Diffusion of nodal signaling activity in the absence of the feedback inhibitor Lefty2.

The role of Lefty2 in left-right patterning was investigated by analysis of mutant mice that lack asymmetric expression of lefty2. These animals exhibited various situs defects including left isomerism. The asymmetric expression of nodal was prolonged and the expression of Pitx2 was upregulated in the mutant embryos. The absence of Lefty2 conferred on Nodal the ability to diffuse over a long distance. Thus, Nodal-responsive genes, including Pitx2, that are normally expressed on the left side were expressed bilaterally in the mutant embryos, even though nodal expression was confined to the left side. These results suggest that Nodal is a long-range signaling molecule but that its range of action is normally limited by the feedback inhibitor Lefty2.

Role of asymmetric signals in left-right patterning in the mouse.

Left-right asymmetric signaling molecules in mammals include three transforming growth factor beta (TGFbeta)-related factors, Nodal, Lefty1 and Lefty2. They are all expressed on the left half of developing mouse embryos. Nodal acts as a left-side determinant by transducing signals through Smad and FAST and by inducing Pitx2 expression on the left side. Lefty proteins are antagonists that inhibit Nodal signaling. There are positive and negative transcriptional regulatory loops between nodal and lefty2 genes. Thus, Nodal activates its own gene and lefty2. Lefty2 protein produced then inhibits Nodal signaling and terminates expression of both genes. This feedback mechanism can restrict the range and duration of Nodal signaling in developing embryos.

The transcription factor FoxH1 (FAST) mediates Nodal signaling during anterior-posterior patterning and node formation in the mouse.

FoxH1 (FAST) is a transcription factor that mediates signaling by transforming growth factor-beta, Activin, and Nodal. The role of FoxH1 in development has now been investigated by the generation and analysis of FoxH1-deficient (FoxH1(-/-)) mice. The FoxH1(-/-) embryos showed various patterning defects that recapitulate most of the defects induced by the loss of Nodal signaling. A substantial proportion of FoxH1(-/-) embryos failed to orient the anterior-posterior (A-P) axis correctly, as do mice lacking Cripto, a coreceptor for Nodal. In less severely affected FoxH1(-/-) embryos, A-P polarity was established, but the primitive streak failed to elongate, resulting in the lack of a definitive node and its derivatives. Heterozygosity for nodal renders the FoxH1(-/-) phenotype more severe, indicative of a genetic interaction between FoxH1 and nodal. The expression of FoxH1 in the primitive endoderm rescued the A-P patterning defects, but not the midline defects, of FoxH1(-/-) mice. These results indicate that a Nodal-FoxH1 signaling pathway plays a central role in A-P patterning and node formation in the mouse.

The retinoic acid-inactivating enzyme CYP26 is essential for establishing an uneven distribution of retinoic acid along the anterio-posterior axis within the mouse embryo.

Retinoic acid (RA), a derivative of vitamin A, plays a pivotal role in vertebrate development. The level of RA may be determined by the balance between its synthesis and degradation. We have examined the role of CYP26, a P450 enzyme that may degrade RA, by generating mutant mice that lack CYP26. CYP26(-/-) mice exhibited anomalies, including caudal agenesis, similar to those induced by administration of excess RA. The concentration of endogenous RA, as revealed by marker gene activity, was markedly increased in the tailbud of the mutant animals, in which CYP26 is normally expressed. Expression of T (Brachyury) and Wnt3a in the tailbud was down-regulated in CYP26(-/-) mice, which may underlie the caudal truncation. The lack of CYP26 also resulted in homeotic transformation of vertebrae as well as in misspecification of the rostral hindbrain associated with anterior expansion of RA-positive domains. These results suggest that local degradation of RA by CYP26 is required for establishing an uneven distribution of RA along the anterio-posterior axis, which is essential for patterning the hindbrain, vertebrae, and tailbud.

Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.

GFR alpha3, a component of the artemin receptor, is required for migration and survival of the superior cervical ganglion.

GFR alpha3 is a component of the receptor for the neurotrophic factor artemin. The role of GFR alpha3 in nervous system development was examined by generating mice in which the Gfr alpha3 gene was disrupted. The Gfr alpha3-/- mice exhibited severe defects in the superior cervical ganglion (SCG), whereas other ganglia appeared normal. SCG precursor cells in the mutant embryos failed to migrate to the correct position, and they subsequently failed to innervate the target organs. In wild-type embryos, Gfr alpha3 was expressed in migrating SCG precursors, and artemin was expressed in and near the SCG. After birth, SCG neurons in the mutant mice underwent progressive cell death. These observations suggest that GFR alpha3-mediated signaling is required both for the rostral migration of SCG precursors and for the survival of mature SCG neurons.

lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal.

lefty-1, lefty-2, and nodal are expressed on the left side of developing mouse embryos and are implicated in left-right (L-R) determination. The role of lefty-1 was examined by analyzing mutant mice lacking this gene. The lefty-1-deficient mice showed a variety of L-R positional defects in visceral organs. Unexpectedly, however, the most common feature of lefty-1-/- mice was thoracic left isomerism (rather than right isomerism). The lack of lefty-1 resulted in bilateral expression of nodal, lefty-2, and Pitx2 (a homeobox gene normally expressed on the left side). These observations suggest that the role of lefty-1 is to restrict the expression of lefty-2 and nodal to the left side, and that lefty-2 or nodal encodes a signal for "leftness."

Pitx2, a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry.

Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

Two closely-related left-right asymmetrically expressed genes, lefty-1 and lefty-2: their distinct expression domains, chromosomal linkage and direct neuralizing activity in Xenopus embryos.

BACKGROUND: Vertebrates have numerous lateral asymmetries in the position of their organs, but the molecular basis for the determination of left-right (L-R) asymmetries remains largely unknown. TGFbeta-related genes such as lefty and nodal are L-R asymmetrically expressed in developing mouse embryos, and may be involved in L-R determination. RESULTS: We have identified two highly conserved genes, lefty-1 and lefty-2, in the mouse genome. These two genes are tightly linked on mouse chromosome 1. lefty-1 and lefty-2 are both expressed in a L-R asymmetric fashion in mouse embryos. However, the major expression domains of the two genes are different: lefty-1 expression is predominantly confied to the left side of ventral neural tube, whereas lefty-2 is strongly expressed in the lateral plate mesoderm on the left side. In embryos homozygous for the iv and inv mutation, which cause situs inversus, the expression sites of both genes are affected, either reversed or bilaterally, indicating that lefty-1 and lefty-2 are downstream of iv and inv. Although Lefty-1 and Lefty-2 prepro-proteins are not readily processed in cultured cells, BMP2-Lefty chimeric proteins can be processed to a secreted form. We have examined the activities of Lefty-1 and Lefty-2 in Xenopus embryos. In animal cap explants, Lefty-1 and Lefty-2 induce neural cells in the absence of mesoderm induction. The direct neuralizing activities of Lefty-1 and Lefty-2 thus seem remarkably similar to those of BMP antagonists such as noggin and chordin, suggesting that the action of Lefty-1 and Lefty-2 may be to locally antagonize BMP (bone morphogenic protein)-mediated signals in tissues positioned on the left side of the mouse embryos. CONCLUSION: There are two lefty genes in mice (lefty-1 and lefty-2), both of which are expressed in a L-R asymmetric fashion and are downstream of iv and inv. Lefty-1 and Lefty-2 possess direct neuralizing activity in Xenopus embryos, resembling the activities of BMP antagonists.

Left-right asymmetric expression of the TGF beta-family member lefty in mouse embryos.

Examples of lateral asymmetry are often found in vertebrates, such as the heart being on the left side, but the molecular mechanism governing the establishment of this left-right (L-R) handedness is unknown. A diffusible morphogen may determine L-R polarity, but a likely molecule has not so far been identified. Here we report on the gene lefty, a member of the transforming growth factor-beta family, which may encode a morphogen for L-R determination. Lefty protein contains the cysteine-knot motif characteristic of this superfamily and is secreted as a processed form of relative molecular mass 25K-32K. Surprisingly, lefty is expressed in the left half of gastrulating mouse embryos. This asymmetric expression is very transient and occurs just before the first sign of lateral asymmetry appears. In the mouse mutants iv and inv, which cause situs inversus, the sites of lefty expression are inverted, indicating that lefty is downstream of iv and inv. These results suggest that lefty may be involved in setting up L-R asymmetry in the organ systems of mammals.

Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor.

BACKGROUND: Oct-3, a pluripotent cell-specific POU transcription factor, appears to be a key regulator in pluripotential early embryonic cells and germ cells. In order to study how pluripotency is maintained, it is essential to know what genes are regulated by Oct-3. RESULTS: By employing a subtraction method, we identified several pluripotent cell-specific genes. Based upon expression patterns in various cell lines lacking or possessing Oct-3 function, about half of the genes were placed downstream of Oct-3. These downstream genes included a previously-known gene (Glut-3: a gene for a glucose transporter) and novel genes (226, 383 and 880). Their expression patterns paralleled that of Oct-3: all of these genes were highly expressed in pluripotent cells such as EC/ES cells, but switched off upon differentiation. More importantly, their expression was rescued in 'revertant' cells that ectopically acquired the Oct-3 transactivating function. Furthermore, the expression profiles of Glut-3, 226 and 383 during mouse development also overlapped that of Oct-3. The Glut-3 gene possessed multiple Oct-3 binding sites in its transcriptional regulatory regions, suggesting that at least one of the downstream genes was a direct target of Oct-3. CONCLUSIONS: A large proportion of pluripotent cell-specific genes appear to be downstream targets of Oct-3.

Synchronous sperm penetration of zona-free mouse eggs in vitro.

To synchronize sperm penetration of zona-free eggs immediately after insemination, zona-free eggs preloaded with Hoechst-33342 were inseminated under various conditions. Insemination was mostly conducted at 10 sperm/microliter. In preliminary experiments, fatty acid-free BSA (FAF) was more satisfactory for sperm penetration than fraction V BSA, and FAF was used in the following experiments. Only 26% of zona-free eggs were fertilized at 10 min after insemination when the eggs were inseminated immediately after zona removal and preloading. However, egg preincubation significantly improved the penetration rate (1 h preincubation: 63%, 2 h preincubation: 82% penetrated 10 min after insemination). Some eggs preincubated for 2 h were already penetrated at 3 min (7%), and the rate gradually increased in a time-dependent manner (3 min: 7%, 5 min: 30%, 10 min: 80%). The rate further improved as the sperm concentration was increased; the maximal level was obtained at 160 sperm/microliters. At 160 sperm/microliters, 54% of the eggs were penetrated at 3 min and 78% were at 5 min. These results indicate that it is possible to synchronize the sperm entry and that not only sperm but zona-free eggs should be preincubated before insemination. These data are considered valuable for investigating early events in fertilization.