Nodal signaling patterns the organizer

Spemann's organizer plays an essential role in patterning the vertebrate embryo. During gastrulation, organizer cells involute and form the prechordal plate anteriorly and the notochord more posteriorly. The fate mapping and gene expression analyses in zebrafish presented in this study reveal that this anteroposterior polarity is already initiated in the organizer before gastrulation. Prechordal plate progenitors reside close to the blastoderm margin and express the homeobox gene goosecoid, whereas notochord precursors are located further from the margin and express the homeobox gene floating head. The nodal-related genes cyclops and squint are expressed at the blastoderm margin and are required for prechordal plate and notochord formation. We show that differential activation of the Nodal signaling pathway is essential in establishing anteroposterior pattern in the organizer. First, overexpression of cyclops and squint at different doses leads to the induction of floating head at low doses and the induction of both goosecoid and floating head at higher doses. Second, decreasing Nodal signaling using different concentrations of the antagonist Antivin inhibits goosecoid expression at low doses and blocks expression of both goosecoid and floating head at higher doses. Third, attenuation of Nodal signaling in zygotic mutants for the EGF-CFC gene one-eyed pinhead, an essential cofactor for Nodal signaling, leads to the loss of goosecoid expression and expansion of floating head expression in the organizer. Concomitantly, cells normally fated to become prechordal plate are transformed into notochord progenitors. Finally, activation of Nodal signaling at different times suggests that prechordal plate specification requires sustained Nodal signaling, whereas transient signaling is sufficient for notochord development. Together, these results indicate that differential Nodal signaling patterns the organizer before gastrulation, with the highest level of activity required for anterior fates and lower activity essential for posterior fates.     

Otx2 and HNF3beta genetically interact in anterior patterning

Patterning the developing nervous system in the mouse has been proposed to depend on two separate sources of signals, the anterior visceral endoderm (AVE) and the node or organizer. Mutation of the winged-helix gene HNF3beta leads to loss of the node and its derivatives, while mutation of the homeobox gene Otx2 results in loss of head structures, apparently at least partially because of defects in the AVE. To investigate the potential genetic interactions between the two signaling centers, we crossed Otx2+/- and HNF3beta+/- mice and found that very few Otx2+/-;HNF3beta+/- double heterozygous mutants survived to weaning. Normal Mendelian ratios of genotypes were observed during gestation, but more than half the double heterozygotes displayed a severe anterior patterning phenotype that would be incompatible with postnatal survival. The phenotype was characterized by varying degrees of holoprosencephaly, cyclopia with proboscis-like structures, and anterior forebrain truncations. Regional marker analysis revealed that ventral forebrain structures of Otx2+/-;HNF3beta+/- mutant embryos were most severely affected. Shh expression was completely absent in the anterior region of Otx2+/-;HNF3beta+/- embryos, suggesting that Otx2 and HNF3beta genetically interact, directly or indirectly, to regulate Shh expression in the anterior midline. In addition, the forebrain truncations suggest an involvement of both genes in anterior patterning, through their overlapping expression domains in either the AVE and/or the prechordal mesoderm.     

Zebrafish Dkk1, induced by the pre-MBT Wnt signaling, is secreted from the prechordal plate and patterns the anterior neural plate

mRNA injection into the ventral blastomeres of Xenopus embryos of mRNA encoding Wnt pathway genes induces a secondary axis with complete head structures. To identify target genes of the pre-MBT dorsalization pathway that might be responsible for head formation in zebrafish, we have cloned zebrafish dickkopf1 (dkk1), which is expressed in tissues implicated in head patterning. We found that dkk1 blocks the post-MBT Wnt signaling and dkk1 is a target of the pre-MBT Wnt signaling. Dkk1 overexpression in the prechordal plate suggests that Dkk1, secreted from the prechordal plate, expands the forebrain at the expense of the midbrain in the anterior neural plate. Furthermore, dkk1 acts in parallel to the homeobox gene bozozok and bozozok is required for the maintenance of dkk1 expression. The nodal gene squint is also required for the maintenance of dkk1 expression. Among the mutually dependent target genes of the pre-MBT Wnt signaling, dkk1 plays an important role in patterning the anterior head of zebrafish.     

moz regulates Hox expression and pharyngeal segmental identity in zebrafish

In vertebrate embryos, streams of cranial neural crest (CNC) cells migrate to form segmental pharyngeal arches and differentiate into segment-specific parts of the facial skeleton. To identify genes involved in specifying segmental identity in the vertebrate head, we screened for mutations affecting cartilage patterning in the zebrafish larval pharynx. We present the positional cloning and initial phenotypic characterization of a homeotic locus discovered in this screen. We show that a zebrafish ortholog of the human oncogenic histone acetyltransferase MOZ (monocytic leukemia zinc finger) is required for specifying segmental identity in the second through fourth pharyngeal arches. In moz mutant zebrafish, the second pharyngeal arch is dramatically transformed into a mirror-image duplicated jaw. This phenotype resembles a similar but stronger transformation than that seen in hox2 morpholino oligo (hox2-MO) injected animals. In addition, mild anterior homeotic transformations are seen in the third and fourth pharyngeal arches of moz mutants. moz is required for maintenance of most hox1-4 expression domains and this requirement probably at least partially accounts for the moz mutant homeotic phenotypes. Homeosis and defective Hox gene expression in moz mutants is rescued by inhibiting histone deacetylase activity with Trichostatin A. Although we find early patterning of the moz mutant hindbrain to be normal, we find a late defect in facial motoneuron migration in moz mutants. Pharyngeal musculature is transformed late, but not early, in moz mutants. We detect relatively minor defects in arch epithelia of moz mutants. Vital labeling of arch development reveals no detectable changes in CNC generation in moz mutants, but later prechondrogenic condensations are mispositioned and misshapen. Mirror-image hox2-dependent gene expression changes in postmigratory CNC prefigure the homeotic phenotype in moz mutants. Early second arch ventral expression of goosecoid (gsc) in moz mutants and in animals injected with hox2-MOs shifts from lateral to medial, mirroring the first arch pattern. bapx1, which is normally expressed in first arch postmigratory CNC prefiguring the jaw joint, is ectopically expressed in second arch CNC of moz mutants and hox2-MO injected animals. Reduction of bapx1 function in wild types causes loss of the jaw joint. Reduction of bapx1 function in moz mutants causes loss of both first and second arch joints, providing functional genetic evidence that bapx1 contributes to the moz-deficient homeotic pattern. Together, our results reveal an essential embryonic role and a crucial histone acetyltransferase activity for Moz in regulating Hox expression and segmental identity, and provide two early targets, bapx1 and gsc, of moz and hox2 signaling in the second pharyngeal arch.     

Development of chick axial mesoderm: specification of prechordal mesoderm by anterior endoderm-derived TGFbeta family signalling

Two populations of axial mesoderm cells can be recognised in the chick embryo, posterior notochord and anterior prechordal mesoderm. We have examined the cellular and molecular events that govern the specification of prechordal mesoderm. We report that notochord and prechordal mesoderm cells are intermingled and share expression of many markers as they initially extend out of Hensen's node. In vitro culture studies, together with in vivo grafting experiments, reveal that early extending axial mesoderm cells are labile and that their character may be defined subsequently through signals that derive from anterior endodermal tissues. Anterior endoderm elicits aspects of prechordal mesoderm identity in extending axial mesoderm by repressing notochord characteristics, briefly maintaining gsc expression and inducing BMP7 expression. Together these experiments suggest that, in vivo, signalling by anterior endoderm may determine the extent of prechordal mesoderm. The transforming growth factor (beta) (TGFbeta) superfamily members BMP2, BMP4, BMP7 and activin, all of which are transiently expressed in anterior endoderm mimic distinct aspects of its patterning actions. Together our results suggest that anterior endoderm-derived TGFbetas may specify prechordal mesoderm character in chick axial mesoderm.     

Chordin and noggin promote organizing centers of forebrain development in the mouse

In this study we investigate the roles of the organizer factors chordin and noggin, which are dedicated antagonists of the bone morphogenetic proteins (BMPs), in formation of the mammalian head. The mouse chordin and noggin genes (Chrd and Nog) are expressed in the organizer (the node) and its mesendodermal derivatives, including the prechordal plate, an organizing center for rostral development. They are also expressed at lower levels in and around the anterior neural ridge, another rostral organizing center. To elucidate roles of Chrd and Nog that are masked by the severe phenotype and early lethality of the double null, we have characterized embryos of the genotype Chrd(-/-);Nog(+/-). These animals display partially penetrant neonatal lethality, with defects restricted to the head. The variable phenotypes include cyclopia, holoprosencephaly, and rostral truncations of the brain and craniofacial skeleton. In situ hybridization reveals a loss of SHH expression and signaling by the prechordal plate, and a decrease in FGF8 expression and signaling by the anterior neural ridge at the five-somite stage. Defective Chrd(-/-);Nog(+/-) embryos exhibit reduced cell proliferation in the rostral neuroepithelium at 10 somites, followed by increased cell death 1 day later. Because these phenotypes result from reduced levels of BMP antagonists, we hypothesized that they are due to increased BMP activity. Ectopic application of BMP2 to wild-type cephalic explants results in decreased FGF8 and SHH expression in rostral tissue, suggesting that the decreased expression of FGF8 and SHH observed in vivo is due to ectopic BMP activity. Cephalic explants isolated from Chrd;Nog double mutant embryos show an increased sensitivity to ectopic BMP protein, further supporting the hypothesis that these mutants are deficient in BMP antagonism. These results indicate that the BMP antagonists chordin and noggin promote the inductive and trophic activities of rostral organizing centers in early development of the mammalian head.     

Synergistic interaction between Gdf1 and Nodal during anterior axis development

Growth and Differentiation Factor 1 (GDF-1) has been implicated in left-right patterning of the mouse embryo but has no other known function. Here, we demonstrate a genetic interaction between Gdf1 and Nodal during anterior axis development. Gdf1-/-;Nodal+/- mutants displayed several abnormalities that were not present in either Gdf1-/- or Nodal+/- single mutants, including absence of notochord and prechordal plate, and malformation of the foregut; organizing centers implicated in the development of the anterior head and branchial arches, respectively. Consistent with these deficits, Gdf1-/-;Nodal+/- mutant embryos displayed a number of axial midline abnormalities, including holoprosencephaly, anterior head truncation, cleft lip, fused nasal cavity, and lack of jaws and tongue. The absence of these defects in single mutants indicated a synergistic interaction between Nodal and GDF-1 in the node, from which the axial mesendoderm that gives rise to the notochord, prechordal plate, and foregut endoderm originates, and where the two factors are co-expressed. This notion was supported by a severe downregulation of FoxA2 and goosecoid in the anterior primitive streak of double mutant embryos. Unlike that in the lateral plate mesoderm, Nodal expression in the node was independent of GDF-1, indicating that both factors act in parallel to control the development of mesendodermal precursors. Receptor reconstitution experiments indicated that GDF-1, like Nodal, can signal through the type I receptors ALK4 and ALK7. However, analysis of compound mutants indicated that ALK4, but not ALK7, was responsible for the effects of GDF-1 and Nodal during anterior axis development. These results indicate that GDF-1 and Nodal converge on ALK4 in the anterior primitive streak to control the formation of organizing centers that are necessary for normal forebrain and branchial arch development.     

Expression patterns of fork head and goosecoid homologues in the mollusc Patella vulgata supports the ancestry of the anterior mesendoderm across Bilateria

We have characterised orthologues of the genes fork head and goosecoid in the gastropod Patella vulgata. In this species, the anterior-posterior (AP) axis is determined just before gastrulation, and leads to the specification of two mesodermal components on each side of the presumptive endoderm, one anterior (ectomesoderm), and one posterior (endomesoderm). Both fork head and goosecoid are expressed from the time the AP axis is specified, up to the end of gastrulation. fork head mRNA is detected in the whole endoderm, as well as in the anterior mesoderm, whereas goosecoid is only expressed anteriorly, in the three germ layers. The two genes are thus coexpressed in the anterior mesoderm, suggesting the latter's homology with vertebrate prechordal mesoderm. In addition, since prechordal plate is known to belong to an anterior, so called "head organiser", and since its inductive role is dependent on the function of the vertebrate fork head and goosecoid orthologues, we further suggest that the anterior mesoderm may also have a role in anterior inductive patterning in Spiralia. Finally, we propose that a mode of axial development involving two organisers, one anterior and one posterior, is ancestral to the Bilateria, and that both organisers evolved from the single head organiser of a putative hydra-like ancestor.     

The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning

Dickkopf1 (dkk1) encodes a secreted WNT inhibitor expressed in Spemann's organizer, which has been implicated in head induction in Xenopus. Here we have analyzed the role of dkk1 in endomesoderm specification and neural patterning by gain- and loss-of-function approaches. We find that dkk1, unlike other WNT inhibitors, is able to induce functional prechordal plate, which explains its ability to induce secondary heads with bilateral eyes. This may be due to differential WNT inhibition since dkk1, unlike frzb, inhibits Wnt3a signalling. Injection of inhibitory antiDkk1 antibodies reveals that dkk1 is not only sufficient but also required for prechordal plate formation but not for notochord formation. In the neural plate dkk1 is required for anteroposterior and dorsoventral patterning between mes- and telencephalon, where dkk1 promotes anterior and ventral fates. Both the requirement of anterior explants for dkk1 function and their ability to respond to dkk1 terminate at late gastrula stage. Xenopus embryos posteriorized with bFGF, BMP4 and Smads are rescued by dkk1. dkk1 does not interfere with the ability of bFGF to induce its immediate early target gene Xbra, indicating that its effect is indirect. In contrast, there is cross-talk between BMP and WNT signalling, since induction of BMP target genes is sensitive to WNT inhibitors until the early gastrula stage. Embryos treated with retinoic acid (RA) are not rescued by dkk1 and RA affects the central nervous system (CNS) more posterior than dkk1, suggesting that WNTs and retinoids may act to pattern anterior and posterior CNS, respectively, during gastrulation.     

The role of gsc and BMP-4 in dorsal-ventral patterning of the marginal zone in Xenopus: a loss-of-function study using antisense RNA.

The dorsal-specific homeobox gene goosecoid (gsc) and the bone morphogenetic protein 4 gene (BMP-4) are expressed in complementary regions of the Xenopus gastrula. Injection of gsc mRNA dorsalizes ventral mesodermal tissue and can induce axis formation in normal and UV-ventralized embryos. On the other hand, BMP-4 mRNA injection, which has a strong ventralizing effect on whole embryos, has been implicated in ventralization by UV, and can rescue tail structures in embryos dorsalized by LiCl. The above-mentioned putative roles for BMP-4 and gsc are based on gain-of-function experiments. In order to determine the in vivo role of these two genes in the patterning of the Xenopus mesoderm during gastrulation, partial loss-of-function experiments were performed using antisense RNA injections. Using marker genes that are expressed early in gastrulation, we show that antisense gsc RNA has a ventralizing effect on embryos, whereas antisense BMP-4 RNA dorsalizes mesodermal tissue. These loss-of-function studies also show a requirement for gsc and BMP-4 in the dorsalization induced by LiCl and in the ventralization generated by UV irradiation, respectively. Thus, both gain- and loss-of-function results for gsc and BMP-4 support the view that these two genes are necessary components of the dorsal and ventral patterning pathways in Xenopus embryos.     

Region-specific requirement for cholesterol modification of sonic hedgehog in patterning the telencephalon and spinal cord

Sonic hedgehog (Shh) secreted from the axial signaling centers of the notochord and prechordal plate functions as a morphogen in dorsoventral patterning of the neural tube. Active Shh is uniquely cholesterol-modified and the hydrophobic nature of cholesterol suggests that it might regulate Shh spreading in the neural tube. Here, we examined the capacity of Shh lacking the cholesterol moiety (ShhN) to pattern different cell types in the telencephalon and spinal cord. In mice expressing ShhN, we detected low-level ShhN in the prechordal plate and notochord, consistent with the notion that ShhN can rapidly spread from its site of synthesis. Surprisingly, we found that low-level ShhN can elicit the generation of a full spectrum of ventral cell types in the spinal cord, whereas ventral neuronal specification and ganglionic eminence development in the Shh(N/-) telencephalon were severely impaired, suggesting that telencephalic patterning is more sensitive to alterations in local Shh concentration and spreading. In agreement, we observed induction of Shh pathway activity and expression of ventral markers at ectopic sites in the dorsal telencephalon indicative of long-range ShhN activity. Our findings indicate an essential role for the cholesterol moiety in restricting Shh dilution and deregulated spread for patterning the telencephalon. We propose that the differential effect of ShhN in patterning the spinal cord versus telencephalon may be attributed to regional differences in the maintenance of Shh expression in the ventral neuroepithelium and differences in dorsal tissue responsiveness to deregulated Shh spreading behavior.