Development of electrical rhythmic activity in early embryonic cultured chick double-heart monitored optically with a voltage-sensitive dye

Double-hearted embryos were produced by whole-embryo culture of chick embryos which were microsurgically cut through the tissue of the anterior intestinal portal at the 1- to 6-somite developmental stage, at the time when the cardiac primordia have not yet fused in the bulboventricular region. The cultured embryos were removed from an incubator usually at the 7- to 10-somite stages of development, and then spontaneous electrical action potentials and/or contractions were optically recorded simultaneously from both the right and left half-hearts, using a 10 X 10- element photodiode matrix array together with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). At the 7- to 8-somite stages, spontaneous action potentials were detected from bilateral prebeating half-hearts or sometimes from one half-heart. In each half-heart, the first spontaneous beating was often observed in the half-heart of the 9 somite embryos. In the beating half-hearts regular activity was always observed, while in the prebeating half-hearts at the 7- to 8-somite stages, both the regular and irregular rhythms of action potentials were detected, and the incidence of occurrence of regular activity significantly outnumbered that of the irregular rhythm. The heart rate in the left half-heart was faster than that in the right half-heart in the great majority of the prebeating and beating double-hearted embryos.     

Retinoid signaling is required to complete the vertebrate cardiac left/right asymmetry pathway

Vitamin A-deficient (VAD) quail embryos have severe abnormalities, including a high incidence of reversed cardiac situs. Using this model we examined in vivo the physiological function of vitamin A in the left/right (L/R) cardiac asymmetry pathway. Molecular analysis reveals the expression of early asymmetry genes activin receptor IIa, sonic hedgehog, Caronte, Lefty-1, and Fgf8 to be unaffected by the lack of retinoids, while expression of the downstream genes nodal-related, snail-related (cSnR), and Pitx2 is altered. In VAD embryos nodal expression in left lateral plate mesoderm (LPM) is severely downregulated and the expression domain altered during neurulation. Similarly, the expression of cSnR in the right LPM and of Pitx2 in the left side posterior heart-forming region (HFR) is downregulated in the VAD embryos. The lack of retinoids does not cause randomization or ectopic expression of nodal, cSnR, or Pitx2. At the six- to eight-somite stage nodal is expressed transiently in the left posterior HFR of normal quail embryos; this expression is missing in VAD embryos and may be linked to the loss of Pitx2 expression in this region of VAD quail embryos. Administration of retinoids to VAD embryos prior to the six-somite stage rescues the expression of nodal, cSnR, and Pitx2 as well as the randomized VAD cardiac phenotype. There is an absolute requirement for retinoids at the four- to five-somite developmental window for cardiogenesis and cardiac L/R specification to proceed normally. We conclude that retinoids do not regulate the left/right-specific sidedness assignments for expression of genes on the vertebrate cardiac asymmetry pathway, but are required during neurulation for the maintenance of adequate levels of their expression and for the development of the posterior heart tube and a loopable heart. Cardiac asymmetry may be but one of several critical events regulated by retinoid signaling in the retinoid-sensitive developmental window.     

The outflow tract of the heart is recruited from a novel heart-forming field.

As classically described, the precardiac mesoderm of the paired heart-forming fields migrate and fuse anteriomedially in the ventral midline to form the first segment of the straight heart tube. This segment ultimately forms the right trabeculated ventricle. Additional segments are added to the caudal end of the first in a sequential fashion from the posteriolateral heart-forming field mesoderm. In this study we report that the final major heart segment, which forms the cardiac outflow tract, does not follow this pattern of embryonic development. The cardiac outlet, consisting of the conus and truncus, does not derive from the paired heart-forming fields, but originates separately from a previously unrecognized source of mesoderm located anterior to the initial primitive heart tube segment. Fate-mapping results show that cells labeled in the mesoderm surrounding the aortic sac and anterior to the primitive right ventricle are incorporated into both the conus and the truncus. Conversely, if cells are labeled in the existing right ventricle no incorporation into the cardiac outlet is observed. Tissue explants microdissected from this anterior mesoderm region are capable of forming beating cardiac muscle in vitro when cocultured with explants of the primitive right ventricle. These findings establish the presence of another heart-forming field. This anterior heart-forming field (AHF) consists of mesoderm surrounding the aortic sac immediately anterior to the existing heart tube. This new concept of the heart outlet's embryonic origin provides a new basis for explaining a variety of gene-expression patterns and cardiac defects described in both transgenic animals and human congenital heart disease.     

The restriction of the heart morphogenetic field in Xenopus laevis

We have examined the spatial restriction of heart-forming potency in Xenopus laevis embryos, using an assay system in which explants or explant recombinates are cultured in hanging drops and scored for the formation of a beating heart. At the end of neurulation at stage 20, the heart morphogenetic field, i.e., the area that is capable of heart formation when cultured in isolation, includes anterior ventral and ventrolateral mesoderm. This area of developmental potency does not extend into more posterior regions. Between postneurula stage 23 and the onset of heart morphogenesis at stage 28, the heart morphogenetic field becomes spatially restricted to the anterior ventral region. The restriction of the heart morphogenetic field during postneurula stages results from a loss of developmental potency in the lateral mesoderm, rather than from ventrally directed morphogenetic movements of the lateral mesoderm. This loss of potency is not due to the inhibition of heart formation by migrating neural crest cells. During postneurula stages, tissue interactions between the lateral mesoderm and the underlying anterior endoderm support the heart-forming potency in the lateral mesoderm. The lateral mesoderm loses the ability to respond to this tissue interaction by stages 27-28. We speculate that either formation of the third pharyngeal pouch during stages 23-27 or lateral inhibition by ventral mesoderm may contribute to the spatial restriction of the heart morphogenetic field.     

The restriction of the heart morphogenetic field in Xenopus laevis

We have examined the spatial restriction of heart-forming potency in Xenopus laevis embryos, using an assay system in which explants or explant recombinates are cultured in hanging drops and scored for the formation of a beating heart. At the end of neurulation at stage 20, the heart morphogenetic field, i.e., the area that is capable of heart formation when cultured in isolation, includes anterior ventral and ventrolateral mesoderm. This area of developmental potency does not extend into more posterior regions. Between postneurula stage 23 and the onset of heart morphogenesis at stage 28, the heart morphogenetic field becomes spatially restricted to the anterior ventral region. The restriction of the heart morphogenetic field during postneurula stages results from a loss of developmental potency in the lateral mesoderm, rather than from ventrally directed morphogenetic movements of the lateral mesoderm. This loss of potency is not due to the inhibition of heart formation by migrating neural crest cells. During postneurula stages, tissue interactions between the lateral mesoderm and the underlying anterior endoderm support the heart-forming potency in the lateral mesoderm. The lateral mesoderm loses the ability to respond to this tissue interaction by stages 27–28. We speculate that either formation of the third pharyngeal pouch during stages 23–27 or lateral inhibition by ventral mesoderm may contribute to the spatial restriction of the heart morphogenetic field.     

Flow on the right side of the gastrocoel roof plate is dispensable for symmetry breakage in the frog Xenopus laevis

Leftward flow of extracellular fluid breaks the bilateral symmetry of most vertebrate embryos, manifested by the ensuing asymmetric induction of Nodal signaling in the left lateral plate mesoderm (LPM). Flow is generated by rotational beating of polarized monocilia at the posterior notochord (PNC; mammals), Kupffer's vesicle (KV; teleost fish) and the gastrocoel roof plate (GRP; amphibians). To manipulate flow in a defined way we cloned dynein heavy chain genes dnah5, 9 and 11 in Xenopus. dnah9 expression was closely related to motile cilia from neurulation onwards. Morphant tadpoles showed impaired epidermal ciliary beating. Leftward flow at the GRP was absent, resulting in embryos with loss of asymmetric marker gene expression. Remarkably, unilateral knockdown on the right side of the GRP did not affect laterality, while left-sided ablation of flow abolished marker gene expression. Thus, flow was required exclusively on the left side of the GRP to break symmetry in the frog. Our data suggest that the substrate of flow is generated within the GRP and not at its margin, disqualifying Nodal as a candidate morphogen.     

Pleiotropic effects of the Cardiac-lethal gene in the axolotl (Ambystoma mexicanum)

Embryos of the axolotl affected with the cardiac-lethal mutation form hearts that never begin to beat. A number of other traits characteristic of the mutant phenotype, including edema, underdeveloped gills, shorter stature, and aphagia (the inability to feed), were believed to be secondary effects of the absence of circulation. We have recently demonstrated that the pre-cardiac mesoderm is directly affected by the c gene, making it unresponsive to normal inductive signals. In this study, we replaced part or all of the mutant pre-cardiac mesoderm with wild-type tissue, to produce embryos with normally beating hearts and circulation. As expected, most of the other mutant characteristics were also corrected. However, otherwise normal individuals remained aphagic. All embryos with beating hearts containing mutant tissue also suffered from an unexpected circulatory arrest some time after the onset of circulation. This apparently indicates that there are at least two tissues other than the myocardium which appear to be directly affected by the c gene. These previously unsuspected pleiotropic effects of the mutation may involve poorly-characterized mesodermal-neural crest inductive interactions and may also lead to a greater understanding of the link between congenital heart defects and feeding difficulties in humans. © 1993Wiley-Liss, Inc.     

Signals from trunk paraxial mesoderm induce pronephros formation in chick intermediate mesoderm

We used Pax-2 mRNA expression and Lim 1/2 antibody staining as markers for the conversion of chick intermediate mesoderm (IM) to pronephric tissue and Lmx-1 mRNA expression as a marker for mesonephros. Pronephric markers were strongly expressed caudal to the fifth somite by stage 9. To determine whether the pronephros was induced by adjacent tissues and, if so, to identify the inducing tissues and the timing of induction, we microsurgically dissected one side of chick embryos developing in culture and then incubated them for up to 3 days. The undisturbed contralateral side served as a control. Most embryos cut parallel to the rostrocaudal axis between the trunk paraxial mesoderm and IM before stage 8 developed a pronephros on the control side only. Embryos manipulated after stage 9 developed pronephric structures on both sides, but the caudal pronephric extension was attenuated on the cut side. These results suggest that a medial signal is required for pronephric development and show that the signal is propagated in a rostral to caudal sequence. In manipulated embryos cultured for 3 days in ovo, the mesonephros as well as the pronephros failed to develop on the experimental side. In contrast, embryos cut between the notochord and the trunk paraxial mesoderm formed pronephric structures on both sides, regardless of the stage at which the operation was performed, indicating that the signal arises from the paraxial mesoderm (PM) and not from axial mesoderm. This cut also served as a control for cuts between the PM and the IM and showed that signaling itself was blocked in the former experiments, not the migration of pronephric or mesonephric precursor cells from the primitive streak. Additional control experiments ruled out the need for signals from lateral plate mesoderm, ectoderm, or endoderm. To determine whether the trunk paraxial mesoderm caudal to the fifth somite maintains its inductive capacity in the absence of contact with more rostral tissue, embryos were transected. Those transected below the prospective level of the fifth somite expressed Pax-2 in both the rostral and the caudal isolates, whereas embryos transected rostral to this level expressed Pax-2 in the caudal isolate only. Thus, a rostral signal is not required to establish the normal pattern of Pax-2 expression and pronephros formation. To determine whether paraxial mesoderm is sufficient for pronephros induction, stage 7 or earlier chick lateral plate mesoderm was cocultured with caudal stage 8 or 9 quail somites in collagen gels. Pax-2 was expressed in chick tissues in 21 of 25 embryos. Isochronic transplantation of stage 4 or 5 quail node into caudal chick primitive streak resulted in the generation of ectopic somites. These somites induced ectopic pronephroi in lateral plate mesoderm, and the IM that received signals from both native and ectopic somites formed enlarged pronephroi with increased Pax-2 expression. We conclude that signals from a localized region of the trunk paraxial mesoderm are both required and sufficient for the induction of the pronephros from the chick IM. Studies to identify the molecular nature of the induction are in progress.     

Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm

De novo assembly of myofibrils was investigated in explants of precardiac mesoderm from quail embryos to address a controversy about different models of myofibrillogenesis. The sequential expression of sarcomeric components was visualized in double- and triple-stained explants before, during, and just after the first cardiomyocytes began to beat. In explants from stage 6 embryos, cultured for 10 h, ectoderm, endoderm, and the precardiac mesoderm displayed arrays of stress fibers with alternating bands of the nonmuscle isoforms of alpha-actinin and myosin IIB. With increasing time in culture, mesoderm cells contained fibrils composed of actin, nonmuscle myosin IIB, and sarcomeric alpha-actinin. Several hours later, before beating occurred, both nonmuscle and muscle myosin II localized in some of the fibrils in the cells. Concentrations of muscle myosin began as thin bundles, dispersed in the cytoplasm, often overlapping one another, and progressed to small, aligned A-band-sized aggregates. The amount of nonmuscle myosin decreased dramatically when Z-bands formed, the muscle myosin became organized into A-bands, and the cells began beating. The sequential changes in protein composition of the fibrils in the developing muscle cells supports the model of myofibrillogenesis in which assembly begins with premyofibrils and progresses through nascent myofibrils to mature myofibrils.     

Well-defined growth factors promote cardiac development in axolotl mesodermal explants

The effect of growth factors on the formation of cardiac mesoderm in the urodele, Ambystoma mexicanum (axolotl), has been examined using an in vitro explant system. It has previously been shown that cardiac mesoderm is induced by pharyngeal endoderm during neurula stages in urodeles. In this study, explants of prospective cardiac mesoderm from early neurula stage embryos rarely formed beating cardiac tissue in culture. When transforming growth factor beta-1 (TGF-beta 1) or platelet-derived growth factor BB (PDGF) was added to such explants, the frequency of heart tissue formation increased markedly. The addition of other growth factors to these explants did not enhance cardiac mesoderm formation. The addition of basic fibroblast growth factor (bFGF) to prospective heart mesoderm derived from later stage embryos resulted in a decreased tendency to form cardiac tissue. These results suggest that growth factors analogous to TGF-beta 1, PDGF, and bFGF may regulate the initial stages of vertebrate cardiac development in vivo.     

Fate map of early avian cardiac progenitor cells

Cardiogenic fate maps are used to address questions on commitment, differentiation, morphogenesis and organogenesis of the heart. Recently, the accuracy of classical cardiogenic fate maps has been questioned, raising concerns about the conclusions drawn in studies based on these maps. We present accurate fate maps of the heart-forming region (HFR) in avian embryos and show that the putative cardiogenic molecular markers Bmp2 and Nkx2.5 do not govern the boundaries of the HFR as suggested in the literature. Moreover, this paper presents the first fate map of the HFR at stage 4 and addresses a void in the literature concerning rostrocaudal patterning of heart cells between stages 4 and 8.     

The effect of 5-bromodeoxyuridine (BrdU) on cardiac muscle differentiation

Cultured cardiac muscle cells undergo cell division and form beating progeny. Incorporation of BrdU into the nuclei of daughter cells does not suppress their ability to beat and form cross-striated myofibrils. Fluorescence microscopy of clones derived from single beating cells fed with BrdU-treated medium for over 2 weeks reveal cytoplasmic fibrils stainable with fluorescein-labeled antimyosin. The effect of BrdU on the emergence of cardiac muscle phenotype was also investigated by utilizing cardiac myogenic precursor cells from precardiac mesoderm in early embryos (stage 4–stage 9). These studies show that the cardiac myogenic cells fall into the following categories with respect to their ability to express the differentiated phenotype in the presence of BrdU: (1) precardiac mesodermal cells that are inhibited; (2) precardiac mesodermal cells that are not inhibited; and (3) beating cardiac muscle cells that are not inhibited. The entry of precardiac cells from the first category to the second and to the third appears to be unsynchronized.     

THE METACHRONAL WAVE OF LATERAL CILIA OF MYTILUS EDULIS

The form of beat of cilia and the structure of the metachronal wave on the lateral gill epithelium of Mytulus edulis have been studied on living material by interference-contrast microscopy and stroboscopic illumination, and compared with the same features in rapid-fixed preparations studied by light microscopy and with the scanning electron microscope. The most striking finding is that the beat of the cilia is not planar, as previously assumed, but involves a sideways movement in the recovery stroke Previous reports on nonplanar ciliary beating from protozoan examples describe a planar effective stroke and a counterclockwise rotation in the recovery stroke; in this molluscan example there is a clockwise rotation in the recovery stroke The lateral inclination of the cilia in the recovery stroke is in the same direction as the propagation of the waves, and the orientation of cilia in the recovery stroke is thought to determine whether the waves move to the left or right of the direction of the effective stroke     

Loss of late primitive streak mesoderm and interruption of left-right morphogenesis in the Ednrb(s-1Acrg) mutant mouse

This study characterizes defects associated with abnormal mesoderm development in mouse embryos homozygous for the induced Ednrb(s-1Acrg) allele of the piebald deletion complex. The Ednrb(s-1Acrg) deletion results in recessive embryonic lethality and mutant embryos exhibit a truncated posterior body axis. The primitive streak and node become disfigured, consistent with evidence that cell migration is impaired in newly formed mesoderm. Additional defects related to mesoderm development include notochord degeneration, somite malformations, and abnormal vascular development. Arrested heart looping morphogenesis and a randomized direction of embryonic turning indicate that left-right development is also perturbed. The expression of nodal and leftb, Tgf-beta-related genes involved in a left-determinant signaling pathway, is variably lost in the left lateral plate mesoderm. Mutational analysis has demonstrated that Fgf8 and Brachyury (T) are required for normal mesoderm and left-right development and the asymmetric expression of nodal and leftb. Fgf8 expression in nascent mesoderm exiting the primitive streak is dramatically reduced in mutant embryos, and diminished T expression accompanies the progressive loss of paraxial, lateral, and primitive streak mesoderm. In contrast, axial mesoderm persists and T and nodal appear to be appropriately expressed in their specific domains in the node and notochord. We propose that this mutation disrupts a morphogenetic pathway, likely involving FGF signaling, important for the development of streak-derived posterior mesoderm and lateral morphogenesis.     

BMP signaling positively regulates Nodal expression during left right specification in the chick embryo

Exogenous application of BMP to the lateral plate mesoderm (LPM) of chick embryos at the early somite stage had a positive effect on Nodal expression. BMP applications into the right LPM were followed by a rapid activation of Nodal, while applications into the left LPM resulted in expansion of the normal domain of Nodal expression. Conversely, blocking of BMP signaling by Noggin in the left LPM interfered with the activation of Nodal expression. These results support a positive role for endogenous BMP on Nodal expression in the LPM. We also report that BMP positively regulates the expression of Caronte, Snail and Cfc in both the left and right LPM. BMP-treated embryos had molecular impairment of the midline with downregulation of Lefty1, Brachyury and Shh but we also show that the midline defect was not sufficient to induce ectopic Nodal expression. We discuss our findings in the context of the known molecular control of the specification of left-right asymmetry.     

Development of left/right handedness in the chick heart.

The chick heart tube develops from the fusion of the right and left areas of precardiac mesoderm and in almost all cases loops to the embryo's right-hand side. We have investigated whether any intrinsic difference exists in the right and left areas of precardiac mesoderm, that influences the direction of looping of the heart tube. Chick embryos incubated to stages 4,5 and 6 were cultured by the New method. Areas of precardiac mesoderm were exchanged between donor and host embryos of the same stage and different stages to form control, double-right and double-left sided embryos. Overall, double-right sided embryos formed many more left-hand loops than double-left sided embryos. At stages 4 and 5 a small percentage of double-right embryos formed left-hand loops (13%) whereas at stage 6 almost 50% of hearts had left-hand loops. Control embryos formed right-hand loops in 97% of cases. The stability of right-hand heart looping by double-left sided embryos, may be related to the process of 'conversion', whereas the direction of looping by double-right sided embryos has become randomised. There is some indication that an intrinsic change occurred in the precardiac mesoderm between stages 5 and 6 that later influenced the direction of looping of the heart tube. The direction of body turning is suggested to be linked to the direction of heart looping.