Development of the Murine Pulmonary Veinand Its Relationship to the Embryonic Venous Sinus

SANDRA WEBB,1* NIGEL A. BROWN,1 ANDY WESSELS,2 AND ROBERT H. ANDERSON3

1Department of Anatomy and Developmental Biology,St. George’s Hospital Medical School, London, UK

2Department of Cell Biology and Anatomy, Medical Universityof South Carolina, Charleston, South Carolina

3Section of Paediatrics, Imperial College School of Medicine,National Heart & Lung Institute, Imperial College School of Medicine, London, UK

ABSTRACT Background: Arguments concerning the development ofthe pulmonary vein, and its relationship to the embryonic venous sinus(sinus venosus) have continued for well over a century. Recently, attentionhas again been focused on the origin of the pulmonary vein. It has beensuggested that, whereas the pulmonary vein originates from the leftatrium in humans, in all other vertebrates it originates from the venoussinus, with subsequent transfer to the left atrium. The nature of thistransfer has not, however, been elucidated, although there is speculationthat the pulmonary vein is ‘‘pinched off’’ from the left side of the embry-onic venous sinus.

Methods: We studied closely staged hearts of normal mouse embryosfrom a C57BL/6 x CBA cross days 10 and 11 of gestation (plug day 5 day 1).Two series of embryos were collected and fixed in 2% glutaraldehyde, 1%formaldehyde, buffered with 0.05 M sodium cacodylate pH 7.4 (adjusted to330 mOsm with NaCl). One series was wax embedded, serially sectioned,and stained with Masson’s trichrome. The second series was subject tomicrodissection and scanning electron microscopy.

Results: The atrial component of the heart tube is attached to the body ofthe embryo by reflections of the atrial myocardial wall. The attachmentcan be considered, from the outset, as the heart stalk, with the myocardial-mesodermal connections forming a horseshoe of tissue that projectsventrally into the lumen of the atrium, surrounding a single evaginationin the midline of the embryo. This heart stalk is cranial to the connectionsof the tributaries of the embryonic venous sinus and ventral to theforegut. When traced through its developmental stages, the evagination inthe centre of the stalk, which we describe as the pulmonary pit, is seen tobecome the portal of entry for the developing pulmonary vein.

Conclusions: The heart stalk, representing the area used by the pulmo-nary vein to gain access to the heart, and analogous to the dorsalmesocardium, is, from the outset, discrete from the area occupied by theorifices of the horns of the embryonic venous sinus. The pulmonary veindoes not, in the mouse, develop from the tissues that form the walls of thetributaries of the systemic venous sinus. Comparisons with other studiessuggest that early events in the development of the pulmonary vein arelikely to be the same in all mammals, including humans. Anat. Rec.250:325-334, 1998. r 1998 Wiley-Liss, Inc.

Key words: pulmonary vein; mouse; sinus venosus; cardiac development

Contract grant sponsor: British Heart Foundation; Contract grantnumber PG/96114; Contract grant sponsor: NIH; Contract grantnumber: HL52813; Contract grant sponsor: the Joseph Levy Founda-tion; Contract grant sponsor: U.K. Medical Research Council.

*Correspondence to: S. Webb, Department of Anatomy and Develop-mental Biology, St. George’s Hospital Medical School, Cranmer Ter-race, London, SW17 0RE, UK.

Received 23 June 1997; Accepted 14 November 1997

THE ANATOMICAL RECORD 250:325–334 (1998)

r 1998 WILEY-LISS, INC.

Arguments concerning the development of the pulmo-nary vein, and its relationship to the embryonic venoussinus (sinus venosus), have continued for well over acentury. In 1880, His suggested that the pulmonaryvein joined the heart through that part of the atrialcomponent of the primary tube that was anchored tothe body wall. At the same time, he described anotherstructure involved in atrial development, which hecalled the spina vestibuli (spine of the vestibule),arguing that development of the spine walled thegrowing pulmonary vein into the definitive left atrium.Subsequent to this work, it became generally acceptedthat the pulmonary vein, in humans, did indeed origi-nate within the atrial segment of the heart, rather thangrowing from the embryonic venous sinus. Over thesame period, nonetheless, it had been argued that, atleast in mammals apart from humans, the pulmonaryvein was derived from the venous sinus (Goette, 1875;Boas, 1880, 1883; Hochstetter, 1903, 1908).

Auer (1941, 1948) subsequently expressed doubt thata reasoned argument could be made for the veingrowing from the embryonic venous sinus. He backedup his contentions with reconstructions of serial sec-tions of the developing atrial septum, these supportingthe view of His (1880) that the developing vein used thecontiguities of the myocardium of the heart tube withthe mesenchyme of the body wall as the portal of entryto the eventual left atrium. These myocardial-mesoder-mal connections surrounded the area, which for pur-poses of this discussion we call the heart stalk. Others,including Auer (1941, 1948), have described this area asthe dorsal mesocardium.

Recently the debate has surfaced again, with newsuggestions that the pulmonary vein is derived fromthe venous sinus rather than the atrial component ofthe embryonic heart (DeRuiter et al., 1995; Tasaka etal., 1996). How can this be? First, interpretation ofserial sections of the developing heart is far from easy,even when backed up with reconstructions, so differ-ences in interpretation are not unexpected. Second,without significant prior knowledge, it is difficult toappreciate the features demonstrated in the reconstruc-tions made by Auer (1941). Third, and perhaps mostimportantly, there are no morphological landmarksduring early development to delineate the boundariesof embryonic venous sinus from the atrial component ofthe primary heart tube. Because of this, many of theapparent disagreements relate to differences in nomen-clature rather than conflicts in observations. We havenow performed studies using both serial sections anddissections of embryos viewed by scanning electronmicroscopy. Our findings attest to the accuracy ofAuer’s (1941) reconstructions and support strongly hisinterpretations and those of His (1880), along with themore recent account of Bliss and Hutchins (1995). Wepresent here evidence showing that, from the outset,the stalk of the heart tube is connected to the body wallaround the area that will, in time, become the pulmo-nary venous portal. At the same time, our studies offerfurther information concerning the nature of the tis-sues that, eventually, wall the pulmonary vein into theleft atrium. They also show that many of the explana-tions put forward to explain the incorporation of theembryonic systemic venous sinus into the definitiveright atrium are unnecessarily complicated.

MATERIALS AND METHODS

The hearts examined for this study were from normalmouse embryos derived using a C57BL/6xCBA cross.The animals were handled in accordance with theGuide for the Care and Use of Laboratory Animals(NIH publication 85-23, revised 1985). Mice were matedovernight, the presence of a copulation plug the nextmorning being taken as evidence of successful mating.This day was then designated as day 1 of gestation.Pregnant dams were sacrificed by cervical dislocationon days 10 and 11 of gestation, the conceptuses ex-planted, and the extra-embryonic membranes removed.

All embryos were examined under a stereomicro-scope for gross morphology. The number of pairs ofsomites were counted for the purposes of staging.Embryos were then immediately fixed by immersion ina mixture of 2% glutaraldehyde and 1% formaldehyde,buffered with 0.05 M sodium cacodylate pH 7.4 (ad-justed to 330 mOsm with NaCl) for 2 hours at roomtemperature.

Scanning Electron Microscopy

Microdissection was done by hand under a stereomi-croscope using iridectomy scissors. After fixation for 2hours, embryos were dissected to give various views ofthe atrial region and the sinus horns and their connec-tion to the body in the region of the venous sinus.Dissection was followed by overnight immersion in thesame fixative at room temperature. All samples werepostfixed in 1% OsO4, dehydrated through a gradedalcohol series, critical point dried (using liquid CO2 asthe transition fluid), mounted on stubs, then goldsputter coated. Samples were viewed on a Zeiss SM940scanning electron microscope at an accelerating voltageof 25KV.

Serial Sections

Hearts for sectioning were fixed overnight at roomtemperature by immersion in 2% glutaraldehyde and1% formaldehyde, buffered with 0.05 M sodium cacodyl-ate pH 7.4 (adjusted to 330 mOsm with NaCl). Afterfixation, embryos were rinsed, embedded in 2% agarose(to facilitate orientation), dehydrated, embedded inparaffin wax, and serially sectioned at a nominal thick-ness of 5 µm. Sections were dewaxed, rehydrated,stained with Masson’s trichrome stain, dehydrated,and coverslipped using DPX mounting medium. Micro-graphs of the sections were taken using a Zeiss D-7082transmitted-light stereo photomicroscope.

RESULTS

At the earliest stages examined (embryos between19-22 somites), the heart was unseptated and tubular,with the atrioventricular canal positioned to the left ofthe midline The primary dorsal mesocardium, whichinitially joined the myocardial heart tube to the body ofthe embryo over most of its length, had broken down,leaving the greater part of the myocardial tube as afree-standing structure within the pericardial cavity. Incontrast, at the midline of the atrial component, thewalls of the heart remained in continuity with themesoderm ventral to the foregut. This connection,which we call the heart stalk, also has been describedas the dorsal mesocardium. When viewed from outside

326 S. WEBB ET AL.

the heart, the cranial and lateral margins of the heartstalk are surrounded by coelomic space. Viewed fromthe luminal aspect, the heart stalk was visible as ahorseshoe-shape elevation (Figs. 1A, 2, 3A) that sur-rounded a depression in the dorsal surface of theprimary atrial component of the tube (Figs. 1A whitearrow, 1B black arrow, 2). We call this depression the

pulmonary pit. More caudally, the limbs of the horse-shoe were confluent with the area where the cavities ofthe right and left horns of the embryonic venous sinusopened into the lumen of the primary atrial component,the venous orifices being lateral and inferior to thepulmonary pit. The walls of the heart tube in this areawere directly continuous with the walls of the sinus

Fig. 1. A. Scanning electron micrograph of a 24 somite embyro,showing the connection of the cardiac atrial segment to the body viathe heart stalk. The pulmonary pit is indicated by the white arrow.B-D. Histological sections from a 22 somite embryo, cut in a cranial-caudal direction to complement the scanning electron micrograph. Bdemonstrates the pulmonary pit (solid black arrow), flanked on eitherside by the reflected atrial myocardium which forms the right and left

pulmonary ridges. The walls of the heart are directly continuous withthe somatic mesoderm to either side of the pit. When traced dorsally,the area merges with the horns of the venous sinus, which extenddirectly into the somatic mesoderm of the mediastium. C and D showthe entry of the horns of the venous sinus to be caudal to the area of thepulmonary pit. The bifurcation between the horns forms the sinusseptum (open arrow). Scale bars 5 50 µm.

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horns, which were embedded within the somatic mesen-chyme. This arrangement was markedly different fromthat of the sleeve of myocardium surrounding thecranial and lateral margins of the pit itself (Fig. 1:compare B with D).

Apart from these differences, it was not possible atthis stage to identify, from inside the heart, boundariesbetween the systemic venous orifices and the area

surrounding the pulmonary pit (Fig. 3A,B). It could beseen, nonetheless, that the tissue between the mouthsof the right and left horns (the so-called sinus septum)was simply the bifurcation of the two venous channels(open arrow Fig. 1D). This structure separating thehorns was, at this stage, a midline structure, relative tothe body of the embryo. The mouths of the tributaries ofthe embryonic venous sinus were themselves bilater-ally symmetrical, being positioned caudally in theprimary atrium relative to the myocardial elevations ofthe heart stalk that surround and enclose the prospec-tive pulmonary portal. The pit itself was an ovaldepression floored by the somatic mesoderm of the bodywall. The slopes of the pit were prominent laterally,forming two ridges (the pulmonary ridges) that at thisstage were also symmetrical about the midline (Figs.1A,B,2,3A). The two ridges were continuous cranially.The ‘‘horseshoe’’ of tissue thus formed enclosed the pitand protruded into the cavity of the heart (Figs. 2, 3A).The pit was closely associated dorsally with the tracheo-bronchial groove of the oesophagus, although the lungshad not budded from the groove at this stage (Fig. 1).

The bifurcation of the sinus horns was still in linewith the long axis of the pit in a 27 somite embryo (solidarrow Fig. 4A). By 28 somites, changes were to be foundin the location and dimensions of the sinus horns. Themouth of the left sinus horn was by now smaller thanthat of the right horn (Fig. 4B). The righthand pulmo-nary ridge was also continuous, caudally, with thebifurcation of the sinus horns within the venous sinus.The primary component of the atrial septum (theseptum primum) was now evident, developing as abroad flange running in a cranio-caudal direction,immediately cranial to the pulmonary ridges (Fig. 4C).Its position relative to the horseshoe produced an in-verted Y-shape structure in the dorsal atrial wall, withthe pulmonary ridges forming the bifurcated arms ofthe Y and the stem of the Y being the primary atrialseptum. Histologic sections showed that from the time

Fig. 2. Scanning electron micrograph of a 24 somite embryo,transected (lower half removed), and with the ventral surface of theatrium removed to show the pulmonary ridges either side of thepulmonary pit in the back of the common atrium. Scale bar 5 50 µm.

Fig. 3. This series of embryos was dissected to show the changesinvolving the pulmonary pit. The dissection of a 26 somite embryo (A)is made by removing the heart tube at the level of the atrioventricularcanal. The slit–like pit is seen in the atrial floor (arrow) flanked by theright and left pulmonary ridges (RPR, LPR; ep - epicardial organ). Bythe stage of 29 somites (B), the pulmonary ridges are asymmetric. By32 somites (C), the venous valves (VV) have become evident, nowdistinguishing the venous sinus from the primary atrial component.The pulmonary pit (arrowed) now opens to the left side of thedeveloping atrium. The left sinus horn is now identifiable as the leftsuperior caval vein (LCV), with its walls forming a discrete venouschannel, which is separate from the atrioventricular junction. By 34somites (D), the venous valves are prominent structures, with theright valve (RVV) much longer than the left (LVV). The right end of thevalves forms the septum spurium (SS). The left end is continuous withthe primary septum (SP) and the right pulmonary ridge. Note themarked endocardial elevation at the junction of these structures—thespina vestibuli (asterisk). By the stage of 35 somites (E), this endocar-dial mound on the right pulmonary ridge has become particularlyprominent at the union of the left venous valve with the primaryseptum (PAS). Note that the pulmonary pit, flanked by the leftpulmonary ridge (LPR), now points to the left side of the developingatrium. Note also the location of the left superior caval vein (LCV). F.In this 38 somite embryo, the venous valves and the primary atrialseptum are well seen. The pulmonary pit, flanked by the left pulmo-nary ridge, is opening directly above the cut edge of the inferiorendocardial cushion. Note again how the left caval vein (LCV)encircles the atrioventricular junction, with its walls discrete andseparate from those of the heart. Scale bar 5 50 µm.

328 S. WEBB ET AL.

Fig. 3.

of its initial appearance, the muscular septum wascapped by an aggregation of mesenchymal tissue compa-rable in appearance to the atrioventricular endocardialcushions.

By 29 somites, a slight asymmetry was seen betweenthe pulmonary ridges, with the righthand ridge beingmore prominent than the left (Fig. 3B). The left sinushorn, identifiable as the left superior caval vein by thestage of 32 somites, extended as a free-standing venouschannel running beneath the primary atrium andencircled the heart stalk (Fig. 3C). As the left hornelongated, its mouth continued to be reduced in sizewhen compared with the orifice of the right horn, andthe tissue separating the horns at the site of theirbifurcation became more pronounced. By this stage,anatomic boundaries had begun to form between the

mouths of the sinus horns and the remainder of theatrial component—the so-called venous valves. Thesehad become increasingly prominent, demarcating thevenous sinus within the developing right atrium. By 34somites, the right and left valves had come togethercranially to form the septum spurium (Fig. 3D). Theright pulmonary ridge was now particularly pro-nounced, projecting forward as a pronounced spurtoward the opening of the atrioventricular canal. Thisright ridge appears to correlate with the structureidentified by His (1880) as the spina vestibuli (asteriskFig. 3D,E). The pulmonary pit now faced leftward. At 35somites, the two pulmonary ridges were even moreasymmetric, with the prominent right pulmonary ridgecontinuous with the left venous valve, which was muchshorter than its right counterpart (Fig. 3E).

Fig. 4. A. A normal 27 somite embryo dissected to show therelationship of the mouths of the sinus horns to the pulmonary pit,which is flanked by the pulmonary ridges (RPR, LPR). The sinusseptum (solid black arrow) is the bifurcation of the left and right sinushorns. B. This view of a 28 somite embryo shows the diminution in sizeof the left horn (LSH) as compared to the right (RSH). The sinusseptum (black arrow), at this stage, is in line with the pulmonary pit(solid white arrow) and continuous with the right pulmonary ridge.

C. The atrial component of a 28 somite embryo, viewed from the left,showing the primary atrial septum (PAS) growing from the atrial roofas the stem of an inverted Y. Note also the remnants of the atrioven-tricular endocardial cushions (SC, IC). The solid white arrow showsthe pulmonary pit. D. The atrial component of a 38 somite embryo,viewed from the right, showing the primary atria septum growingtoward the AV junction. Note that the venous valve (VV) is an infoldingof the atrial wall. Scale bars 5 50 µm.

330 S. WEBB ET AL.

By the stage of 38 somites, the primary atrial septumcould be seen growing down toward the orifice of theatrioventricular canal. The right pulmonary ridge ap-peared less prominent, but was continuous with the leftvenous valve and the caudal aspect of the primaryatrial septum (Fig. 3F). The right venous valve was nowcontinuous with the inferior endocardial cushion. Thevenous sinus itself had completed its rotation to theright, so that the right superior and inferior caval veins,together with the left caval vein (which persists in themouse) opened within the confines of the venous valves.The primary atrial septum was seen growing towardthe atrioventricular canal, with its cranial margin incontinuity with the superior atrioventricular endocar-dial cushion (Fig. 4D).

A sagittal section of an embryo at this same stageshowed that the right pulmonary ridge, forming theright margin of putative pulmonary vein, was inter-posed between the leftward margin of the inferiorendocardial cushion and the base of the primary atrialseptum. Serial histolological sections taken in a frontalplane showed that the spina vestibuli consisted of adense whorl of cells that could be traced back throughthe heart stalk into the substance of the somaticmesoderm (Fig. 5A). The structure of this whorl wasmarkedly different from the mesenchymal cap on themuscular primary atrial septum. During the growththrough these stages, the luminised vasculature of thedeveloping pulmonary vein became visible, being posi-tioned consistently within the heart stalk to the left ofthe spina vestibuli. By the stage of 40 somites, the heartstalk had become the portal of entry of the developingpulmonary vein to the atrial cavity. The vein itself nowextended dorsally into the mesoderm of the body wall(the mediastinum) and bifurcated toward the develop-ing lung buds (Figs. 5B,6). The mesenchyme that

covered the right pulmonary ridge within the heart hadbegun to fuse with the inferior endocardial cushion at43 somites, thus confining the entry of the pulmonaryvein to the definitive left atrium (Fig. 5C).

DISCUSSION

To comprehend the formation of the pulmonary vein,it is essential to understand the nature of the tissues inwhich the vessel must develop, namely, the mesodermbetween the inflow portion of the heart tube and thelung buds that evaginate from the foregut into somaticmesoderm. When the heart tube first forms (but beforeit loops), it is attached throughout its length to themesoderm of the body wall. This anchorage is theprimary dorsal mesocardium (Chang, 1931), whichsuspends the tube within the developing pericardialcavity. It has been described in humans as ‘‘a pairedlamella of loose structure in which can be seen inclu-sions and cysts’’ (Davis, 1927). Once the primary dorsalmesocardium has broken down, the heart tube is free-standing within the pericardial cavity over most of itslength. The atrial component of the heart, however,continues to be attached to the body of the embryo andremains so throughout development. This persistingatrial connection has also been termed the ‘‘dorsalmesocardium,’’ which we believe misleading because itis of a different structure and has an entirely differentfate, compared to the initial, more transient attach-ment of the heart tube. The atrium is connected byreflections of the myocardium that abut the somaticmesoderm dorsal to the heart. This connection forms adistinct pedicle of tissue, which we call the heart stalk(Fig.6). It is ventral to the foregut and the area in whichthe lung buds will develop, and we show here that itencloses the putative pulmonary portal, as also de-scribed by Bliss and Hutchins (1995) in the human.

Fig. 5. A. A 38 somite embryo sectioned in a frontal plane. Thissection reveals the dense tissue of the right pulmonary ridge (RPR).The pulmonary pit is marked by a solid arrow. B. A 40 somite embryosectioned in a transverse plane, showing the luminized pulmonaryvein (PV) passing back into the somatic mesoderm and bifurcating asit approaches the lung buds (LB). The right pulmonary ridge abuts the

inferior endocardial cushion (IC). C. A transverse section through a 43somite embryo just caudal to the opening of the pulmonary vein,showing the mesenchymal cap covering the right pulmonary ridgefusing with the inferior endocardial cushion. Other abbreviations:LCV - left caval vein; LPR - left pulmonary ridge; PAS - primary atrialseptum; SC - superior endocardial cushion. Scale bars 5 200 µm.

331DEVELOPMENT OF MURINE PULMONARY VEIN

To clarify disagreements concerning the pulmonaryvein, it is important to distinguish the features of theattachment of the atrium to the somatic mesoderm inthe area of the heart stalk from the features of thejunctions of the systemic venous tributaries to theheart. When viewed from within the atrial cavity, themyocardial components of the heart stalk form a ‘‘horse-shoe’’ of tissue that surrounds the pulmonary pit. Theatrial folds to the left and right of the pit form markedlyraised ridges, which were named the pulmonary ridgesby Auer (1941, 1948). When viewed from outside the

heart, the margins of the heart stalk are surrounded bycoelomic space. In contrast, the venous horns aresimply extensions of the lumen of the heart tuberunning into the somatic mesoderm. The bifurcation ofthe horns forms the sinus septum, which can be likenedto the crotch in a pair of trousers.

At early stages, therefore, there are no clear ana-tomic boundaries that demarcate the venous sinus fromthe mouths of the venous horns, nor any surfacedistinction of the lumen between the venous horns andthe remainder of the primary atrial component of the

Fig. 6. A. Midline sagittal sectionthrough a 45 somite embryo toshow the connection of the atrialsegment of the heart to the body ofthe embryo via the heart stalk.The boxed area has been enlarged(B) and demonstrates the full ex-tent of this connection (betweenarrows), with the pulmonary veinextending back toward the develop-ing lung buds. Other abbreviations:LCV - left caval vein; PAS - pri-mary atrial septum; SC - superiorendocardial cushion; IC - inferiorendocardial cushion; OP - ostiumprimum; OS - ostium secundum,T - trachea; PV - pulmonary vein.Scale bars 5 200 µm.

332 S. WEBB ET AL.

heart tube. Nevertheless, the area of direct atrialattachment surrounding the pulmonary pit (the heartstalk) is always cranial to the extensions of the horns ofthe embryonic venous sinus into the body wall (thelatter becoming the future mediastium). In addition tobeing cranial to the venous sinus, the pulmonary pit isventral to the foregut (the area in which the lung budsand their associated vasculature develop). As we havealready indicated, it eventually becomes the portal ofentry of the pulmonary vein. The vein itself lumenizeswithin the somatic mesenchyme concomitant with theformation of the lung buds (Rammos et al., 1990;DeRuiter et al., 1993a). Thus from the outset, in termsof its connections with the somatic mesoderm, thepulmonary portal is distinct from the orifices of thesystemic venous channels (the sinus venosus), evenprior to the formation of the venous valves (Fig. 7). Thisis not to deny the existence of extracardiac communica-tions between the developing pulmonary and splanch-nic venous plexuses. At the level of the heart itself,nonetheless, the heart stalk is present prior to theluminization of the pulmonary vein.

As a necessary prelude to atrial septation, the hornsof the embryonic venous sinus can be considered asmoving around the fixed point of anchorage at the heartstalk, eventually coming to open between the venousvalves in the right side of the developing atrium.Concomitant with this rotational change, the tissues atthe junction of the sinus horns (the sinus septum) movefrom being parallel to the long axis of the pulmonaryridges to being almost at right angles to them. This

shift brings the orifice of the coronary sinus into theright side of the atrium. This process we describe in themouse is similar to that described in humans (Stedinget al. 1990), except that in the mouse, the coronarysinus is a large vessel that retains its connection withthe left caval vein (Webb et al., 1996). As in humans, theright venous valve is a continuous structure dividedinto two parts, one initially derived from the caudal foldof the orifice of the right sinus horn and one derivedfrom the caudal fold of the orifice of the left sinus horn.It is the sinus septum that marks the junction of thesetwo parts. Crucially, the walls of the left horn are, fromthe outset, discrete from the developing left atrioven-tricular junction. They are always separated from theleft atrial wall. Indeed, the mouth of the coronary sinusbecomes incorporated as the posterior margin of thedeveloping atrioventricular septum. The pulmonaryvein is not ‘‘pinched off’’ from the venous sinus withsubsequent transfer to the left atrium (DeRuiter et al.1995). Consequently, there is no need to invoke theformation of a left sinuatrial fold when consideringdevelopment of the coronary sinus.

Subsequent to its rotation, the venous sinus is to theright of the developing muscular primary atrial sep-tum. The opening of the venous valves can then belikened to the lips of the mouth, with two corners. Therightsided cranial corner is formed by the septumspurium. The leftsided caudal corner is continuous withthe elevation noted on the right pulmonary ridge andwith the developing primary septum. It is the elevationon the right pulmonary ridge that represents an areawhere somatic mesoderm appears to grow into theheart from the body wall and that, according to ourinterpretation, His (1880) identified as the ‘‘spina ves-tibuli.’’ Others, however, have correlated His’s spinavestibuli with the entire mesenchymal cap on theleading edge of the developing primary atrial septum.This cap is evident from the time of the initial appear-ance of the primary atrial septum (Lopez Rodriguez,1951; Puerta Fonolla et al., 1978; Asami et al., 1995).Fonalla and Llorca (1978) described the elevation re-lated to the right pulmonary ridge (the spina vestibuliof His) as the ‘‘crista prima,’’ following the precedent ofLos (1960). Our findings suggest that the two struc-tures have a separate developmental origin. The mesen-chymal cap on the leading edge of the primary atrialseptum may originate as the result of localised epithe-lial-mesenchymal transformation, in much the sameway as the endocardial cushions develop in the atrioven-tricular canal (Arrechedera et al., 1987; Gerety andWatanabe, 1997). Its structure is remarkably similar,when examined histologically, to that of the atrioven-tricular endocardial cushions. The tissue related tothe right pulmonary ridge, in contrast, stains moredarkly and represents an area where somatic mesen-chyme grows into the heart from the posterior bodywall.

It has been shown, using a monoclonal antibodyagainst endothelial and their precursors in the quail,that the pulmonary and bronchial vasculature developfrom the same vascular plexus (DeRuiter et al., 1993a).Lumenization of these vessels within the somatic meso-derm, as demonstrated in our histological sections,occurs concomitant with development of the lung buds,with the midpharyngeal strand giving rise to the

Fig. 7. This diagram illustrates the fundamental difference in therelationships of cardiac and somatic mesoderm in the region of thepulmonary pit versus the tributaries of the systemic venous sinus. Atthe pulmonary pit, the walls of the atrium (stippled) become confluentwith the mesoderm of the body (cross-hatched), thus enclosing thepulmonary pit as a blind-ending pouch. In contrast, at the mouths ofthe venous sinus the walls of the atrium are directly continuous withthose of the veins, enclosing the venous lumens (stippled areas). Thesite of division of the two venous horns forms the sinus septum. Thedifferences between the arrangement of the pulmonary pit and thesystemic venous tributaries are shown in the inset.

333DEVELOPMENT OF MURINE PULMONARY VEIN

pulmonary vein (DeRuiter et al., 1993b). Our studyshows that the developing pulmonary vein uses theheart stalk as its portal of entry to the atrial segment ofthe heart, being confined to the left atrium when themesenchyme capping the right pulmonary ridgefuses with the atrioventricular endocardial cushions.When the pulmonary vein first becomes evident as ananatomic entity, therefore, the embryonic venous sinusis already demarcated by the venous valves and isanatomically separate from the body of the atrium. His(1880) did state that the pulmonary vein opened intothe left portion of the venous sinus in a human embryo.Importantly, however, the opening of the vein was saidto be situated to the left of the left venous valve. If,therefore, the venous valves are taken as delineatinganatomically the extent of the systemic venous sinus,the vein cannot logically be described at this stage asoriginating from the systemic venous sinus.

Some 50 years have passed since Auer (1948) de-scribed the development of the pulmonary vein ‘‘. . . .asa conical evagination of the lumen into the dorsalmesocardium. This evagination, the stem of the pulmo-nary vein, is surrounded by well defined ridges whichhave been named the right and left pulmonary ridges.It has also been revealed that the right ridge finallymerges with the septum primum of which the primor-dium is a single ridge on higher levels. This continua-tion of the septum primum and of the right pulmonaryridge in a cranio-caudal direction answers the questionas to how the pulmonary vein finally empties into theleft atrium.’’ Our findings endorse totally this concept.These findings are themselves all consistent with thedata presented by those who suggest that the pulmo-nary vein is derived from the embryonic systemicvenous sinus (DeRuiter et al., 1995; Tasaka et al.,1996). It is the interpretation of the findings that differ.When account is taken of the anatomical continuitybetween the myocardial heart tube and the mesoder-mal body wall at the heart stalk, in contrast to theextensions of the lumens of the venous tributaries intothe mediastinum of the developing embryo, it is clearthat the surrounds of the pulmonary pit are, from theoutset, destined to form the orifice of the definitivepulmonary vein. This area is always discrete from thehorns of the embryonic systemic venous sinus. Fromthe outset, the pulmonary ridges may be defined on thebasis of their topographical relationship to the pulmo-nary pit. The nature and extent of the tissues enteringthe heart from extracardiac mesoderm through thestalk remains to be elucidated. Many of the argumentsconcerning these developmental events seem to us to beunnecessarily clouded by inconsistent nomenclature, inparticular to the use of ‘‘dorsal mesocardium.’’ We havechosen to use the term ‘‘heart stalk’’ to describe thestructure that connects, throughout development, theatrial segment of the heart tube to the body wall. Thisstalk is discrete from the venous tributaries that be-come the systemic venous sinus. The heart stalk is alsodiscrete from the large region of somatic mesodermventral to the foregut in which the lung buds develop.The stalk represents the persisting component of theinitially extensive dorsal mesocardium. Distinction ofthese various components will aid those who continueto seek the mechanisms underscoring anomalous devel-opment of the pulmonary vein.

ACKNOWLEDGMENTS

We thank Ian Harragan and Ray Moss for theirtechnical expertise. This work was supported by theBritish Heart Foundation, grant number PG/96114.R.H.A. is supported by the British Heart Foundationand the Joseph Levy Foundation. A.W. is supported byNIH grant HL52813. Additional funding to N.A.B. isfrom the U.K. Medical Research Council.

LITERATURE CITED

Arrechedera, H., M. Alvarez, M. Strauss, and C. Ayesta 1987 Origin ofmesenchymal tissue in the septum primum: a structural andultrastructural study. J. Molec. Cell. Cardiol., 19:641–651.

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