A role of the cryptic gene in the correct establishment of the left-right axis

During vertebrate embryogenesis, a left-right axis is established. The heart, associated vessels and inner organs adopt asymmetric spatial arrangements and morphologies. Secreted growth factors of the TGF-beta family, including nodal, lefty-1 and lefty-2, play crucial roles in establishing left-right asymmetries [1] [2] [3]. In zebrafish, nodal signalling requires the presence of one-eyed pinhead (oep), a member of the EGF-CFC family of membrane-associated proteins [4]. We have generated a mutant allele of cryptic, a mouse EGF-CFC gene [5]. Homozygous cryptic mutants developed to birth, but the majority died during the first week of life because of complex cardiac malformations such as malpositioning of the great arteries, and atrial-ventricular septal defects. Moreover, laterality defects, including right isomerism of the lungs, right or left positioning of the stomach and splenic hypoplasia were observed. Nodal gene expression in the node was initiated in cryptic mutant mice, but neither nodal, lefty-2 nor Pitx2 were expressed in the left lateral plate mesoderm. The laterality defects observed in cryptic(-/-) mice resemble those of mice lacking the type IIB activin receptor or the homeobox-containing factor Pitx2 [6] [7] [8] [9], and are reminiscent of the human asplenic syndrome [10]. Our results provide genetic evidence for a role of cryptic in the signalling cascade that determines left-right asymmetry.     

Heart and gut chiralities are controlled independently from initial heart position in the developing zebrafish

A fundamental problem in developmental biology is how left-right (LR) asymmetry is generated, both on the whole organism level and at the level of an individual organ or structure. To investigate the relationship of organ sidedness to organ chirality, we examined 12 zebrafish mutants for initial heart tube position and later heart looping direction (chirality). Anomalous initial heart position was found in seven mutants, which also demonstrated loss of normal LR asymmetry in lateral plate mesoderm (LPM) antivin/lefty-1 and Pitx2 expression. Those with a relatively normal notochord (cyc(b16), din, and spt) displayed a predictive correlation between initial heart position and heart chirality, whereas initial heart position and heart chirality were independently randomized in those with a defective notochord (flh, boz, ntl, and mom). The predictability of heart chirality in spt, din, and b16 embryos, even in the absence of normal antivin/lefty-1 and Pitx2 expression, strongly suggests that heart chirality is controlled by a process distinct from that which controls appropriate left-sided LPM expression of antivin-Pitx2 signaling pathway molecules. In addition, there was correlation of initial heart position with gut chirality (and also between heart chirality and gut chirality) in the first class of mutants with normal notochord, but not in the second class, which appears to model human heterotaxy syndrome.     

Smad5 is essential for left-right asymmetry in mice

Left-right (L-R) asymmetry of the vertebrate body plan is established from an originally morphologically symmetric embryo. Recent studies have implicated several TGF-beta family signaling proteins (i.e., nodal, lefty-1, lefty-2, activin receptor type IIB, and Smad2) in L-R axis determination in the mouse. However, the genetic pathways underlying L-R patterning are still unclear. Smad5 is a downstream component in the TGF-beta family signaling cascade, and lack of Smad5 results in embryonic lethality between E9.5 and E11.5. In this report, we demonstrate that Smad5 mutant embryos have defects in heart looping and embryonic turning which are the first signs of L-R asymmetry in mice. To gain more insights into the molecular basis of the laterality defects in the Smad5-deficient embryos, we examined the expression of lefty-1, lefty-2, nodal, and Pitx2 since the asymmetric expression of these genes always closely correlates with the direction of heart looping and embryonic turning. In the absence of Smad5, lefty-1 was expressed at very low or undetectable levels, while nodal, lefty-2, and Pitx2 were expressed bilaterally. These data suggest that Smad5 is upstream of lefty-1, nodal, and lefty-2, and as a consequence also of Pitx2, and Smad5 is essential for L-R axis determination.     

Chick CFC controls Lefty1 expression in the embryonic midline and nodal expression in the lateral plate

Members of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left-right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger-Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left-right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory.     

Retinoic acid signaling sequentially controls visceral and heart laterality in zebrafish

During zebrafish development, the left-right (LR) asymmetric signals are first established around the Kupffer vesicle (KV), a ciliated organ generating directional fluid flow. Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm. Although numerous molecules and signaling pathways are involved in controlling LR asymmetry, mechanistic difference and concordance between different organs during LR patterning are poorly understood. Here we show that RA signaling regulates laterality decisions at two stages in zebrafish. Before the 2-somite stage (2So), inhibition of RA signaling leads to randomized visceral laterality through bilateral expression of nodal/spaw in the lateral plate mesoderm, which is mediated by increases in cilia length and defective directional fluid flow in KV. Fgf8 is required for the regulation of cilia length by RA signaling. Blockage of RA signaling before 2So also leads to mild defects of heart laterality, which become much more severe through perturbation of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. At this stage, visceral laterality and the left-sided Nodal remain unaffected. These findings suggest that RA signaling controls visceral laterality through the left-sided Nodal signal before 2So, and regulates heart laterality through cardiac bmp4 mainly after 2So, first identifying sequential control and concordance of visceral and heart laterality.     

Retinoic-acid signalling in node ectoderm and posterior neural plate directs left-right patterning of somitic mesoderm

Somitogenesis requires bilateral rhythmic segmentation of paraxial mesoderm along the antero-posterior axis. The location of somite segmentation depends on opposing signalling gradients of retinoic acid (generated by retinaldehyde dehydrogenase-2; Raldh2) anteriorly and fibroblast growth factor (FGF; generated by Fgf8) posteriorly. Retinoic-acid-deficient embryos exhibit somite left-right asymmetry, but it remains unclear how retinoic acid mediates left-right patterning. Here, we demonstrate that retinoic-acid signalling is uniform across the left-right axis and occurs in node ectoderm but not node mesoderm. In Raldh2(-/-) mouse embryos, ectodermal Fgf8 expression encroaches anteriorly into node ectoderm and neural plate, but its expression in presomitic mesoderm is initially unchanged. The late stages of somitogenesis were rescued in Raldh2(-/-) mouse embryos when the maternal diet was supplemented with retinoic acid until only the 6-somite stage, demonstrating that retinoic acid is only needed during node stages. A retinoic-acid-reporter transgene marking the action of maternal retinoic acid in rescued Raldh2(-/-) embryos revealed that the targets of retinoic-acid signalling during somitogenesis are the node ectoderm and the posterior neural plate, not the presomitic mesoderm. Our findings suggest that antagonism of Fgf8 expression by retinoic acid occurs in the ectoderm and that failure of this mechanism generates excessive FGF8 signalling to adjacent mesoderm, resulting initially in smaller somites and then left-right asymmetry.     

Xenopus Brachyury regulates mesodermal expression of Zic3, a gene controlling left-right asymmetry

The Brachyury gene has a critical role in the formation of posterior mesoderm and notochord in vertebrate development. A recent study showed that Brachyury is also responsible for the formation of the left-right (L-R) axis in mouse and zebrafish. However, the role of Brachyury in L-R axis specification is still elusive. Here, it is demonstrated that Brachyury is involved in L-R specification of the Xenopus laevis embryo and regulates expression of Zic3, which controls the L-R specification process. Overexpression of Xenopus Brachyury (Xbra) and dominant-negative type Xbra (Xbra-EnR) altered the orientation of heart and gut looping, concomitant with disturbed laterality of nodal-related 1 (Xnr1) and Pitx2 expression, both of which are normally expressed in the left lateral plate mesoderm. Furthermore, activation of inducible type Xbra (Xbra-GR) induces Zic3 expression within 20 min. These results suggest that a role of Brachyury in L-R specification may be the direct regulation of Zic3 expression.     

Rotation and asymmetric development of the zebrafish heart requires directed migration of cardiac progenitor cells

We have used high-resolution 4D imaging of cardiac progenitor cells (CPCs) in zebrafish to investigate the earliest left-right asymmetric movements during cardiac morphogenesis. Differential migratory behavior within the heart field was observed, resulting in a rotation of the heart tube. The leftward displacement and rotation of the tube requires hyaluronan synthase 2 expression within the CPCs. Furthermore, by reducing or ectopically activating BMP signaling or by implantation of BMP beads we could demonstrate that BMP signaling, which is asymmetrically activated in the lateral plate mesoderm and regulated by early left-right signals, is required to direct CPC migration and cardiac rotation. Together, these results support a model in which CPCs migrate toward a BMP source during development of the linear heart tube, providing a mechanism by which the left-right axis drives asymmetric development of the vertebrate heart.     

Maintenance of asymmetric nodal expression in Xenopus laevis

Vertebrate species display consistent left-right asymmetry in the arrangement of their internal organs. This asymmetry reflects the establishment of the left-right axis and the alignment of the organs along this axis during development. Members of the TGF-β family of molecules have been implicated in both the establishment and signaling of left-right axis information. Asymmetric expression of one member, nodal (called Xnr-1 in the frog, Xenopus laevis), is highly conserved among species. The nodal-related genes are normally expressed in the left lateral plate mesoderm prior to the development of morphologic asymmetry. Expression patterns of nodal have been correlated with heart situs in mouse, chick, and frog and our previous work has implicated the dorsal midline structures in the regulation of nodal expression and cardiac laterality. In this study, three approaches were used to address the embryologic and molecular basis of asymmetric Xnr-1 expression. First, notochord and lateral plate recombinants were performed and showed that notochord can repress Xnr-1 expression in lateral plate mesoderm explants derived from either the left or the right side. Second, lateral plate mesoderm grafts indicated that Xnr-1 expression is specified but not determined at neurula stages and can subsequently be repatterned. These experiments suggest that a repressive signal from the notochord is required for maintenance of asymmetric Xnr-1 expression and that Xnr-1 expression is regulated by signals outside of the lateral plate mesoderm. Third, candidate molecules were injected to test for their ability to alter Xnr-1 expression pattern in the lateral plate. Late injection of activin protein on the right side of the embryo induced ectopic Xnr-1 expression and randomized cardiac orientation. This suggests that activin or a related TGF-β molecule is involved in the proximal regulation of asymmetric Xnr-1 expression. Dev. Genet. 23:194–202, 1998. © 1998 Wiley-Liss, Inc.     

The role of the dorsal lip in the induction of heart mesoderm in Xenopus laevis

We have examined the tissue interactions responsible for the expression of heart-forming potency during gastrulation. By comparing the specification of different regions of the marginal zone, we show that heart-forming potency is expressed only in explants containing both the dorsal lip of the blastopore and deep mesoderm between 30 degrees and 45 degrees lateral to the dorsal midline. Embryos from which both of these 30 degrees-45 degrees dorsolateral regions have been removed undergo heart formation in two thirds of cases, as long as the dorsal lip is left intact. If the dorsal lip is removed along with the 30 degrees-45 degrees regions, heart formation does not occur. These results indicate that the dorsolateral deep mesoderm must interact with the dorsal lip in order to express heart-forming potency. Transplantation of the dorsal lip into the ventral marginal zone of host embryos results in the formation of a secondary axis; in over half of cases, this secondary axis includes a heart derived from the host mesoderm. These findings suggest that the establishment of heart mesoderm is initiated by a dorsalizing signal from the dorsal lip of the blastopore.     

XCR2, one of three Xenopus EGF-CFC genes, has a distinct role in the regulation of left-right patterning

Members of the EGF-CFC family facilitate signaling by a subset of TGFbeta superfamily ligands that includes the nodal-related factors and GDF1/VG1. Studies in mouse, zebrafish, and chick point to an essential role for EGF-CFC proteins in the action of nodal/GDF1 signals in the early establishment of the mesendoderm and later visceral left-right patterning. Antisense knockdown of the only known frog EGF-CFC factor (FRL1), however, has argued against an essential role for this factor in nodal/GDF1 signaling. To address this apparent paradox, we have identified two additional Xenopus EGF-CFC family members. The three Xenopus EGF-CFC factors show distinct patterns of expression. We have examined the role of XCR2, the only Xenopus EGF-CFC factor expressed in post-gastrula embryos, in embryogenesis. Antisense morpholino oligonucleotide-mediated depletion of XCR2 disrupts left-right asymmetry of the heart and gut. Although XCR2 is expressed bilaterally at neurula stage, XCR2 is required on the left side, but not the right side, for normal left-right patterning. Left-side expression of XNR1 in the lateral plate mesoderm depends on XCR2, whereas posterior bilateral expression of XNR1 does not, suggesting that distinct mechanisms maintain XNR1 expression in different regions of neurula-tailbud embryos. Ectopic XCR2 on the right side initiates premature right-side expression of XNR1 and XATV, and can reverse visceral patterning. This activity of XCR2 depends on its co-receptor function. These observations indicate that XCR2 has a crucial limiting role in maintaining a bistable asymmetry in nodal family signaling across the left-right axis.     

The Oak Ridge Polycystic Kidney (orpk) disease gene is required for left-right axis determination

Analysis of several mutations in the mouse is providing useful insights into the nature of the genes required for the establishment of the left-right axis during early development. Here we describe a new targeted allele of the mouse Tg737 gene, Tg737(Delta)2-3(beta)Gal), which causes defects in left-right asymmetry and other abnormalities during embryogenesis. The Tg737 gene was originally identified based on its association with the mouse Oak Ridge Polycystic Kidney (orpk) insertional mutation, which causes polycystic kidney disease and other defects. Complementation tests between the original orpk mutation and the new targeted knock-out mutation demonstrate that Tg737(Delta)2-3(beta)Gal) behaves as an allele of Tg737. The differences in the phenotype between the two mutations suggest that the orpk mutation is a hypomorphic allele of the Tg737 gene. Unlike the orpk allele, where all homozygotes survive to birth, embryos homozygous for the Tg737(Delta)2-3(beta)Gal) mutation arrest in development at mid-gestation and exhibit neural tube defects, enlargement of the pericardial sac and, most notably, left-right asymmetry defects. At mid-gestation the direction of heart looping is randomized, and at earlier stages in development lefty-2 and nodal, which are normally expressed asymmetrically, exhibit symmetrical expression in the mutant embryos. Additionally, we determined that the ventral node cells in mutant embryos fail to express the central cilium, which is a characteristic and potentially functional feature of these cells. The expression of both Shh and Hnf3(beta) is downregulated in the midline at E8.0, indicating that there are significant alterations in midline development in the Tg737(Delta)2-3(beta)Gal) homozygous embryos. We propose that the failure of ventral node cells to fully mature alters their ability to undergo differentiation as they migrate out of the node to contribute to the developing midline structures. Analysis of this new knockout allele allows us to define a critical role for the Tg737 gene during early embryogenesis. We have named the product of the Tg737 gene Polaris, which is based on the various polarity related defects associated with the different alleles of the Tg737 gene.     

Zic3 is involved in the left-right specification of the Xenopus embryo

Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.     

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.     

Defective Nodal and Cerl2 expression in the Arl13b(hnn) mutant node underlie its heterotaxia

Specification of the left-right axis during embryonic development is critical for the morphogenesis of asymmetric organs such as the heart, lungs, and stomach. The first known left-right asymmetry to occur in the mouse embryo is a leftward fluid flow in the node that is created by rotating cilia on the node surface. This flow is followed by asymmetric expression of Nodal and its inhibitor Cerl2 in the node. Defects in cilia and/or fluid flow in the node lead to defective Nodal and Cerl2 expression and therefore incorrect visceral organ situs. Here we show the cilia protein Arl13b is required for left right axis specification as its absence results in heterotaxia. We find the defect originates in the node where Cerl2 is not downregulated and asymmetric expression of Nodal is not maintained resulting in symmetric expression of both genes. Subsequently, Nodal expression is delayed in the lateral plate mesoderm (LPM). Symmetric Nodal and Cerl2 in the node could result from defects in either the generation and/ or the detection of Nodal flow, which would account for the subsequent defects in the LPM and organ positioning.     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.