The Burnham Institute, Center for Neuroscience and Aging, La Jolla, CA 92037, USA
Harvard Medical School, Department of Neurobiology, Boston, MA 02115, USA
* Present address: The Queens Medical Center, Honolulu, HI 96813, USA
Author for correspondence (e-mail: nnakanishi{at}burnham.org)
Accepted April 12, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Left-right axis formation, Vertebrate, Embryogenesis, Patterning, Chick
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite the ability of PKI to potently inhibit PKA activity, its physiological roles are not well understood. In adult heart, the level of PKI is 20% of that necessary to inhibit fully activated PKA (Walsh et al., 1990). Therefore, the effect of PKI on PKA activity after cAMP production may be marginal. For this reason, it has been suggested that PKI may act as a regulator of basal PKA activity, or the activity that persists independent of adenylate cyclase activation (Walsh et al., 1990). In addition to the pseudo-substrate sequence, PKI contains a nuclear export signal (NES) (Wen et al., 1994; Wen et al., 1995). The binding of the C subunit to PKI exposes the NES, triggering its exit from the nucleus. Thus, it is possible that PKI may also regulate the intracellular distribution of C subunits.
PKA activity has been implicated in signal transduction pathways that underlie cell-type specification in invertebrate and vertebrate embryos. In Drosophila imaginal discs, cells that lack the C subunit exhibit pattern respecifications similar to those generated by ectopic hedgehog (hh) expression (Jiang and Struhl, 1995; Lepage et al., 1995; Li et al., 1995; Pan and Rubin, 1995; Strutt et al., 1995). In zebrafish, expression of a variant PKA-R subunit that constitutively inhibits PKA activity induces the expansion of proximal fates in the eye, ventral fates in the brain, and adaxial fates in somites and head mesenchyme (Hammerschmidt et al., 1996; Concordet et al., 1996). In embryonic limb, BMP-2-mediated stimulation of chondrogenesis is dependent on PKA (Lee and Chuong, 1997), and a pharmacological agent that inhibits PKA activity stimulates renal branching morphogenesis (Gupta et al., 1999). These experiments demonstrate that ectopic inhibitions of PKA activity can lead to alterations in cell-type specificity.
Recent studies have identified molecules involved in the formation of L-R asymmetry in the vertebrate embryo (Varlet and Robertson, 1997; Levin, 1998; Yost, 1999; Capdevila et al., 2000). Several genes are asymmetrically expressed along the L-R axis. In the chick, these include Sonic hedgehog (Shh), activin receptor IIa (ActRIIa), Nodal (Levin et al., 1995), lefty (Meno et al., 1996), SnR (Isaac et al., 1997), Pitx2 (Logan et al., 1998; Yoshioka et al., 1998; Piedra et al., 1998; Ryan et al., 1998), FGF8 (Boetgger et al., 1999; Meyers and Martin, 1999), Caronte (Rodriguez-Esteban et al., 1999; Yokouchi et al., 1998), NKX3.2 (Schneider et al., 1999), N-cadherin (Garcia-Castro et al., 2000), FGF18 (Ohuchi et al., 2000) and BMP4 (Monsoro-Burq and Le Douarin, 2000). Between stages 4 and 6, Shh is expressed on the left side of the node, while the expression of ActRIIa and FGF8 is confined to the right. Ectopic Shh delivered to the right side of the node induces expression of a TGFß family member Nodal (Levin et al., 1995), and anti-Shh antibody placed on the left side of the node abolishes Nodal expression (Pagán-Westphal and Tabin, 1998). Furthermore, Nodal seems to suppress expression of the right-sided gene SnR, a zinc-finger molecule, in lateral plate mesoderm (Pagán-Westphal and Tabin, 1998). SnR, in turn, is a repressor of another left-sided gene Pitx2 (Patel et al., 1999). These experiments suggest that side-specific gene expression is established by positive and negative interactions among the molecules. Finally, experimental manipulations of these side-specific genes lead to abnormal morphogenesis along the L-R axis (reviewed by Capdevila et al., 2000).
We hypothesized that PKI might play a role in cell patterning through the inhibition of PKA activity. To test this idea, we examined the expression pattern of PKI in chick embryos. PKI
expression is developmentally and regionally restricted in chick embryos. Interestingly, PKI
is expressed more strongly on the right side of Hensens node than the left side between stages 6 and 7+. Treatment of embryos with antisense PKI
oligonucleotides led to an increase in the incidence of reversed heart looping and bilateral expression of Nodal and Pitx2. The same treatment suppressed the expression of SnR on the right side of the posterior lateral plate. Similarly, treatment with PKA activators induced bilateral expression of Nodal. Ectopic activin induced PKI
expression on the left side of the node, while ectopic Shh and anti-Shh antibody had no effect on PKI
expression. We propose a model in which PKI
interferes with the propagation of the Shh-Nodal pathway on the right side of the embryo.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antisense oligonucleotide application
Phosphorothioate oligodeoxynucleotides were used in this study. The sequence of antisense oligonucleotides (shown in Fig. 3B) corresponds to nucleotides 374 to 355, numbered from the A of the start methionine codon of chick PKI. We chose this sequence for antisense targeting, based on the thermodynamic algorithm developed by Toagosei, Tokyo, Japan. Oligonucleotide applications were performed as described in Nieto et al. (Nieto et al., 1994). Briefly, blastoderms were detached from their membranes, incubated with 80 µm oligonucleotides in protein-free medium (50% Hanks balanced salt solution/50% L-15 tissue culture medium with Ca2+ and Mg2+) for 3 hours at 37°C, and placed back on the original membranes. The blastoderms reattached and continued to spread on the membranes. In situ hybridization was performed to analyze expression of Shh, Nodal and Pitx2, using embryos at stages 6, 9, and 10 respectively. Heart looping was observed at stage 12.
|
Bead implantation
Activin bead implantation was performed as described in Levin et al. (Levin et al., 1995). Affigel blue beads were washed with PBS and soaked in activin for 1 hour on ice. Then beads were picked up and implanted between the epiblast and endoderm of stage 4 embryo in New culture (New, 1955). The materials for bead implantation using anti-Shh monoclonal antibody 5E1 and Shh protein have been described by Pagán-Westphal and Tabin (Pagán-Westphal and Tabin, 1998). Control beads were soaked in the buffer 5 mM NaP/150 mM NaCl/0.5 mM DTT (pH 5.5) in which Shh was diluted.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Nodal appears to suppress expression of a right-sided gene SnR in lateral plate mesoderm (Pagán-Westphal and Tabin, 1998). Thus, we examined the effect of PKI antisense oligonucleotides on the expression of SnR. If PKI
is upstream of Nodal, as indicated from the results shown above, then antisense treatment would suppress right-sided expression of SnR. In embryos treated with sense oligonucleotides, SnR expression is bilateral within somites, while it is asymmetric in lateral plate mesoderm (Fig. 3A). This expression pattern is similar to those in normal embryos (Isaac et al., 1997; Pagán-Westphal and Tabin, 1998). Antisense oligonucleotides drastically reduced the expression of SnR in the posterior area of lateral mesoderm (Fig. 3B).
In order to assess the effect of oligonucleotides, we examined the expression of PKI in embryos treated with PKI
sense and antisense oligonucleotides. Antisense treatment drastically diminished the signal for PKI
mRNA (Fig. 2N), while sense treatment had no effect (Fig. 2M). This finding is consistent with the previous report that antisense oligodeoxynucleotides can lead to degradation of mRNAs by RNaseH-like activity (Dash et al., 1987). Next, we synthesized mutant antisense oligonucleotides with one (M1), three (M3), or five (M5) mismatches (Fig. 4) in order to verify the specificity of the antisense oligonucleotides. We then examined the effect of these oligonucleotides on bilateral expression of Nodal at stage 9. As shown in Fig. 4, antisense oligonucleotides with increased mismatches exhibited decreased effects on bilateral expression of Nodal. These data suggest that the antisense oligonucleotides perturbed L-R development in a sequence-specific manner. Taken together, endogenous PKI
appears to suppress the Nodal-Pitx2 pathway in the right lateral plate mesoderm.
|
In the first set of experiments, we treated stage 4+ embryos with medium only (no drug), 1,9-forskolin, forskolin, Rp-cAMPS and Sp-cAMPS. We then performed in situ hybridization to analyze Nodal expression at stage 9. Forskolin and Sp-cAMPS induced bilateral expression of Nodal, while an inactive compound 1,9-forskolin and a PKA inhibitor Rp-cAMPS did not alter the left-sided expression of Nodal (Fig. 5A). In the second set of experiments, we examined the direction of heart looping in chick embryos treated with forskolin and 1,9-forskolin. Forskolin induced reversed heart looping at the frequency significantly higher than that by 1,9-forskolin (Fig. 5B). These results suggest that activation of PKA in whole embryos leads to abnormal L-R development, and are consistent with the notion that PKI acts upstream of PKA in the signal transduction pathway leading to the suppression of the Nodal-Pitx2 pathway.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Recently, it was reported that N-cadherin is expressed on the right side of the node and left side of the primitive streak (Garcia-Castro et al., 2000). This asymmetric expression is seen between stages 3+ and 5 at the primitive streak, and between stages 4+ and 5 at the node. Blockade of N-cadherin function by a monoclonal antibody resulted in the altered expression of Snail and Pitx2, but had no effect on Nodal. Based on these data, it has been suggested that N-cadherin is a component of a pathway parallel to Nodal. As PKI appears upstream of Nodal, it is unlikely that N-cadherin and PKI
are in the same pathway.
Our results also indicate that PKI is not downstream of Shh. Nonetheless, the two molecules that we found were regulated by PKI
are Nodal and Pitx2, and these genes are downstream of Shh. It has been noted that hh signaling pathways interact with PKA activity. In Drosophila, loss of the C subunit of PKA leads to an increase in the level of full-length Cubitus interruptus, as well as the induction of the hh target genes dpp, wg and ptc (Chen et al., 1998). Paradoxically, transgenic PKA potentiates the activation of these genes, suggesting that PKA could regulate hh signaling pathways both positively and negatively (Ohlmeyer and Kalderon, 1997; Chen et al., 1998). Furthermore, it has been suggested that the regulation of the hh pathway by PKA in Drosophila is not mediated by cAMP (Li et al., 1995; Jiang and Struhl, 1995; Ohlmeyer and Kalderon, 1997). Our finding that PKI
regulates Nodal and Pitx2 raises the possibility that a putative Drosophila homolog of PKI may mediate the interaction of PKA and hh pathways.
One might predict that the treatment with Rp-cAMPS would result in the suppression of Nodal in both sides of embryos. However, we did not observe such events at a significant frequency. It is possible that the inhibition of PKA activity is not sufficient to suppress Nodal expression. It is also possible that Rp-cAMPS does not mimic PKI functions, owing to the difference in mechanisms by which these two molecules inhibit PKA. Rp-cAMPS is an analog of cAMP, and inhibits PKA activity by inactivating the R subunit. However, PKI inactivates the C subunit by binding it as a pseudosubstrate. It has been suggested that there may be a greater number of C subunits relative to R subunits in some cells (Walsh et al., 1990). The PKA activity carried out by these C subunits can be suppressed by PKI, but not by Rp-cAMPs. Such PKA activity could constitute the basal activity, which may play a role in the establishment of L-R axis.
Several asymmetric genes along the L-R axis are also involved in the formation of the dorsoventral and anteroposterior axes (Danos and Yost, 1995; Danos and Yost, 1996; Yost, 1995). PKI is expressed in the dorsal region of somites around stage 10 (M. K., unpublished). Inhibition of PKA activity by a transgenic dominant-negative R subunit led to abnormal patterning in the mesoderm including somitic myotomes (Hammerschmidt et al., 1996; Concordet et al., 1996). Hence, PKI
may be a component of signal transduction pathways repeatedly employed in pattern formation.
Features of PKI-mediated PKA regulation
Our findings suggest that an extracellular signal(s) could regulate PKA activity by controlling the expression of PKI. What are the distinct features of PKA modulation governed by PKI when compared with cAMP-mediated regulation? First, PKI directly interacts with the PKA-C subunit, and could thus inhibit the PKA previously activated by cAMP. Second, unlike cAMP (which is a small diffusible molecule), the intracellular distribution of PKI could be localized to certain structures or organelles. This would allow a spatially specific activation/inactivation of PKA within a single cell. Finally, the kinetics of PKA regulation based on the modulation of PKI gene expression are likely be drastically different from those mediated by cAMP. Turnover of cAMP is rapid, while transcriptional regulation of PKI would occur with much slower kinetics. Regulation of the PKI gene may thus modify basal PKA activity for lengthy periods during events such as pattern formation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Boetgger, T., Wittler, L. and Kessel, M. (1999). FGF8 functions in the specification of the right body side of the chick. Curr. Biol. 9, 277-280.[Medline]
Capdevila, J., Vogan, K. J., Tabin, C. J. and Izpisua Belmonte, J. C. (2000). Mechanisms of left-right determination in vertebrates. Cell 101, 9-21.[Medline]
Chen, Y., Gallaher, N., Goodman, R. H. and Smolik, S. M. (1998). Protein kinase A directly regulates the activity and proteolysis of cubitus interruptus. Proc. Natl. Acad. Sci. USA 95, 2349-2354.
Collins, S. P. and Uhler, M. D. (1997). Characterization of PKI, a novel isoform of the protein kinase inhibitor of cAMP-dependent protein kinase. J. Biol. Chem. 272, 18169-18178.
Concordet, J.-P., Lewis, K., Moore, J., Goodrich, L. V., Johnson, R. L., Scott, M. P. and Ingham, P. W. (1996). Spatial regulation of a zebrafish Patched homologue reflects the roles of Sonic hedgehog and protein kinase A in neural tube and somite patterning. Development 122, 2835-2846.
Danos, M. C. and Yost, H. J. (1995). Linkage of cardiac left-right asymmetry and dorsal-anterior development in Xenopus. Development 121, 1467-1474.
Danos, M. C. and Yost, H. J. (1996). Role of notochord in specification of cardiac left-right orientation zebrafish and Xenopus. Dev. Biol. 177, 96-103.[Medline]
Dash, P., Lotan, I., Knapp, M., Kandel, E. R. and Goelet, P. (1987). Selective elimination of nRNAs in vivo: complementary oligodeoxynucleotides promote RNA degradation by an RNase H-like activity. Proc. Natl. Acad. Sci. USA 84, 7896-7900.[Abstract]
Fan, C.-M., Porter, J. A., Chiang, C., Chang, D. T., Beachy, P. A. and Tessier-Lavigne, M. (1995). Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway. Cell. 81, 457-465.[Medline]
Frey, U., Huang, Y.-Y. and Kandel, E. R. (1993). Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons. Nature 260, 1661-1664.
Garcia-Castro M. I., Vielmetter E. and Bronner-Fraser M. (2000). N-Cadherin, a cell adhesion molecule involved in establishment of embryonic left-right asymmetry. Science 288, 1047-1051.
Gupta, I. R., Piscione, T. D., Grisaru, S., Phan, T., Macias-Silva, M., Zhou, X., Whiteside, C., Wrana, J. L. and Rosenblum, N. D. (1999). Protein kinase A is a negative regulator of renal branching morphogenesis and modulates inhibitory and stimulatory bone morphogenetic proteins. J. Biol. Chem. 274, 26305-26314.
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Exp. Morphol. 88, 49-92.
Hammerschmidt, M., Bitgood, M. J. and McMahon, A. P. (1996). Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev. 10, 647-658.[Abstract]
Isaac, A., Sargent, M. G. and Cooke, J. (1997). Control of vertebrate left-right asymmetry by a Snail-related zinc finger gene. Science 275, 1301-1304.
Jiang, J. and Struhl, G. (1995). Protein kinase A and hedgehog signaling in Drosophila limb development. Cell. 80, 563-572.[Medline]
Lee, Y.-S. and Chuong, C.-M. (1997). Activation of protein kinase A is a pivotal step involved in both BMP-2- and cyclic AMP-induced chondrogenesis. J. Cell Physiol. 170, 153-165.[Medline]
Lepage, T., Cohen, S. M., Diaz-Benjumea, F. J. and Parkhurst, S. M. (1995). Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning. Nature 373, 711-715.[Medline]
Levin, M. (1998). Left-right asymmetry and the chick embryo. Semin. Cell Dev. Biol. 9, 67-76.[Medline]
Levin, M., Johnson, R. L., Stern, C. D., Kuehn, M. R. and Tabin, C. J. (1995). A Molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 82, 803-814.[Medline]
Li, W., J.T., O., Lane, M. E. and Kalderon, D. (1995). Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 80, 553-562.[Medline]
Logan, M., Pagán-Westphal, S. M., Smith, D. M., Paganessi, L. and Tabin, C. J. (1998). The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell 94, 307-317.[Medline]
Marchetto, G. S. and Henry, H. L. (1995). Cloning and sequencing of the cDNA encoding the avian kidney cAMP-dependent protein kinase inhibitor protein. Gene 158, 303-304.[Medline]
Meno, C., Saijoh, Y., Fujii, H., Ikeda, M., Yokoyama, T., Yokoyama, M., Toyoda, Y. and Hamada, H. (1996). Left-right asymmetric expression of the TGFß-family member lefty in mouse embryos. Nature 381, 151-155.[Medline]
Meyers, E. N. and Martin, G. R. (1999). Differences in left-right axis pathways in mouse and chick: functions of FGF8 and SHH. Science 285, 403-406.
Monsoro-Burq, A. and Le Douarin, N. (2000). Left-right asymmetry in BMP4 signalling pathway during chick gastrulation. Mech. Dev. 97, 105-108.[Medline]
New, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol. 3, 326-331.
Nieto, M. A., Sargent, M. G., Wilkinson, D. G. and Cooke, J. (1994). Control of cell behavior during vertebrate development by Slug, a zinc finger gene. Science 264, 835-839.[Medline]
Nomura, M. and Li, E. (1998). Smad2 role in mesoderm formation, left-right patterning and craniofacial development. Nature 393, 786-790.[Medline]
Oh, S. P. and Li, E. (1997). The signaling pathway mediated by the type IIB activin receptor controls axial patterning and lateral asymmetry in the mouse. Genes Dev. 11, 1812-1826.[Abstract]
Ohlmeyer, J. T. and Kalderon, D. (1997). Dual pathways for induction of wingless expression by protein kinase A and Hedgehog in Drosophila embryos. Genes Dev. 11, 2250-2258.
Ohuchi H., Kimura S., Watamoto M. and Itoh N. (2000). Involvement of fibroblast growth factor (FGF)18-FGF8 signaling in specification of left-right asymmetry and brain and limb development of the chick embryo. Mech. Dev. 95, 55-66.[Medline]
Olsen, S. R. and Uhler, M. D. (1991). Isolation and characterization of cDNA clones for an inhibitor protein of cAMP-dependent protein kinase. J. Biol. Chem. 266, 11158-11162.
Pagán-Westphal, S. M. and Tabin, C. J. (1998). The transfer of left-right positional information during chick embryogenesis. Cell. 93, 25-35.[Medline]
Pan, D. and Rubin, G. M. (1995). cAMP-dependent protein kinase and hedgehog act antagonistically in regulating decapentaplegic transcription in Drosophila imaginal discs. Cell 80, 543-552.[Medline]
Patel, K., Isaac, A. and Cooke, J. (1999). Nodal signalling and the roles of the transcription factors SnR and Pitx2 in vertebrate left-right asymmetry. Curr. Biol. 9, 609-612.[Medline]
Piedra, M. E., Icardo, J. M., Albajar, M., Rodriguez-Rey, J. C. and Ros, M. A. (1998). Pitx2 participates in the late phase of the pathway controlling left-right asymmetry. Cell. 94, 319-324.[Medline]
Rodriguez-Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A. and Izpisua Belmonte, J. C. (1999). The novel cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry. Nature 401, 243-251.[Medline]
Ryan, A. K., Blumberg, B., Rodriguez-Esteban, C., Yonei-Tamura, S., Tamura, K., Tsukui, T., de la Pena, J., Sabbagh, W., Greenwald, J., Choe, S., Norris, D. P. et al. (1998). Pitx2 determines left-right asymmetry of internal organs in vertebrates. Nature 394, 545-551.[Medline]
Schneider, A., Mijalski, T., Schlange, T., Dai, W., Overbeek, P., Arnold, H. H. and Brand, T. (1999). The homeobox gene NKX3.2 is a target of left-right signaling and is expressed on opposite sides in chick and mouse embryos. Curr. Biol. 9, 911-914[Medline]
Scott, J. D., Fischer, E. H., Demaille, J. G. and Krebs, E. G. (1985). Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 82, 4379-4383.[Abstract]
Seasholtz, A. F., Gamm, D. M., Ballestero, R. P., Scarpetta, M. A. and Uhler, M. D. (1995). Differential expression of mRNAs for protein kinase inhibitor isoforms in mouse brain. Proc. Natl. Acad. Sci. USA 92, 1734-1738.[Abstract]
Strutt, D. I., Wiersdorff, V. and Mlodzik, M. (1995). Regulation of furrow progression in the Drosophila eye by cAMP-dependent protein kinase A. Nature 373, 705-709.[Medline]
Van Patten, S. M., Ng, D. C., Thng, J. P. H., Angelos, K. L., Smith, A. J. and Walsh, D. A. (1991). Molecular cloning of a rat testis form of the inhibitor protein of cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 88, 5383-5387.[Abstract]
Van Patten, S. M., Howard, P., Walsh, D. A. and Maurer, R. A. (1992). The - and ß-isoforms of the inhibitor protein of the 3', 5, cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression. Mol. Endocrinol. 6, 2114-2122.[Abstract]
Varlet, I. and Robertson, E. J. (1997). Left-right asymmetry in vertebrates. Curr. Opin. Genet. Dev. 7, 510-523.
Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E. H. and Krebs, E. G. (1971). Purification and characterization of a protein inhibitor of adenosine 3',5'-monophosphate-dependent protein kinases. J. Biol. Chem. 246, 1977-1985.
Walsh, D. A., Angelos, K. L., Van Patten, S. M., Glass, D. B. and Garetto, L. P. (1990). Peptides and Protein Phosphorylation (Kemp, B.E., ed), pp. 43-84. Boca Raton: CRC Press.
Wen, W., Harootunian, A. T., Adamus, S. R., Feramisco, J., Tsien, R. Y., Meinkoth, J. L. and Taylor, S. S. (1994). Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal. J. Biol. Chem. 269, 32214-32220.
Wen, W., Meinkoth, J. L., Tsien, R. Y. and Taylor, S. S. (1995). Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463-473.[Medline]
Yokouchi, Y., Vogan, K. J., Pearse, R. V., II and Tabin, C. J. (1998). Antagonistic signaling by Caronte, a novel Cerberus-related gene, establishes left-right asymmetric gene expression. Cell 98, 573-583.
Yoshioka, H., Meno, C., Koshiba, K., Sugihara, M., Itoh, H., Ishimaru, Y., Inoue, T., Ohuchi, H., Semina, E. V., Murray, J. C. et al. (1998). Pitx2, a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry. Cell 94, 299-305.[Medline]
Yost, H. J. (1995). Vertebrate left-right development. Cell 82, 689-692.[Medline]
Yost, H. J. (1999). Diverse initiation in a conserved left-right pathway? Curr. Opin. Genet. Dev. 9, 422-426.[Medline]