved in most vertebrates for PCV formation.

As described in the previous stage, the reticular hepatic sinusoids rapidly develop, and the HeV begins to sprout from the ventral edge of the sinusoid at this stage (Fig. 5A). In dye-injected specimens, the reticular sinusoids are seldom detected because of their narrow calibers at this stage (Fig. 5C,D). Although a small sprout for the pancreatic sinusoids (PS) can be seen on the SIA, the sinusoids develop at later stages (Fig. 5C,D).

Both hemi-loops connecting to the hyaloid vessels extend craniocaudally within the optic capsule (Fig. 5B). The cerebral arterial system remains as that of the previous stage, exclusive of the slight elongation of the BA. The PHSs come on line on both sides and become thicker along the ventral sides of the otic capsules, while the PHBCs become thinner. Thus, most blood from the cranial region takes the PHS route to empty into the ACV on each side. From the dorsal tips of the MCeV, sprouts extend caudally to connect the sprouts that appeared from stage 27 as the PCeVs (Fig. 5A and see Fig. 6A–C). The PHBC, which empties into the ACV, begins to extend more caudally along the ventrolateral wall of the hindbrain. The CCVs and MV meander more intensely on the yolk surface.

Stage 30 (82 hr)

Although the AA1s are still robust, the AA3s are superseding the main aortic (branchial) arches (Fig. 6A,C; in a few specimens, second branchial arches [AA4s] are also detected). In the trunk, the DLAV on each side connects several Seps and Sess longitudinally above the spinal chord (Fig. 6A,D,E). The Sess are formed by the loss of their connection with the DA in odd paths (see Fig. 4A; Sep+Ses). Then the PCV has more Sess, although the left PCV has not yet come on line (boxed portion in Figs. 5A, 6A,D,E). As a consequence, at this stage, the blood stream starts in the initial trunk circulatory unit consisting of DA, intersegmental artery (Sep), DLAV, intersegmental vein (Ses), and PCV (Fig. 6A,D,E). With the full caudal extension of both CA and CV, blood cells in the CA make a U-turn directly into the CV.

The blood flow becomes steady and robust in the digestive vascular system. With the connection of the HeV to the left CCV by the end of this stage, the hepatic portal route, which consists of the SIV, DAV, ventral route beneath the fore- and mid-gut boundary, HPVp, reticular hepatic sinusoids, HeV, and left CCV, has been established (Fig. 6A,D,E). In the SIV, however, blood cells flow caudally to empty into the MV on the yolk sac and then into the SV in these early stages. With the regression of the HPVp at the later stage and that of the MV caused by the complete consumption of yolk by the posthatching stages, blood cells in the SIV are forced to drain into the liver via the hepatic portal route, which consists of the SIV, DAV, ventral route beneath the fore- and mid-gut boundary, and then into the HeV. After the posthatching stage, it takes still more time for the hepatic portal system of the adult fish to begin to function.

Because of the increase in the number of melanocytes in the eye capsule, it becomes very difficult to observe the blood circulation there. The BA extends more caudally and branches out laterally to drain into the PHBCs. The anterior mesencephalic central artery (AMCtA), which penetrates deep into the brain substance, projects rostrally from the BCA, irrigates the frontal region of the midbrain, and then drains into the PMBC (Fig. 6A). In some specimens, the posterior mesencephalic central artery (PMCtA) is detected (Fig. 6B,C). The PCeVs complete their formation, and consequently blood from the MtA begins to have another route into the ACV along the dorsolateral wall of the hindbrain.


Before the onset of blood circulation, the primary vascular system arises via vasculogenesis, which is characterized by in situ differentiation of selected mesodermal cells into endothelial cell precursors, or angioblasts. Classic morphogenetic studies have shown that the pattern of the circulatory system is species-specifically characteristic and very constant in each species (Ura,1943,1949,1956; Aoyama,1956; Ishida,1956; Kawanishi,1956; Tada,1956; Miki,1968; Saito,1984). These studies also revealed that the common vascular pattern, which is composed of ventral aorta, aortic arches, dorsal aorta, transverse vessels (so-called vitelline arteries), vitelline capillary plexus, and primary subintestinal vein (marginal vein), is highly conserved throughout the vertebrates (Fig. 7A). Even mammals, which abandoned yolk storage and developed a circulatory system involving a placenta, hold this common pattern (Fig. 7B). Recent studies have revealed that major embryonic vessels are assembled to generate the “hard-wired” circulatory system prior to the initiation of blood flow, and that genetically programmed extrinsic cues guide this patterning (reviewed in Weinstein,1999). It should be noted that the celiac and the superior and the inferior mesenteric arteries, and the hepatic portal system of humans originate embryologically from the transverse vessels and primary subintestinal veins, respectively (Tada,1956; Arey,1966; Miki,1968; Romer and Parsons,1977). In addition, the posterior cardinal veins, which are essential to form the inferior vena cava (McClure and Butler,1925), develop vasculogenically along the pronephric ducts as the pronephric portal veins at the next developmental step (Meier,1980; Hirakow and Hiruma,1981; Poole and Coffin,1988). Only Teleosts, as seen in the zebrafish, lack the transverse vessels, vitelline capillary plexus, and primary subintestinal vein because of the presence of the intermediate cell mass of Oellacher rather than the blood island. On behalf of those vessels, the zebrafish uses the common cardinal veins for yolk absorption, and forces the posterior cardinal vein to develop and act as the afferent vein into the common cardinal veins at early stages (Fig. 7C; Isogai et al.,2001). Therefore, it had been thought that teleosts that had acquired this peculiar pattern might not be suitable for genetic and experimental studies on primary vascular development. In certain teleosts, however, blood cells are formed not only in the intermediate cell mass but also on the yolk sac (Colle-Vandevelde,1963; Iuchi and Yamamoto,1983; Isogai and Horiguchi,1997). So we searched for another genetically and experimentally accessible species among the teleost fishes that conserves the common developmental pattern of the primary vascular system of vertebrates.

For yolk absorption, the medaka uses the MV like most vertebrates, and in addition, adopts the CCVs similar to the zebrafish (Fig. 7D). Of interest, in the medaka, these vessels do not develop any vascular plexuses on the yolk surface. Instead, the MV has some sprouts by stage 28, and at later stages the MV and CCVs meander to secure greater contact with the yolk. These variations are often detected in each species. These differences might be derived from extension and/or omission of the basic plan to accommodate specialized circulatory requirements. Another reason for these differences might be due to adaptation to rapid development. In the medaka, the heart starts to beat at stage 24 (approximately 44 hr postfertilization; 26°C), the embryo hatches at stage 39 (approximately 9 days postfertilization; 26°C), and the larva can swim and feed immediately. It might be necessary to abbreviate some processes of the common plan to establish the functional circulatory system during such a brief period. The formation process of medaka yolk veins (CCVs and MV, then vitellocaudal vein) before the onset of circulation was described previously (Koh et al.,2004). The expression of embryonic transglutaminase showed a branching pattern around the MV at stage 23, and then its branching expression was assimilated into a large vessel at stage 25. These results support that the route for the yolk absorption of medaka is homologous to that of all vertebrates. Focusing upon the TVs, one or some TVs can act to connect the DA with the MV across the left gut wall (the right gut wall in some exceptional specimens), but by stage 26, they lose their direct connection with the DA; therefore, the MV receives its blood from the CV via the PCV and TV. With this process, in the medaka, the arterial supply to the vitelline vascular system is replaced by a venous one, which might be caused by the unique teleostean arrangements of the yolk and the intermediate cell mass (Ura,1943; Ballard,1964; Balinsky,1976; Portmann,1976).

In the early 20th century, great efforts revealed that the posterior cardinal veins are essential for the formation of the inferior (caudal) vena cava of mammals (McClure and Butler,1925). In the developing embryos of amphibians, reptiles, birds, and mammals, the right and left posterior cardinal veins were demonstrated to sprout from the respective common cardinal veins and to extend caudally along the pronephric ducts as a pronephric portal vein (Takaoka,1956; Abe,1960; Inui,1960; Miura,1960). More recent studies on the chicken have confirmed this process and revealed the posterior cardinal veins to be formed by vasculogenesis (Meier,1980; Hirakow and Hiruma,1981; Poole and Coffin,1988). In teleosts, the forming process of the posterior cardinal veins in the rainbow trout was described in detail and shown to conform to the common pattern (Isogai and Horiguchi,1997). On the contrary, the formation plan for the posterior cardinal veins of the zebrafish is different from that of the vertebrates (Isogai et al.,2001). The posterior cardinal veins are already completed at the onset of circulation, because the zebrafish does not have any transverse vessels and marginal vein, which cause the posterior cardinal veins to be necessary for the drainage from the trunk and the tail circulation. In the medaka, as noted above, the PCVs extend from the CCVs along the pronephric ducts, and in addition, the caudal portion of the vein is seen to be elongated from the origin of the active TV. Thus, the formation process of the PCVs in the medaka as well as in the rainbow trout retains a morphogenetic process similar to that observed in most vertebrates. Therefore, medaka vascular mutants whose phenotypes show various defects in the common and essential vessels of all vertebrate embryos are highly desirable for study.

As is well known to be the case in the human brain, the two internal carotid arteries supply the greater part of the cerebral hemispheres and the eyes, and a considerable part of brain is supplied by the two vertebral arteries. The circulus arteriosus cerebri (circle of Willis) and the basilar artery anastomose these paired arteries: internal carotid arteies and vertebral arteries (Williams et al.,1995). Embryological studies have shown that the PHBC receives venous blood from the eye and forebrain, the middle region of the brain including the cerebellum, and the remainder of the hindbrain, and then drains into the anterior cardinal vein just behind the otic vesicle near the vagus nerve. This venous system is situated within the cranial cavity, the so-called medial head vein. However, the PHBC soon becomes more or less completely replaced by another lateral longitudinal vein, the PHS (so-called lateral head vein), which develops outside the cranial wall and flows into the anterior cardinal vein behind the vagus nerve (Sabin,1917; Padget,1948; Goodrich,1958). A study using microangiography has already confirmed that the cranial vascular formation of the zebrafish holds to this common wiring plan (Isogai et al.,2001). Although we have not studied the full formation process yet, the medaka closely follows these arterial and venous plans from stage 24 through 30.

In vertebrates, the mesenteric arteries and the hepatic venous portal system are derived from the vasculature that originates from the blood islands on the yolk sac. However, the zebrafish totally lacks the transverse vessel (vitelline artery), vitelline capillary plexus, and primary subintestinal vein (marginal vein), because whole hemangioblasts cohere intraembryonically beneath the notochord in the intermediate cell mass of Oellacher. (The SIV of zebrafish, which does not originate from the blood islands, appears at a relatively late stage [2 days post fertilization] and might be generated by angiogenesis [Isogai et al.,2001]. We designate it as the secondary SIV to distinguish it from the primary one.) This situation suggests that the mesenteric arteries and the hepatic venous portal system have different endothelial sources and formation mechanisms in the zebrafish, although they have not been uncovered yet. Even vestigially, the medaka still conserves the transverse vessels, vitelline capillary plexus, and marginal vein. Moreover, it seems to form the hepatic portal system by the same mechanisms as in other vertebrates, which use the vascular sources on the yolk sac. The mesenteric arteries remain in the initial developmental phase in 24–30 stages, and so, detailed analysis of their formation after stage 30 needs to be made.

All of our results suggest that the medaka is useful as a fish model complementary to the zebrafish to study the primary vasculature of the vertebrates as well as to investigate the essential vessels that are extremely fundamental in humans. To fully exploit the advantages in the study of the vascular development using diminutive teleosts, the accurate primary vascular wiring plan must be understood and compared not only among teleosts but also among vertebrates including human beings. This study and further analysis of the vascular anatomy at later stages in the medaka should be helpful for both comparative and genetic vascular studies.


Medaka Methods

Medaka (Oryzias Latipes) embryos were obtained from natural spawning of the Qurt inbred strain (Wada et al.,1998). Fish were raised under artificial reproductive conditions (10 hr dark, 14 hr light; 26–28°C). Embryos were incubated at 28 ± 2°C, which is in general, and staged from morphology as described (Iwamatsu,1994,2004). The developmental times at 28°C are different from those at 26°C, which condition was performed by Iwamatsu (Yamamoto,1975). We indicated the developmental times next to the developmental stage numbers in the results section due to Iwamatsu's description for reference.

Confocal Microangiography

The confocal microangiography method was developed and explained previously (Weinstein et al.,1995; Isogai et al.,2001). A suspension of fluoresceinated carboxylated latex beads (Molecular Probes) mixed with 2% bovine serum albumin (Sigma) was sonicated by a homogenizer (VP-55 ULTRA5 HOMOGENIZER: TAITEC), centrifuged for 1 min in an Eppendorf microcentrifuge. Its supernatant was used as the contrast medium. Glass microneedles were prepared from 1-mm capillaries (GC-1: Narishige) by using a vertical pipette puller (MODEL PC-10: Narishige). Staged embryos were dechorionated by the hatching enzyme and anesthetized with 0.84% tricaine (Sigma) solution. Then, for the injection, the cardiac area of an embryo was held in the “up” position by embedding the embryo in 1% low melting temperature agarose. For embryos, a tip-broken glass microneedle was inserted obliquely into the sinus venosus, and the bead solution was injected. Rapidly after the injection, the embryos were reoriented in 1% low melting temperature agarose, and their vascular wiring images were collected by a confocal microscope (YOKOGAWA CSU10 confocal scanner unit attached to a Zeiss Axioskop) using the argon laser line (IRC-003 PNEUM Omnichrome 10N Laser Power Supply). On average, 40 optical consecutive sections were collected with a spacing distance of 5 μm. The microangiograms shown in all figures were reconstructed at a single angle of 0 degrees by using IPLab3.5.5 and Voxblast2.2 software.

Dye Injection

As a stable blood flow is essential for confocal microangiography, this method is not suitable for examining the wiring patterns of early embryos before and around the initiation of circulation. In addition, the confocal microangiography method has difficulties with imaging deep or overlapping vascular regions. These limitations were compensated by the use of Berlin-blue dye (Wako) -injected embryos (Ura,1944). For dye injection, an embryo was anesthetized, the heart was incised to allow blood drainage, and the yolk sac was torn for ease of manipulation. The 0.75% Berlin-blue solution was injected into the dorsal aorta with a tip-broken glass microneedle, and then the embryo was fixed in 4% paraformaldehyde. The specimens were observed through the stereomicroscope.

Diagrams of Vascular System

Z-series stacks of every region from various angles at each stage were investigated in detail at each optical section level, and vascular wiring patterns were confirmed. Then, local diagrams of vascular system were drawn by tracing the reconstructed images with a 0.2- or 0.3-mm technical pen (Isograph). The diagram of each stage of whole embryos was completed by combining separate reconstructions.

Vessel Nomenclature

The designated names and abbreviations of all vessels named in the diagrams and text of this paper were listed in the Abbreviations List. The vessel nomenclature was basically followed that of the rainbow trout (Isogai and Horiguchi,1997) and the zebrafish (Isogai et al.,2001). All vessels were given names generalized as much as possible for comparison with vessels of any other vertebrates.


We thank Dr. Brant M. Weinstein (NIH) for critical reading of the manuscript.