Multiple pathways in the midline regulate concordant brain, heart and gut left-right asymmetry.

The embryonic midline in vertebrates has been implicated in left-right development, but the mechanisms by which it regulates left-right asymmetric gene expression and organ morphogenesis are unknown. Zebrafish embryos have three domains of left-right asymmetric gene expression that are useful predictors of organ situs. cyclops (nodal), lefty1 and pitx2 are expressed in the left diencephalon; cyclops, lefty2 and pitx2 are expressed in the left heart field; and cyclops and pitx2 are expressed in the left gut primordium. Distinct alterations of these expression patterns in zebrafish midline mutants identify four phenotypic classes that have different degrees of discordance among the brain, heart and gut. These classes help identify two midline domains and several genetic pathways that regulate left-right development. A cyclops-dependent midline domain, associated with the prechordal plate, regulates brain asymmetry but is dispensable for normal heart and gut left-right development. A second midline domain, associated with the anterior notochord, is dependent on no tail, floating head and momo function and is essential for restricting asymmetric gene expression to the left side. Mutants in spadetail or chordino give discordant gene expression among the brain, heart and gut. one-eyed pinhead and schmalspur are necessary for asymmetric gene expression and may mediate signaling from midline domains to lateral tissues. The different phenotypic classes help clarify the apparent disparity of mechanisms proposed to explain left-right development in different vertebrates.

Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling.

The Lefty subfamily of TGFbeta signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left-right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left-right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFbeta family during left-right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFbeta family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left-right morphogenesis of the gut and heart.

Mesendoderm and left-right brain, heart and gut development are differentially regulated by pitx2 isoforms.

The pitx2 gene is a member of the bicoid-homeodomain class of transcription factors that has been implicated in the control of left-right asymmetry during organogenesis. Here we demonstrate that in zebrafish there are two pitx2 isoforms, pitx2a and pitx2c, which show distinct expression patterns and have non-overlapping functions during mesendoderm and asymmetric organ development. pitx2c is expressed symmetrically in presumptive mesendoderm during late blastula stages and in the prechordal plate during late gastrulation. pitx2a expression is first detected at bud stage in the anterior prechordal plate. The regulation of early mesendoderm pitx2c expression is dependent on one-eyed pinhead (EGF-CFC-related gene) and spadetail (tbx-transcription factor) and can be induced by ectopic goosecoid expression. Maintenance of pitx2c midline expression is dependent on cyclops (nodal) and schmalspur, but not no tail (brachyury). Ectopic expression of pitx2 isoforms results in distinct morphological and molecular phenotypes, indicating that pitx2a and pitx2c have divergent regulatory functions. Both isoforms downregulate goosecoid on the dorsal side, but in contrast to earlier reports that nodal and lefty are upstream of pitx2, ectopic pitx2c in other regions induces cyclops, lefty2 and goosecoid expression. Asymmetric isoform expression occurs in non-overlapping domains, with pitx2c in left dorsal diencephalon and developing gut and pitx2a in left heart primordium. Targeted asymmetric expression in Xenopus shows that both isoforms can alter left-right development, but pitx2a has a slightly stronger effect on heart laterality. Our results indicate that distinct genetic pathways regulate pitx2a and pitx2c isoform expression, and each isoform regulates different downstream pathways during mesendoderm and asymmetric organ development.

Overexpression of thyroid hormone receptor alpha 1 during zebrafish embryogenesis disrupts hindbrain patterning and implicates retinoic acid receptors in the control of hox gene expression.

Nuclear receptors play key roles in anterior/posterior (A/P) axis formation during vertebrate embryogenesis. Within this gene family, retinoic acid receptors and retinoic acid itself have profound influences on the establishment of the A/P axis. Thyroid hormone receptors are expressed during early periods of development, long before the establishment of the thyroid gland, and are able to interact with retinoic acid receptors. Here we examined the ability of the thyroid hormone receptor alpha 1 to affect early embryonic development by mRNA injection of either repressor or activator forms of the thyroid hormone receptor. Overexpression of either the thyroid hormone receptor alpha 1 or a constitutive repressor form, v-erbA, caused a swelling in the rostral hindbrain. These defects were associated with disorganization and loss of rhombomere borders as well as an increase in the number of acetylcholine esterase positive cells. This phenotype correlated with a reduction in hoxa1 expression during gastrulation. Furthermore, injection of either thyroid hormone receptor alpha 1 or v-erbA mRNA repressed a reporter gene that contained a retinoic acid response element, demonstrating the ability of the thyroid hormone receptor alpha 1 to repress retinoic acid signaling during gastrulation. In contrast, embryos treated with retinoic acid alone or embryos injected with thyroid hormone receptor alpha 1 and treated with the thyroid hormone analog TRIAC displayed a similar set of defects, including loss of the midbrain-hindbrain border and severe disruption of the rostral hindbrain. These studies support the involvement of retinoic acid and its receptors in the direct control of Hox gene expression and the early patterning of the zebrafish central nervous system.

Regulation of midline development by antagonism of lefty and nodal signaling.

The embryonic midline is crucial for the development of embryonic pattern including bilateral symmetry and left-right asymmetry. In zebrafish, lefty1 (lft1) and lefty2 (lft2) have distinct midline expression domains along the anteroposterior axis that overlap with the expression patterns of the nodal-related genes cyclops and squint. Altered expression patterns of lft1 and lft2 in zebrafish mutants that affect midline development suggests different upstream pathways regulate each expression domain. Ectopic expression analysis demonstrates that a balance of lefty and cyclops signaling is required for normal mesendoderm patterning and goosecoid, no tail and pitx2 expression. In late somite-stage embryos, lft1 and lft2 are expressed asymmetrically in the left diencephalon and left lateral plate respectively, suggesting an additional role in laterality development. A model is proposed by which the vertebrate midline, and thus bilateral symmetry, is established and maintained by antagonistic interactions among co-expressed members of the lefty and nodal subfamilies of TGF-beta signaling molecules.

The zebrafish thyroid hormone receptor alpha 1 is expressed during early embryogenesis and can function in transcriptional repression.

Nuclear receptors are a large family of ligand dependent transcription factors which participate in many diverse processes during development. In this report, we describe the cloning of the zebrafish thyroid hormone receptor alpha 1 (TR alpha 1) gene, the cellular counterpart of the viral oncogene v-erbA. TR alpha 1 is expressed during oogenesis and maternally supplied to the embryo. TR alpha 1 is expressed again after the mid blastula transition. By examining the effects of increased expression of TR alpha 1 on expression of a reporter gene which responds to both TR alpha 1 and retinoic acid receptors (RARs), we show that the zebrafish TR alpha 1 can act as a repressor during early zebrafish development before thyroid hormone is present in the embryo. In addition, our data suggest that TR alpha 1 can repress retinoic acid (RA)-signaling during early development. We propose that TR alpha 1 functions during early development as a transcriptional repressor, similar to the constitutive repressor activity of its viral counterpart v-erbA, which regulates anterior-posterior (A/P) patterning by repressing RA-signaling.

Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos.

Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.