Adrenergic neurotransmitters and calcium ionophore-induced situs inversus viscerum in Xenopus laevis embryos

Xenopus laevis embryos at the blastula–early tail bud stage were exposed to norepinephrine or octopamine dissolved in culture saline until they reached the larval stage. The left–right asymmetry of the heart and gut was then examined. We found that these adrenergic neurotransmitters induced situs inversus in the heart and/or gut in up to 35% of tested neurula embryos. Norepinephrine-induced situs inversus was blocked by the α-1 adrenergic antagonist prazosin. Furthermore, A23187, a calcium ionophore, also increased the incidence of situs inversus up to 54% when late-neurula embryos were exposed to the solution. A23187 treatment initiated before neural groove formation was less effective. The incidence of situs inversus induced by these reagents decreased towards the control level (2.2%, 25 untreated embryos out of 1127 embryos in total) in embryos past the stage of neural tube closure. In the present experiments we obtained 22 gut-only situs inversus embryos having an inverted gut and a normal heart. In contrast, such embryos were not observed among the 1127 untreated embryos. An adrenergic signal mediated by an increase in intracellular free calcium may be involved in the asymmetrical visceral morphogenesis of Xenopus embryos.     

Calmodulin binds to inv protein: Implication for the regulation of inv function

Establishment of the left-right asymmetry of internal organs is essential for the normal development of vertebrates. The inv mutant in mice shows a constant reversal of left-right asymmetry and although the inv gene has been cloned, its biochemical and cell biological functions have not been defined. Here, we show that calmodulin binds to mouse inv protein at two sites (IQ1 and IQ2). The binding of calmodulin to the IQ2 site occurs in the absence of Ca(2+) and is not observed in the presence of Ca(2+). Injection of mouse inv mRNA into the right blastomere of Xenopus embryos at the two-cell stage randomized the left-right asymmetry of the embryo and altered the patterns of Xnr-1 and Pitx2 expression. Importantly, inv mRNA that lacked the region encoding the IQ2 site was unable to randomize left-right asymmetry in Xenopus embryos, implying that the IQ2 site is essential for inv to randomize left-right asymmetry in Xenopus. These results suggest that calmodulin binding may regulate inv function. Based on our findings, we propose a model for the regulation of inv function by calcium-calmodulin and discuss its implications.     

Development of transgenic Xenopus laevis with a high C-src gene expression

Xenopus laevis larvae with an elevated expression of c-src were generated by mating a transgenic X. laevis male frog carrying proviral Rous sarcoma virus (RSV) long terminal repeat (LTR) and most of the pol gene sequences in its sperm DNA and a normal X. laevis female frog. Offspring (15–20%) with a higher dosage of c-Src, detected in disorganized myotomal musculature and in cerebral and spinal neuronal cells by immunohistochemical analysis, developed abnormally, with edemas (in most cases), head deformities, and eye and axial system defects. In the remaining embryos, a small increase in c-src expression seemed to be compatible with normal embryogenesis. The dosage of c-Src correlated with the dosage of RSV LTR integrated in frog DNA as revealed by Southern and polymerase chain reaction (PCR) analyses. Authenticity of the integrated RSV LTR including enhancer sequence was proved by sequencing. Probing of total RNA from aberrant larvae demonstrated several times higher dosage of c-src mRNA in their tissues than in control tadpoles. We hypothesize that the integrated RSV regulatory sequences can stimulate the expression of c-src proto-oncogene of X. laevis above a treshold that interferes with the early developmental program of frog embryos. Mol. Reprod. Dev. 50:410–419, 1998. © 1998 Wiley-Liss, Inc.     

Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.     

Anteriorward shifting of asymmetric Xnr1 expression and contralateral communication in left-right specification in Xenopus

Transient asymmetric Nodal signaling in the left lateral plate mesoderm (L LPM) during tailbud/early somitogenesis stages is associated in all vertebrates examined with the development of stereotypical left-right (L-R) organ asymmetry. In Xenopus, asymmetric expression of Nodal-related 1 (Xnr1) begins in the posterior L LPM shortly after the initiation of bilateral perinotochordal expression in the posterior tailbud. The L LPM expression domain rapidly shifts forward to cover much of the flank of the embryo before being progressively downregulated, also in a posterior-to-anterior direction. The mechanisms underlying the initiation and propagation of Nodal/Xnr1 expression in the L LPM, and its transient nature, are not well understood. Removing the posterior tailbud domain prevents Xnr1 expression in the L LPM, consistent with the idea that normal embryos respond to a posteriorly derived asymmetrically acting positive inductive signal. The forward propagation of asymmetric Xnr1 expression occurs LPM-autonomously via planar tissue communication. The shifting is prevented by Nodal signaling inhibitors, implicating an underlying requirement for Xnr1-to-Xnr1 induction. It is also unclear how asymmetric Nodal signals are modulated during L-R patterning. Small LPM grafts overexpressing Xnr1 placed into the R LPM of tailbud embryos induced the expression of the normally L-sided genes Xnr1, Xlefty, and XPitx2, and inverted body situs, demonstrating the late-stage plasticity of the LPM. Orthogonal Xnr1 signaling from the LPM strongly induced Xlefty expression in the midline, consistent with recent findings in the mouse and demonstrating for the first time in another species conservation in the mechanism that induces and maintains the midline barrier. Our findings suggest that there is long-range contralateral communication between L and R LPM, involving Xlefty in the midline, over a substantial period of tailbud embryogenesis, and therefore lend further insight into how, and for how long, the midline maintains a L versus R status in the LPM.     

Calcium transients triggered by planar signals induce the expression of ZIC3 gene during neural induction in Xenopus

In intact Xenopus embryos, an increase in intracellular Ca(2+) in the dorsal ectoderm is both necessary and sufficient to commit the ectoderm to a neural fate. However, the relationship between this Ca(2+) increase and the expression of early neural genes is as yet unknown. In intact embryos, studying the interaction between Ca(2+) signaling and gene expression during neural induction is complicated by the fact that the dorsal ectoderm receives both planar and vertical signals from the mesoderm. The experimental system may be simplified by using Keller open-face explants where vertical signals are eliminated, thus allowing the interaction between planar signals, Ca(2+) transients, and neural induction to be explored. We have imaged Ca(2+) dynamics during neural induction in open-face explants by using aequorin. Planar signals generated by the mesoderm induced localized Ca(2+) transients in groups of cells in the ectoderm. These transients resulted from the activation of L-type Ca(2+) channels. The accumulated Ca(2+) pattern correlated with the expression of the early neural precursor gene, Zic3. When the transients were blocked with pharmacological agents, the level of Zic3 expression was dramatically reduced. These data indicate that, in open-face explants, planar signals reproduce Ca(2+) -signaling patterns similar to those observed in the dorsal ectoderm of intact embryos and that the accumulated effect of the localized Ca(2+) transients over time may play a role in controlling the expression pattern of Zic3.     

Ciliation and gene expression distinguish between node and posterior notochord in the mammalian embryo

The mammalian node, the functional equivalent of the frog dorsal blastoporal lip (Spemann's organizer), was originally described by Viktor Hensen in 1876 in the rabbit embryo as a mass of cells at the anterior end of the primitive streak. Today, the term "node" is commonly used to describe a bilaminar epithelial groove presenting itself as an indentation or "pit" at the distal tip of the mouse egg cylinder, and cilia on its ventral side are held responsible for molecular laterality (left-right) determination. We find that Hensen's node in the rabbit is devoid of cilia, and that ciliated cells are restricted to the notochordal plate, which emerges from the node rostrally. In a comparative approach, we use the organizer marker gene Goosecoid (Gsc) to show that a region of densely packed epithelium-like cells at the anterior end of the primitive streak represents the node in mouse and rabbit and is covered ventrally by a hypoblast (termed "visceral endoderm" in the mouse). Expression of Nodal, a gene intricately involved in the determination of vertebrate laterality, delineates the wide plate-like posterior segment of the notochord in the rabbit and mouse, which in the latter is represented by the indentation frequently termed "the node." Similarly characteristic ciliation and nodal expression exists in Xenopus neurula embryos in the gastrocoel roof plate (GRP), i.e., at the posterior end of the notochord anterior to the blastoporal lip. Our data suggest that (1) a posterior segment of the notochord, here termed PNC (for posterior notochord), is characterized by features known to be involved in laterality determination, (2) the GRP in Xenopus is equivalent to the mammalian PNC, and (3) the mammalian node as defined by organizer gene expression is devoid of cilia and most likely not directly involved in laterality determination.     

Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region

In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.     

Developmental expression of Xenopus myosin 1d and identification of a myo1d tail homology that overlaps TH1

Xenopus laevis myosin 1d (XlMyo1d) is a member of the myosin I class, subclass 4. Members of this class are single headed, bind calmodulin light chains and have lipid binding domains in their tails. The rat myo1d homologue has been implicated in endosome vesicle recycling in epithelial cells. Mutations in the Drosophila myosin 1d homologue cause situs inversus in the abdomen. The XlMyo1d cDNA has been cloned and the derived amino acid sequence is 80% identical to the rat and human homologues. Sequence comparison revealed a novel isoform‐specific tail homology embedded in the Tail Homology 1 (TH1) domain characteristic of myosin I isoforms. Western blot analysis using a polyclonal antibody raised against an isoform‐specific peptide showed that the protein is present in eggs and levels increase at early neurula through tadpole stages. Whole mount in situ hybridization using a probe containing the 5′UTR (untranslated region) showed that XlMyo1d mRNA is expressed in neural tube, pre‐somitic mesoderm, somites and all three segments of cranial neural crest cells during their migration. Sections of the in situ hybridizations revealed that during somitogenesis, XlMyo1d mRNA was localized to a stripe overlapping the nuclear region of somites during early tadpole stages.     

IP3 signaling is required for cilia formation and left-right body axis determination in Xenopus embryos

Vertebrate left–right (LR) body axis is manifested as an asymmetrical alignment of the internal organs such as the heart and the gut. It has been proposed that the process of LR determination commonly involves a cilia-driven leftward flow in the mammalian node and its equivalents (Kupffer’s vesicle in zebrafish and the gastrocoel roof plate in Xenopus). Recently, it was reported that Ca²⁺ flux regulates Kupffer’s vesicle development and is required for LR determination. As a basis of Ca²⁺ flux in many cell types, inositol 1,4,5-trisphosphate (IP₃) receptor-mediated calcium release from the endoplasmic reticulum (ER) plays important roles. However, its involvement in LR determination is poorly understood. We investigated the role of IP₃ signaling in LR determination in Xenopus embryos. Microinjection of an IP₃ receptor-function blocking antibody that can inhibit IP₃ calcium channel activity randomized the LR axis in terms of left-sided Pitx2 expression and organ laterality. In addition, an IP₃ sponge that could inhibit IP₃ signaling by binding IP₃ more strongly than the IP₃ receptor impaired LR determination. Examination of the gastrocoel roof plate revealed that the number of cilia was significantly reduced by IP₃ signal blocking. These results provide evidence that IP₃ signaling is involved in LR asymmetry formation in vertebrates.