Atrioventricular Cushions Research Articles

Clinical anatomy of the atrial septum with reference to its developmental components.

Knowledge of development is of crucial importance and can help clarify mechanisms of maldevelopment, but it must be properly validated. Concepts of development must be consistent with the anatomy seen in postnatal life. Such consistency is not always achieved. We have reviewed new and old accounts of cardiac embryology with regard to the definitive structure of the atrial septum. The key to understanding is to distinguish between folds of the atrial wall and true interatrial partitions. The flap valve of the oval foramen, and its inferior rim, are true septal structures, whereas the other rims, particularly the antero-superior rim, are infoldings enclosing extracardiac fat. During embryonic life, the systemic venous tributaries must achieve entrance only to the right side of the primary atrium. Development of the pulmonary venous component is a late event, with the canalizing vein using the dorsal mesocardium to gain access to the left side of the atrium. Once the systemic venous tributaries have achieved their rightward shift, the primary septum, together with the mesenchymal cap, grows between the systemic and pulmonary venous orifices. Closure of the primary foramen is achieved by fusion of the mesenchymal cap of the primary septum with the atrioventricular endocardial cushions and the vestibular spine (an additional mesenchymal structure carried on the right side of the pulmonary venous orifice). The superior margin of the newly formed secondary foramen is produced by an infolding of the atrial walls. Historically these mechanisms received appropriate recognition, but not all receive their proper due in current writings.

An autocrine function for transforming growth factor beta 3 in the atrioventricular endocardial cushion tissue formation during chick heart development.

An autocrine function for transforming growth factor beta 3 in the atrioventricular endocardial cushion tissue formation during chick heart development.

Diminished growth of atrioventricular cushion tissue in stage 24 retinoic acid-treated chicken embryos.

Stage 34 chicken hearts have shown a spectrum of looping disturbances, changed hemodynamics, and changed growth of both right ventricular myocardium and atrioventricular cushion tissue after retinoic acid treatment. To obtain more information about the onset of the malformations we studied stage 24, the stage between the previously studied stage 34 and the moment of treatment. Sixteen stage 24 chicken embryos were examined after treatment with 1 microg all-trans retinoic acid at stage 15 and compared with 6 sham operated embryos. Morphological examination was supported by graphic reconstructions. Absolute volumes of atrial, atrioventricular, and ventricular myocardia were measured by a point counting method. The absolute volumes of the endocardial cushions were measured as well. Fifteen (15/16) retinoic acid-treated hearts did not show marked malformations as far as could be detected with our current macroscopic and microscopic techniques. One (1/16) retinoic acid-treated heart showed an abnormal tubular C-shape with a less bended inner curvature and with an abnormal horizontally oriented atrioventricular canal. The dorsal cushion tissue of this atrioventricular canal was discontinuous with the dorsal mesocardium and covered the malpositioned myocardial border between the atrium and the atrioventricular canal. The volume measurements did show a difference between retinoic acid treatment and sham operations. The retinoic acid-treated hearts showed a significant volume decrease of the atrioventricular cushions. No significant differences were found in the volumes of the ventricular myocardium compared to the sham operated embryos. We hypothesize that, between stages 15 and 24, retinoic acid directly affects the myocardial wall and the cushion tissue formation. In the present material this has resulted in decreased atrioventricular cushion growth, in changed hemodynamics, and in a severe looping disturbance of one embryo. We further hypothesize that, between stages 24 and 34, the malformations with minor looping disturbances will become apparent. Thus, development beyond stage 24 would result in the spectrum of looping disturbances as has been found at stage 34. These latter morphological malformations would lead to increasing hemodynamic changes, resulting in changes in growth as a secondary effect.

Development of the atrioventricular valve tension apparatus in the human heart.

Using various microscopical techniques we studied the development of the atrioventricular valves in human hearts between 5 and 19 weeks of development. Within the atrioventricular cushions two different layers could be recognized that remained present in all ages studied. The atrial layer, being present at the side of the atrioventricular orifice, was positive for laminin while the ventricular layer, that was connected to the myocardium, was positive for fibronectin and collagen III. Fate-mapping of these two layers, morphometrics, and scanning electron microscopy, supplemented with in vivo labeling of cushion tissue in chicken hearts have lead to new insights in the process of valve development. The cushions became freely movable prevalvular leaflets by delamination of ventricular myocardium underneath the cushion tissue. This myocardium gradually retracted towards annulus and papillary muscles and finally disappeared, resulting in fibrous, non-myocardial valves. The atrial layer of the cushions remained present as a jelly-like surface on the valve leaflets while the ventricular layer of the cushions became the compact fibrous tissue of the leaflets and the chords. Chordal development was first visible at 10 weeks of development when gaps were formed in the ventricular layer of the cushions on top of the papillary muscles. These gaps enlarged into the interchordal spaces while the cushion tissue in between the gaps lengthened to form the chords. We conclude that the leaflets as well as the chords of the atrioventricular valves are derived from atrioventricular cushion tissue. Myocardium is only important for loosening of the leaflets while keeping connection with the developing papillary muscles. Errors in delamination or retraction of myocardium or remodeling of cushion tissue into chords form the basis for various congenital valve anomalies.

Diminished growth of atrioventricular cushion tissue in stage 24 retinoic acid‐treated chicken embryos

Diminished growth of atrioventricular cushion tissue in stage 24 retinoic acid‐treated chicken embryos

Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions.

The epicardium and dorsal mesocardium are known to be the source of structures that form the wall of the coronary vessels. Because mouse knockout studies have shown that proper epicardial formation is also essential for myocardial development, we have studied in detail the migration and differentiation of epicardium-derived cells (EPDCs) within the developing heart. We constructed chicken-quail chimeras by grafting the quail epicardial organ, including a piece of primordial liver, at essentially stages 16 and 17. The embryos were studied at stages 25 to 43. To detect quail-derived EPDCs, an anti-quail nucleus antibody was used in combination with several differentiation markers, eg, for muscle actin, for vascular smooth muscle cells, for procollagen-I, for quail endothelium, and for Purkinje fibers. At stages 25 to 31, EPDCs are encountered in the myocardial wall and the subendocardial region. The latter deposition is spatially facilitated as the endocardium protrudes through transient discontinuities in the myocardium to contact the subepicardial layer. Later on, at stages 32 to 43, EPDCs invaded, by way of the atrioventricular sulcus, the atrioventricular cushion tissue. The localization is apparent at the interface with the myocardium, as well as subendocardially, but never within the endocardial lining. The origin of endothelium, smooth muscle cells, and fibroblasts of the coronary vessel wall from the epicardial graft were confirmed in accordance with already published data. The functional role of the novel EPDCs in the subendocardium, myocardium, and atrioventricular cushions remains to be investigated. A close positional relationship is found with the differentiating Purkinje fibers. Furthermore, a regulatory role is postulated in the process of endocardial-mesenchymal transformation. The ultimate fate of EPDCs seems to be a cardiac fibroblast cell line involved in the formation of the fibrous heart skeleton.

Formation of the atrioventricular septal structures in the normal mouse.

It is sometimes thought that formation of the atrioventricular septum is equated with fusion of the endocardial cushions and that failure of fusion can explain all deficiencies of atrioventricular septation. Clearly, this is simplistic, but the exact contribution of different primordia to atrioventricular septation is not well understood. To clarify this, we studied normal mouse embryos (days 10 to 15 of gestation), which were serially sectioned and examined by light microscopy. Another group of embryos was examined by scanning electron microscopy after microdissection. Our results show that development of the atrioventricular septal area is highly complex. Proper formation requires the following: remodeling of the inner heart curvature, rotation of the horns of the systemic venous sinus around the pulmonary portal, expansion of the right atrioventricular junction, formation of the muscular atrial and ventricular septa, bridging by the dextrodorsal outflow ridge and the superior endocardial cushion, fusion with the inferior margins of the venous valves, and formation of the mouth of the coronary sinus from the cranial muscular wall of the left sinus horn. Multiple primordia contribute to a central mesenchymal mass (the "septum intermedium"), including the mesenchyme on the leading edge of the primary atrial septum, the atrioventricular endocardial cushions, and the cap of mesenchyme on the spina vestibuli. Fusion of these components closes the ostium primum, completing atrial and atrioventricular septation. Additionally, the spina vestibuli has a mesodermal core, which contributes to the muscularization of the lower margin of the oval fossa. This contrasts with the formation of the upper rim, which occurs as a result of an infolding of the atrial wall itself.

Expression of type VI collagen in the developing mouse heart

During development, the embryonic atrioventricular (AV) endocardial cushions undergo a morphogenic process to form mature valve leaflets and the membranous septa in the heart. Several extracellular matrix (ECM) proteins are expressed in the developing AV endocardial cushions, but it remains to be established if any specific ECM proteins are necessary for normal cushion morphogenesis. Abnormal development of the cardiac AV valves is a frequent cause of congenital heart defects, particularly in infants with trisomy 21 (Down syndrome). The genes encoding the α1 and α2 chains of type VI collagen are located on human chromosome 21 within the region thought to be critical for congenital heart defects in trisomy 21 infants. This suggests that the type VI collagen α1(VI) and α2(VI) chains may be important in normal AV valve morphogenesis. As a first step in understanding the role of type VI collagen in valve development, the authors examined the normal spatial and temporal expression patterns of mRNA and protein for type VI collagen in the embryonic mouse heart. Ribonuclease protection assay analysis demonstrates cardiac expression of the type VI collagen for α1(VI), α2(VI), and α3(VI) transcripts beginning at embryonic days 11–11.5 of mouse development. In situ hybridization studies demonstrate a coordinated pattern of cardiac expression within the AV valves for each type VI collagen chain from embryonic day 11.5 through the neonatal period. Immunohistochemical studies confirm a concentrated type VI collagen localization pattern in the endocardial cushions from the earliest stages of valve development through the neonatal period. These data indicate that type VI collagen is expressed in the developing AV canal in a pattern consistent with cushion tissue mesenchymal cell migration and proliferation, and suggest that type VI collagen plays a role in the morphogenesis of the developing cardiac AV endocardial cushions into the valve leaflets and membranous septa of the heart. Dev. Dyn. 1998;211:248–255. © 1998 Wiley-Liss, Inc.

Expression of type VI collagen in the developing mouse heart.

During development, the embryonic atrioventricular (AV) endocardial cushions undergo a morphogenic process to form mature valve leaflets and the membranous septa in the heart. Several extracellular matrix (ECM) proteins are expressed in the developing AV endocardial cushions, but it remains to be established if any specific ECM proteins are necessary for normal cushion morphogenesis. Abnormal development of the cardiac AV valves is a frequent cause of congenital heart defects, particularly in infants with trisomy 21 (Down syndrome). The genes encoding the alpha1 and alpha2 chains of type VI collagen are located on human chromosome 21 within the region thought to be critical for congenital heart defects in trisomy 21 infants. This suggests that the type VI collagen alpha1(VI) and alpha2(VI) chains may be important in normal AV valve morphogenesis. As a first step in understanding the role of type VI collagen in valve development, the authors examined the normal spatial and temporal expression patterns of mRNA and protein for type VI collagen in the embryonic mouse heart. Ribonuclease protection assay analysis demonstrates cardiac expression of the type VI collagen for alpha1(VI), alpha2(VI), and alpha3(VI) transcripts beginning at embryonic days 11-11.5 of mouse development. In situ hybridization studies demonstrate a coordinated pattern of cardiac expression within the AV valves for each type VI collagen chain from embryonic day 11.5 through the neonatal period. Immunohistochemical studies confirm a concentrated type VI collagen localization pattern in the endocardial cushions from the earliest stages of valve development through the neonatal period. These data indicate that type VI collagen is expressed in the developing AV canal in a pattern consistent with cushion tissue mesenchymal cell migration and proliferation, and suggest that type VI collagen plays a role in the morphogenesis of the developing cardiac AV endocardial cushions into the valve leaflets and membranous septa of the heart.

The Distribution of Tenascin in Rat Embryos with Normal Heart and Cardiovascular Anomalies Induced by Bis‐Diamine

AbstractThe mesenchyme in the truncal ridge and aortico‐pulmonary (A‐P) septum in the developing heart expresses tenascin (TN), a component of extracellular matrices. We observed immunohistochemically the localization of TN with reference to fibronectin (FN) distribution in rat embryonic hearts at gestation days 12. 13 and 14 and compared the results with those in bis‐diamine‐induced cardiovascular anomalies. In control rat embryonic hearts, TN was distributed in the dorsal mesocardium and the distal part of the truncal ridge at gestation day 12, and in part of the conal and truncal ridges, A‐P septum and proximal wall of aortic arch arteries at gestation days 13 and 14. The conotruncal regions with TN expression may correspond to the areas where cardiac neural crest cells migrate in the heart. In bis‐diamine‐treated rat hearts showing conotruncal anomalies, TN was localized in incompletely fused and hypoplastic conotruncal ridges, but the intensity and area of staining were decreased suggesting a decreased neural crest cell migration. FN was diffusely distributed in both the normal and anomalous embryonic hearts, including atrioventricular cushion tissue in addition to the region expressing TN. TN was densely distributed in the fusing area of the conal and truncal ridges in comparison to FN, suggesting that TN may be mainly expressed during the time of fusion of conal and truncal ridges to form the A‐P septum in the embryonic heart. In conclusion, bis‐diamine may cause the poor development of neural crest derived tissue, subsequently bringing about hypoplasia of the A‐P septum and truncal and conal ridges.

Heart defects in connexin43-deficient mice.

Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the D configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a D loop, but when it persists, a "crisscross" configuration develops, with the atrioventricular cushions rotated 90 degrees, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the D configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development of intertrabecular pouches during subsequent development.

Anomalous looping, atrioventricular cushion dysplasia, and unilateral ventricular hypoplasia in the mouse embryos with right isomerism induced by retinoic acid.

Visceroatrial heterotaxy syndrome is characterized by abnormality of visceral laterality and complex cardiovascular anomalies usually involving both the outflow and inflow tract. Morishima et al. (1995) showed that mouse embryos treated with all-trans retinoic acid at embryonic day 6.5 (primitive streak stage) induces this syndrome. To investigate the morphogenetic process of visceroatrial heterotaxy syndrome, we examined retinoic acid-treated mouse embryos at embryonic days 9-15 using scanning electron microscopy. The sinoatrial connection was first distinguished for the determination of situs as early as at embryonic day 10.5. Normal visceroatrial situs was found in 57% of all treated embryos, and the rest had abnormal situs, in which right isomerism was found in 81%. In the right-isomeric mouse, the cardiac morphology was characterized by abnormal looping together with dysplasia of the inflow and outflow tract cushion; that is, the primitive right ventricle was usually deviated cranially to various degrees, the atrioventricular cushion appeared trilobed in a half of them, and unilateral ventricular hypoplasia was noted in about one-third of them after embryonic day 14.5. An anomalous relation between the atrioventricular cushions and the interventricular septum appeared to have caused a restrictive inflow to the unilateral ventricle, leading to ventricular chamber hypoplasia on the ipsilateral side. Thus, we clarified that retinoic-acid treatment at the primitive streak stage disturbed cardiac looping and formation of atrioventricular cushion development, which secondarily influenced ventricular chamber development.

Production of a monoclonal antibody by in vitro immunization that recognizes a native chondroitin sulfate epitope in the embryonic chick limb and heart.

We report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22-23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart.

Anatomy and development of the sinoatrial valves in the dogfish Scyliorhinus canicula

ABSTRACT Background: We describe the adult anatomy and the development of the cardiac sinoatrial valves in the dogfish (Scyliorhinus canicula). Methods We use scanning electron microscopy, histological and histochemical techniques in 39 hearts from embryos and adult specimens. Results The sinoatrial valvular set of the adult dogfish is composed of two transverse valves laterally attached to the sinoatrial junction at their bases. Both valves are composed of two muscular layers, the sinusal and the atrial, whose histological features are similar to the cardiac wall which they face. Collagen bundles, elastic fibers and fibroblasts are present between the muscular layers. The extracellular matrix between the valvular layers also contains sulphated and non-sulphated glycosaminoglycans. The sinoatrial valves develop from two lateral infoldings of the cardiac wall. The left fold is deeper than the right, causing a shift of the sinoatrial communication to the right. The epicardium progressively covers the outer sinoatrial groove and the space between the folds becomes populated by mesenchymal cells. The posterior atrioventricular endocardial cushion is in contact with the base of the left fold until the embryo has about 40 mm TL. Conclusions The sinoatrial valves, in the dogfish, develop from lateral infoldings of the cardiac wall. This origin results in histological and histochemical differences between the two muscular layers which constitute the valves of the adult. The comparison of the sinoatrial valve morphogenesis between the dogfish and some higher vertebrates suggests that the right sinoatrial valve, but not the left, is homologous throughout the vertebrate phylogeny. Anat. Rec. 248:224-232, 1997. © 1997 Wiley-Liss, Inc.

Anatomy and development of the sinoatrial valves in the dogfish (Scyliorhinus canicula).

We describe the adult anatomy and the development of the cardiac sinoatrial valves in the dogfish (Scyliorhinus canicula). We use scanning electron microscopy, histological and histochemical techniques in 39 hearts from embryos and adult specimens. The sinoatrial valvular set of the adult dogfish is composed of two transverse valves laterally attached to the sinoatrial junction at their bases. Both valves are composed of two muscular layers, the sinusal and the atrial, whose histological features are similar to the cardiac wall which they face. Collagen bundles, elastic fibers and fibroblasts are present between the muscular layers. The extracellular matrix between the valvular layers also contains sulphated and non-sulphated glycosaminoglycans. The sinoatrial valves develop from two lateral infoldings of the cardiac wall. The left fold is deeper than the right, causing a shift of the sinoatrial communication to the right. The epicardium progressively covers the outer sinoatrial groove and the space between the folds becomes populated by mesenchymal cells. The posterior atrioventricular endocardial cushion is in contact with the base of the left fold until the embryo has about 40 mm TL. The sinoatrial valves, in the dogfish, develop from lateral infoldings of the cardiac wall. This origin results in histological and histochemical differences between the two muscular layers which constitute the valves of the adult. The comparison of the sinoatrial valve morphogenesis between the dogfish and some higher vertebrates suggests that the right sinoatrial valve, but not the left, is homologous throughout the vertebrate phylogeny.

Stereological study of stage 34 chicken hearts with looping disturbances after retinoic acid treatment: Disturbed growth of myocardium and atrioventricular cushion tissue

Stereological study of stage 34 chicken hearts with looping disturbances after retinoic acid treatment: Disturbed growth of myocardium and atrioventricular cushion tissue

Stereological study of stage 34 chicken hearts with looping disturbances after retinoic acid treatment: disturbed growth of myocardium and atrioventricular cushion tissue.

In a previous study retinoic acid treatment of chicken hearts has resulted in a spectrum of looping disturbances. Because of a decrease in contraction force of these hearts, the myocardial volume was hypothesized to be altered. Because retinoic acid has been suggested to influence endocardial cushion volumes, these were estimated as well. The previously studied hearts were used for estimating the absolute volumes of the atrial and ventricular myocardium and of the endocardial cushions by means of Cavalieri's principle. To measure the surface density of the trabeculations according to the isector method, we used retinoic acid treated hearts, which were perfusion fixed and in which the sections were isotropic uniform random. The volumes and surface densities found in the three morphologically distinguished groups, i.e., intact septum, isolated ventricular septal defect and double outlet right ventricle, were compared with those in shams. A significant volume decrease was found in the right ventricular free wall myocardium of the double outlet right ventricle. No significant differences were found in the surface densities of the trabeculae. The volume of the atrioventricular cushion tissue in the double outlet right ventricle hearts was significantly increased. The morphological spectrum observed previously was also expressed in the right ventricular myocardial volume, which appeared to decrease from the least to the most malformed hearts, and in the volume of the atrioventricular cushion tissue, which appeared to increase. Several studies have shown pathology in myocardial and cushion tissue after retinoic acid treatment. In this study we have found a decreased growth of the right ventricular myocardium and an increased growth of the atrioventricular cushion tissue. We suggest that the previously found looping disturbance causes changed hemodynamics, as reported elsewhere, and that these result in changes in growth. We cannot exclude a direct effect of retinoic acid on the myocardium, which has to explain the looping disturbance.

Temporal and spatial asymmetries in the initial distribution of mesenchyme cells in the atrioventricular canal cushions of the developing chick heart

Temporal and spatial asymmetries in the initial distribution of mesenchyme cells in the atrioventricular canal cushions of the developing chick heart

Temporal and spatial asymmetries in the initial distribution of mesenchyme cells in the atrioventricular canal cushions of the developing chick heart.

We investigated potential early asymmetries in the distribution of mesenchymal cells within the inferior and superior AV cushions in the developing chick heart. Chick embryos stages 16-20 HH were fixed, embedded in polyacrylamide, and the cell nuclei stained with propidium iodide. Cells counts were determined within the cardiac jelly of the atrioventricular canal (AV) by laser confocal microscopy in coronal planes spanning its entire length. Our data show at the different stages studied, 16-20 HH, that the inferior AV cushion invariably contains more cells than the superior AV cushion. In the inferior cushion, the cell distribution is bimodal, i.e., the proximal and distal regions have more mesenchymal cells than the middle part of the AV canal. In the superior cushion, there is a increasing gradient of mesenchymal cells along the longitudinal axis from the atrium to the ventricle. Our findings reveal that the temporal and spatial characteristics of mesenchyme formation in the inferior vs. superior AV cushion are different. This asymmetry suggests several potential hypotheses: (1) the distribution of the inducer molecule or its receptor has a distribution similar to that of mesenchymal cells, (2) the extracellular matrix has a differential composition or regionally-specific physical associations, (3) the endocardium is heterogeneous with respect to transformation capacity, or (4) these patterns result from an earlier inductive event. The potential importance of the observed asymmetries in the distribution of AV mesenchyme is discussed relative to localization patterns of molecules critical to successful cardiac morphogenesis and remodeling.

Carbonic anhydrase II expression pattern in mouse embryonic and fetal heart.

Carbonic anhydrase II localization was studied in mouse embryonic and fetal hearts for better understanding of the functions of this enzyme during cardiac organogenesis. Immunocytolabelling was performed on serial sections of frozen hearts after one night's fixation in 4% paraformaldehyde. In the earliest stages studied, 10, 11 and 12 ed (ed = embryonic day; vaginal plug = day 1), a sharp decrease of labelled cells was observed in the endocardium form which cushion-tissue mesenchyme is derived. During the same period, differences in the decreasing frequencies of labelled cells were also observed between three different cushion-tissue mesenchyme localizations: immunostained cells were abundant in the atrioventricular cushions, less numerous in the proximal part of the conotruncal ridges and rare in their distal part. From 13 ed their repartition was more regular along the conotruncus. From 13 to 16 ed the signal was also present in a peculiar region of the myocardium: the anterior and left walls of the left ventricle. At the 18 and 20 ed labelling was found only in some endothelial cells of coronary vessels, particularly in the interventricular septum. The pattern of expression of carbonic anhydrase II in activated endothelial cells and endothelial-derived mesenchyme cells of the cardiac cushion tissue, strongly suggests that this isoenzyme can be a useful marker for a subpopulation of endothelial cells and cells derived from this endothelium that morphologically express signs of active cell behavior (e.g., invasion, migration, proliferation).