Biased left-right asymmetry is a fascinating and medically important phenomenon. We provide molecular genetic and physiological characterization of a novel, conserved, early, biophysical event that is crucial for correct asymmetry: H + flux. A pharmacological screen implicated the H + -pump H + -V-ATPase in Xenopus asymmetry, where it acts upstream of early asymmetric markers. Immunohistochemistry revealed an actin-dependent asymmetry of H + -V-ATPase subunits during the first three cleavages. H + -flux across plasma membranes is also asymmetric at the four-and eight-cell stages, and this asymmetry requires H + -V-ATPase activity. Abolishing the asymmetry in H + flux, using a dominant-negative subunit of the H + -V-ATPase or an ectopic H + pump, randomized embryonic situs without causing any other defects. To understand the mechanism of action of H + -V-ATPase, we isolated its two physiological functions, cytoplasmic pH and membrane voltage (V mem ) regulation. Varying either pH or V mem , independently of direct manipulation of H + -V-ATPase, caused disruptions of normal asymmetry, suggesting roles for both functions. V-ATPase inhibition also abolished the normal early localization of serotonin, functionally linking these two early asymmetry pathways. The involvement of H + -V-ATPase in asymmetry is conserved to chick and zebrafish. Inhibition of the H + -V-ATPase induces heterotaxia in both species; in chick, H + -V-ATPase activity is upstream of Shh; in fish, it is upstream of Kupffer's vesicle and Spaw expression. Our data implicate H + -V-ATPase activity in patterning the LR axis of vertebrates and reveal mechanisms upstream and downstream of its activity. We propose a pH-and V mem -dependent model of the early physiology of LR patterning. Development 133, 1657Development 133, -1671Development 133, (2006 DEVELOPMENT 1658 necessary to characterize the endogenous behavior of the relevant pumps in embryos and to place their function in the context of known LR patterning mechanisms. Here, we explore the properties of H + -V-ATPase function in several vertebrate embryos. Through endogenous localization of the H + -V-ATPase and gain-and loss-offunction experiments in chick, frog and zebrafish, we identify the H + -V-ATPase as a novel, conserved, obligate component of LR patterning upstream of asymmetric gene expression. KEY WORDS: Left-right asymmetry, H + -V-ATPase, V-ATPase, Xenopus, Chick, Zebrafish, Axial patterning, Cytoplasmic pH, Membrane voltage MATERIALS AND METHODS Animal husbandryXenopus embryos were collected according to standard protocols (Sive et al., 2000) in 0.1ϫ Modified Marc's Ringers (MMR) pH 7.8 + 0.1% Gentamicin. Xenopus embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Chick embryos from Charles River Laboratories, maintained at 38°C, were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1992). Zebrafish embryos (Westerfield, 1995) were maintained at 28.5°C in water containing 1 drop per gallon Methyl Blue. Assaying organ situsXenopus e...

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Abstract-Forward genetic screens in zebrafish have been used to identify mutations in genes with important roles in organogenesis. One of these mutants, small heart, develops a diminutive and severely malformed heart and multiple developmental defects of the brain, ears, eyes, and kidneys. Using a positional cloning approach, we identify that the mutant gene encodes the zebrafish Na ϩ /K ϩ -ATPase ␣1B1 protein. Disruption of Na ϩ /K ϩ -ATPase ␣1B1 function via morpholino "knockdown" or pharmacological inhibition with ouabain phenocopies the mutant phenotype, in a dose-dependent manner. Heterozygosity for the mutation sensitizes embryos to ouabain treatment. Our findings present novel genetic and morphological details on the function of the Na ϩ /K ϩ -ATPase ␣1B1 in early cardiac morphogenesis and the pathogenesis of the small heart malformation. We demonstrate that the reduced size of the mutant heart is caused by dysmorphic ventricular cardiomyocytes and an increase in ventricular cardiomyocyte apoptosis. This study provides a new insight that Na

The notochord normally arises from committed cells in the rostral tip of the primitive streak. After removal of these cells from the avian gastrula, embryos with notochords nevertheless develop in the majority of cases. A region required for the formation of this reconstituted notochord lies lateral to the primitive streak. In the present study we have determined that this region acts as an inducer for more lateral cells in the epiblast, which actually give rise to the reconstituted notochord. The strongest inducing region lies between 0-250 micrometer lateral to the streak and 500-750 micrometer caudal to the rostral end of the streak and chiefly contains cells normally fated to form lateral plate and somitic mesoderm. The responding region is located 250-500 micrometer lateral to the streak and 0-750 micrometer caudal to the rostral end of the streak. This area chiefly contains cells normally fated to form neural ectoderm, although cells normally fated to form lateral plate and somitic mesoderm are also within this area. The inducing and responding areas interact to form reconstituted notochord either when the primitive streak, including its rostral end (Hensen's node), is removed from the cultured blastoderm or when the inducer and responder are grafted together into an ectopic site. Grafting Hensen's node into isolates containing both inducer and responder blocks formation of reconstituted notochord, suggesting that Hensen's node suppresses formation of lateral notochords during normal development. These findings increase our understanding of the early interactions between mesoderm and ectoderm and provide a novel model system that is well defined and accessible for studying inductive events in higher vertebrates.

C-Lmx1 has been shown to be a key regulatory gene for specification of dorsoventral pattern during vertebrate limb development. Here, we describe its earlier pattern of expression during and shortly after neurulation. Transcripts are first expressed in the mesoderm of the head process and rostral tip of the primitive streak at the late gastrula/early neurula stage (stage 4). As neurulation occurs with shaping of the neural plate, C-Lmx1 is expressed in a butterfly-like pattern in the lateral neuroectoderm. During bending of the neural plate, C-Lmx1 expression becomes localized to three areas of the bending neuroectoderm: the median hingepoint (future floor plate of the neural tube) and the paired dorsolateral regions of the neuroepithelium, including the dorsolateral hingepoints and the adjacent neuroectodermal and epidermal ectodermal components of the neural folds. After closure of the neural groove and formation of the primary brain vesicles, C-Lmx1 is expressed in the dorsal neural tube along the entire length of the neuraxis, as well as in the floor plate at the brain but not spinal cord levels. At the midbrain and rostral hindbrain levels, C-Lmx1 is heavily expressed. Here, in addition to expression in the dorsal neural tube and floor plate, it is expressed in the lateral walls of the neural tube, with the exception of the levels of rhombomeres 2 and 4. C-Lmx1 is also expressed in several other discrete domains during and shortly after neurulation, including the prechordal plate and rostral head mesenchyme, foregut endoderm, otic placode and vesicle, dorsal somitic mesoderm, midline endoderm at the level of the caudal spinal cord, mesonephroi and limb bud mesoderm.

The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 microns and a mediolateral extent of less than 250 microns, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate.

The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm.