José M. Icardo

Pitx2 Participates in the Late Phase of the Pathway Controlling Left-Right Asymmetry

Pitx2, a member of the bicoid-related family of homeobox-containing genes, is asymmetrically expressed in the left lateral plate mesoderm and derived tissues during chick and mouse development. Modifications of Pitx2 pattern of expression in the iv mouse mutation correlate with the situs alterations characteristic of the mutation. Misexpression experiments demonstrate that Shh and nodal positively regulate Pitx2 expression. Our results are compatible with a Pitx2 function in the late phase of the gene cascade controlling laterality.

Developmental biology of the vertebrate heart

This paper summarizes the development of the heart from the formation of the heart mesoderm to cardiac septation. A brief account of morphological changes is provided, but attention is focused on mechanisms rather than on morphologic descriptions. Heart induction and differentiation, and the expression of cardiac specific proteins, are reviewed. New developments in these areas include the possible role of cell surface proteins and peptide growth factors in the segregation of the splanchnic mesoderm and in cardiac commitment. Past and recent experiments indicate that the heart morphogenetic information is engraved in the precardiac mesoderm. In spite of this, specific differentiative signals can be overriden experimentally demonstrating the unstability of the cardiac phenotype at the early heart tube stage. The relationship between differentiation and morphogenesis is analyzed. While cardiac differentiation appears to be a prerequisite for morphogenesis, a number of experiments indicate that differentiation can proceed in the absence of any morphogenesis. Formation of the heart loop is separated into two different components; looping itself and the acquisition of handedness. Late heart morphogenesis is explained in terms of differential tissue growth and tissue remodeling. This not only includes morphogenetic changes intrinsic to the heart but the addition of new cell types (neural crest, epicardium, vessels, nerves) that become integrated into the developing heart. The contribution of specific mechanisms to our understanding of heart development, such as cell death and hemodynamics is also analyzed.

Morphologic Study of Ventricular Trabeculation in the Embryonic Chick Heart

This paper presents a morphologic study of ventricular trabeculation in chick embryo hearts between days 2 and 5 of incubation. Trabeculation appears to be the expression of three closely interrelated events: the formation of endocardial outgrowths that eventually invade the myocardium; the development of large intercellular spaces between the myocytes, and the decrease in thickness of the cardiac jelly. Endocardial cells present morphologic differences between trabeculated and nontrabeculated areas of the ventricular region. The elongation of the endocardial cells in the endocardial outgrowths and the presence of mitoses suggest that the endocardium grows out by means of an increase in cell number and by redistribution and elongation of the preexisting endocardial cells. The intercellular spaces of the myocardium appear filled with abundant extracellular material. It is suggested that the continuous synthesis of extracellular material by the myocytes may increase the hydrostatic pressure within the myocardium, inducing the formation and the enlargement of these intercellular spaces. The development and later rupture of endocardium-covered cords is described here. These cords are made up of a core of cardiac jelly material revested by endocardium. The cords may be engaged in the removal of substantial amounts of cardiac jelly during the formation of the trabeculae.

Molecular characterization of the ventricular conduction system in the developing mouse heart: topographical correlation in normal and congenitally malformed hearts

OBJECTIVES Within the adult heart, it is convention to distinguish the conduction system and working (atrial and ventricular) myocardium. The adult conduction system (CS) comprises the sinoatrial (SAN), and atrioventricular (AVN) nodes, the atrioventricular bundle (AVB), the bundle branches and the peripheral Purkinje fibers, each of which display distinct functional properties and distinct profile of gene expression. Characterization of the mouse cardiac conduction system during development is rudimentary at present, even though genetically-modified mice are an increasing source of information regarding cardiac function and embryonic heart development. METHODS We have performed a detailed study of the pattern of expression of myosin heavy chain (MHC), myosin light chain (MLC), troponin I (TnI) isoforms, connexin 43 (Cx43), desmin and alpha-smooth muscle actin (alpha-SMA), in the ventricular conduction system of normal and congenitally malformed mouse hearts (iv background) from embryonic day 14.5 to 19.5. RESULTS The AVN is characterized by co-expression of MHC and MLC isoforms and no detectable expression of Cx43, desmin or alpha-SMA. The AVB expresses betaMHC and MLC2v, but no alphaMHC, MLC2a, Cx43, desmin or alpha-SMA. The right and left bundle branches display enhanced expression of desmin and alpha-SMA but no Cx43. The normal expression profile is maintained in congenitally malformed hearts such as double-outlet right ventricle and common atrioventricular canal. Three-dimensional reconstruction of the conduction system shows normal arrangement of the bundle branches in congenitally malformed hearts, but abnormal location and/or extension of the AVN. CONCLUSIONS Molecular characterization allows to follow the development of the CS in both, normal and malformed mouse hearts. Normal phenotypic expression of the CS is independent of heart situs but shows minor modifications in the presence of heart malformations. It is concluded that the AVN derives from the atrioventricular canal myocardium, the bundle of His from the ventricular myocardium, and the bundle branches from the ventricular trabeculations. Our results do not provide evidence to support an extra-cardiac origin of the ventricular CS.

Bulbus arteriosus of the antarctic teleosts. I. The white-blooded Chionodraco hamatus.

The bulbus arteriosus of teleost fish is a thick‐walled chamber that extends between the single ventricle and the ventral aorta. The functional importance of the bulbus resides in the fact that it maintains a steady blood flow into the gill system through heart contraction. Despite of this, a thorough study of the structure of the bulbus in teleost fish is still lacking. We have undertaken a morphologic study of the bulbus arteriosus in the stenothermal teleosts of the Antarctic sea. The structural organization of the bulbus arteriosus of the icefish Chionodraco hamatus has been studied here by conventional light, scanning, and transmission electron microscopy.

Origin and course of the coronary arteries in normal mice and in iv/iv mice

This paper reports on the origin and distribution of the coronary arteries in normal mice and in mice of the iv/iv strain, which show situs inversus and heterotaxia. The coronary arteries were studied by direct observation of the aortic sinuses with the scanning electron microscope, and by examination of vascular corrosion casts. In the normal mouse, the left and right coronaries (LC, RC) arise from the respective Valsalva sinus and course along the ventricular borders to reach the heart apex. Along this course the coronary arteries give off small branches at perpendicular or acute angles to supply the ventricles. The ventricular septum is supplied by the septal artery, which arises as a main branch from the right coronary. Conus arteries arise from the main coronary trunks, from the septal artery and/or directly from the Valsalva sinus. The vascular casts demonstrate the presence of intercoronary anastomoses. The origin of the coronary arteries was found to be abnormal in 84% of the iv/iv mice. These anomalies included double origin, high take‐off, slit‐like openings and the presence of a single coronary orifice. These anomalies occurred singly or in any combination, and were independent of heart situs. The septal artery originated from RC in most cases of situs solitus but originated predominantly from LC in situs inversus hearts. Except for this anomalous origin no statistical correlation was found between the coronary anomalies and heart situs or a particular mode of heterotaxia. The coronary anomalies observed in the iv/iv mice are similar to those found in human hearts. Most coronary anomalies appear to be due to defective connections between the aortic root and the developing coronaries. iv/iv mice may therefore constitute a good model to study the development of similar anomalies in the human heart.

The heart of the Antarctic icefish as paradigm of cold adaptation

Abstract The stenothermal Antarctic fishes, particularly the hemoglobinless icefish, have developed biochemical, metabolic and morpho-functional features of cardiac performance that can help to decipher some mechanisms underlying cardiac cold adaptation. Examples taken from different levels of cardiac biology in Antarctic fish as a paradigm of cold adaptation include: the function of myoglobin in the icefish species that either express or do not express this pigment; the metabolic and ultrastructural reshaping of the myocardiocytes; and the intrinsic mechanical characteristics of the icefish heart ventricle as a low rate, low pressure and high volume pump.

The structure of the conus arteriosus of the sturgeon (Acipenser naccarii) heart: II. The myocardium, the subepicardium, and the conus-aorta transition.

Sturgeons constitute a family of living “fossil” fish whose heart is related to that of other ancient fish and the elasmobranches. We have undertaken a systematic study of the structure of the sturgeon heart aimed at unraveling the relationship between the heart structure and the adaptive evolutionary changes. In a related paper, data were presented on the conus valves and the subendocardium. Here, the structure of the conus myocardium, the subepicardial tissue, and the conus‐aorta transition were studied by conventional light, transmission, and scanning electron microscopy. In addition, actin localization by fluorescent phalloidin was used. The conus myocardium is organized into bundles whose spatial organization changes along the conus length. The variable orientation of the myocardial cell bundles may be effective in emptying the conus lumen during contraction and in preventing reflux of blood. Myocardial cell bundles are separated by loose connective tissue that contains collagen and elastin fibers, vessels, and extremely flat cells separating the cell bundles and enclosing blood vessels and collagen fibers. The ultrastructure of the myocardial cells was found to be very similar to that of other fish groups, suggesting that it is largely conservative. The subepicardium is characterized by the presence of nodular structures that contain lympho‐hemopoietic (thymus‐like) tissue in the young sturgeons and a large number of lymphocytes after the sturgeons reach sexual maturity. This tissue is likely implicated in the establishment and maintenance of the immune responses. The intrapericardial ventral aorta shows a middle layer of circumferentially oriented cells and internal and external layers with cells oriented longitudinally. Elastin fibers completely surround each smooth muscle cell, and the spaces between the different layers are occupied by randomly arranged collagen bundles. The intrapericardial segment of the ventral aorta is a true transitional segment whose structural characteristics are different from those of both the conus subendocardium and the rest of the ventral aorta. Anat Rec 268:388–398, 2002.

Bulbus arteriosus of the antarctic teleosts. II. The red‐blooded Trematomus bernacchii

The structure of the bulbus arteriosus of the Antarctic teleost, Trematomus bernacchii, has been studied by light, scanning, and transmission electron microscopy. The wall of the bulbus arteriosus is divided into endocardial, subendocardial, middle and external layers. The endocardial endothelium covers the inner surface of the bulbus wall and invaginates into the subendocardium to form solid epithelial cords that show secretory activity. The subendocardial tissue is divided into finger‐like ridges. Ridge cells located under the endocardium appear in niches limited by collagen fibers and thin cell extensions. Away from the endocardium ridge cells cluster into small groups, show some of the characteristics of smooth muscle cells, and appear enmeshed in a filamentous meshwork that lacks collagen and elastin fibers. The middle bulbus layer is formed by typical smooth muscle cells that are enmeshed in a filamentous meshwork similar to that observed in the ridges. The ridges and the middle layer appear to be formed by the same cell type, smooth muscle, with a gradient of differentiation from the endocardium toward the middle layer. In the absence of elastin fibers the filamentous meshwork should confer elastic properties to the bulbus wall. The stretching of the meshwork along the main axis of the middle layer cells, and between different cellular layers, suggests the existence of tensile stress and, hence, the involvement of smooth muscle cells in bulbus wall dynamics. The external layer is formed by numerous cellular types embedded in a collagenous matrix. Among these cellular types, myofibroblasts, macrophages, granulocytes, lymphocytes, dendrite‐like cells, degenerating cells, and plasma cells can be recognized. The subepicardial tissue appears to be a specialized site involved in the production of the humoral immune response and displays many of the morphological characteristics of a germinal center. The outer limiting layer of the bulbus, the visceral pericardium, is formed by epithelial cells that show desmosomes and tight junctions. This suggests a close control of permeability with respect to the pericardial fluid. Anat Rec 256:116–126, 1999.

Structure of the conus arteriosus of the sturgeon (Acipenser naccarii) heart. I: The conus valves and the subendocardium

Sturgeons are bony fish that retain structural traits typical of the more primitive Chondrostei. From an evolutionary viewpoint, sturgeons are considered relic fish. However, they show remarkable ecological plasticity and are well adapted to contemporary environmental conditions. Although development of the cardiovascular system is critical for all organs and systems, and is affected by evolutionary changes, the structure of the sturgeon heart has been mostly overlooked. This is also true for the conus arteriosus, which, as in Chondrostei, is endowed with several rows of valves and a layer of contractile myocardium. This work reports on the structure of the valves, the endocardium, and the subendocardium of the conus arteriosus of the sturgeon (Acipenser naccarii) heart. It is part of a broader study that aims to cover the entire structure of the sturgeon heart. The conus arteriosus of 15 A. naccarii hearts, ranging in age from juveniles to sexually‐differentiated adults, has been studied by conventional light, transmission (TEM), and scanning electron microscopy (SEM). In addition, maceration of the soft tissues with NaOH, and actin localization by fluorescent phalloidin has been used. The conus is a tubular chamber that arises from the right ventricular side and presents two constrictions at the conus‐ventricle and conus‐aorta junctions. The conus is endowed with three rows of valves: one distal and two proximal. The segment of the conus located between the distal and the two proximal rows is devoid of valvular structures. The distal row has four leaflets, while the two proximal rows show the greatest variation in leaflet number, size, and shape. All leaflets have collagenous chordae tendineae arising from the free border and from the parietal side of the leaflets. The endocardium is a flat endothelium which shows a thick, irregular basement membrane. The leaflet body is formed by a loose connective tissue which blends with the subendocardium. The subendocardium is a connective tissue consisting of myofibroblasts, collagen, and elastin. It is divided into two distinct areas: one proximal, which shows little elastin and poorly organized collagen; and one distal, which is rich in elastin, with cells and extracellular fibers organized into layers that are oriented in alternative circumferential and longitudinal directions. The present report is the first systematic analysis of the structure of the sturgeon conus. Descriptions of the conus valves should recognize the existence of three valve rows only. The variability in valve morphology, and the loose structure of the leaflet tissue make it unlikely that the valves play an effective role in preventing blood backflow. In this regard, the ventricle‐conus constriction may act as a sphincter. The subendocardium is an elastic coat capable of actively sustaining the tissue deformation that accompanies the heart contractile cycle. Further comparative studies are needed to provide deeper insight into the structural changes that accompany phyletic diversification. Anat Rec 267:17–27, 2002.