Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod

We report the identification of two distinct noggin genes in the tetrapod Xenopus tropicalis. Noggin functions to antagonize BMP signaling in many developmental contexts, and much work has explored its role in early vertebrate development. We have identified two noggin genes in the tropical clawed frog, X. tropicalis, a diploid anuran which is being explored for its potential as a genetic model system for early vertebrate development. Here we report the cloning and characterization of the Xenopus tropicalis noggin1 and noggin2 genes, which have distinct expression domains in the early embryo with one overlapping domain in the anterior neural tissue. X. tropicalis noggin1 expression is very similar to that of noggin in Xenopus laevis, with expression beginning in the blastula organizer region and continuing through gastrulation and neurulation in the organizer and notochord. Later, it is also expressed in the anterior neural ridge and subsequent forebrain; noggin1 is also expressed in the pharyngeal arches after neural tube closure. At the tadpole stage expression is maintained in the dorsal neural tube and is present in the otic vesicle. However, the expression of noggin2 is much more similar to the expression of noggin2 in D. rerio with expression in the forebrain, hindbrain, and somites, but unlike D. rerio, X. tropicalis noggin2 is expressed in the heart by stage 28. This work presents the first example of a tetrapod with at least two noggin genes.     

Misexpression of Polycomb-group proteins in Xenopus alters anterior neural development and represses neural target genes.

In Drosophila, the Polycomb-group constitutes a set of structurally diverse proteins that act together to silence target genes. Many mammalian Polycomb-group proteins have also been identified and show functional similarities with their invertebrate counterparts. To begin to analyze the function of Polycomb-group proteins in Xenopus development, we have cloned a Xenopus homolog of Drosophila Polycomblike, XPcl1. XPcl1 mRNA is present both maternally and zygotically, with prominent zygotic expression in the anterior central nervous system. Misexpression of Pcl1 by RNA injection into embryos produces defects in the anterior central nervous system. The forebrain and midbrain contain excess neural tissue at the expense of the ventricle and include greatly thickened floor and roof plates. The eye fields are present but Rx2A, an eye-specific marker, is completely repressed. Overexpression of Pcl1 in Xenopus embryos alters two hindbrain markers, repressing En-2 and shifting it and Krox-20 in a posterior direction. Similar neural phenotypes and effects on the En-2 expression pattern were produced by overexpression of three other structurally unrelated Polycomb-group proteins: M33, XBmi-1, and mPh2. These observations indicate an important role for the Polycomb-group in regulating gene expression in the developing anterior central nervous system.     

Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes

During animal development, Hox genes are expressed in characteristic, spatially restricted patterns and specify regional identities along the anterior-posterior (A-P) axis. Polycomb group (PcG) proteins in Drosophila repress Hox expression and maintain the expression patterns during development. Mice deficient for homologues of the Drosophila PcG genes, such as M33, bmi1, mel18, rae28 and eed, show altered Hox expression patterns. In this study, we examined the time course of Hoxb3 expression during late gastrulation and early segmentation of rae28-deficient mice. Hoxb3 was expressed ectopically in pharyngeal arch and hindbrain from embryonic day (E) 9.5 and 10.5, respectively. The anterior boundary of ectopic expression in the hindbrain extended gradually in the rostral direction as development proceeded from E10.5 to E12.5. Expression of kreisler and Krox20, which function as positive regulators of Hoxb3 expression, was not affected in rae28-deficient embryos. Analysis of a neural crest marker, p75, in rae28-deficient mice revealed that the neural crest cells begin to ectopically express Hoxb3 after leaving the hindbrain. Our results suggest that rae28 is not required for the establishment but maintenance of Hoxb3 expression.     

Xenopus XsalF: anterior neuroectodermal specification by attenuating cellular responsiveness to Wnt signaling

Here we show that XsalF, a frog homolog of the Drosophila homeotic selector spalt, plays an essential role for the forebrain/midbrain determination in Xenopus. XsalF overexpression expands the domain of forebrain/midbrain genes and suppresses midbrain/hindbrain boundary (MHB) markers and anterior hindbrain genes. Loss-of-function studies show that XsalF is essential for the expression of the forebrain/midbrain genes and for the repression of the caudal genes. Interestingly, XsalF functions by antagonizing canonical Wnt signaling, which promotes caudalization of neural tissues. XsalF is required for anterior-specific expressions of GSK3beta and Tcf3, genes encoding antagonistic effectors of Wnt signaling. Loss-of-function phenotypes of GSK3beta and Tcf3 mimic those of XsalF while injections of GSK3beta and Tcf3 rescue loss-of-function phenotypes of XsalF. These findings suggest that the forebrain/midbrain-specific gene XsalF negatively controls cellular responsiveness to posteriorizing Wnt signals by regulating region-specific GSK3beta and Tcf3 expression.     

Engrailed and Fgf8 act synergistically to maintain the boundary between diencephalon and mesencephalon

Specification of the forebrain, midbrain and hindbrain primordia occurs during gastrulation in response to signals that pattern the gastrula embryo. Following establishment of the primordia, each brain part is thought to develop largely independently from the others under the influence of local organizing centers like the midbrain-hindbrain boundary (MHB, or isthmic) organizer. Mechanisms that maintain the integrity of brain subdivisions at later stages are not yet known. To examine such mechanisms in the anterior neural tube, we have studied the establishment and maintenance of the diencephalic-mesencephalic boundary (DMB). We show that maintenance of the DMB requires both the presence of a specified midbrain and a functional MHB organizer. Expression of pax6.1, a key regulator of forebrain development, is posteriorly suppressed by the Engrailed proteins, Eng2 and Eng3. Mis-expression of eng3 in the forebrain primordium causes downregulation of pax6.1, and forebrain cells correspondingly change their fate and acquire midbrain identity. Conversely, in embryos lacking both eng2 and eng3, the DMB shifts caudally into the midbrain territory. However, a patch of midbrain tissue remains between the forebrain and the hindbrain primordia in such embryos. This suggests that an additional factor maintains midbrain cell fate. We find that Fgf8 is a candidate for this signal, as it is both necessary and sufficient to repress pax6.1 and hence to shift the DMB anteriorly independently of the expression status of eng2/eng3. By examining small cell clones that are unable to receive an Fgf signal, we show that cells in the presumptive midbrain neural plate require an Fgf signal to keep them from following a forebrain fate. Combined loss of both Eng2/Eng3 and Fgf8 leads to complete loss of midbrain identity, resulting in fusion of the forebrain and the hindbrain primordia. Thus, Eng2/Eng3 and Fgf8 are necessary to maintain midbrain identity in the neural plate and thereby position the DMB. This provides an example of a mechanism needed to maintain the subdivision of the anterior neural plate into forebrain and midbrain.     

FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate.

The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression.     

Conserved expression of<Emphasis Type="Italic"> Hoxa1</Emphasis> in neurons at the ventral forebrain/midbrain boundary of vertebrates

The previously described expression patterns of zebrafish and mouse Hoxa1 genes are seemingly very disparate, with mouse Hoxa1 expressed in the gastrula stage hindbrain and the orthologous zebrafish hoxa1a gene expressed in cell clusters within the ventral forebrain and midbrain. To investigate the evolution of Hox gene deployment within the vertebrate CNS, we have performed a comparative expression analysis of Hoxa1 orthologs in a range of vertebrate species, comprising representatives from the two major lineages of vertebrates (actinopterygians and sarcopterygians). We find that fore/midbrain expression of hoxa1a is conserved within the teleosts, as it is shared by the ostariophysan teleost zebrafish (Danio rerio) and the distantly related acanthopterygian teleost medaka (Oryzias latipes). Furthermore, we find that in addition to the described gastrula stage hindbrain expression of mouse Hoxa1, there is a previously unreported neurula stage expression domain, again located more anteriorly at the ventral fore/midbrain boundary. A two-phase expression profile in early hindbrain and later fore/midbrain is shared by the other tetrapod model organisms chick and Xenopus. We show that the anterior Hoxa1 expression domain is localized to the anterior terminus of the medial longitudinal fasciculus (MLF) in mouse, chick, and zebrafish. These findings suggest that anterior expression of Hoxa1 is a primitive characteristic that is shared by the two major vertebrate lineages. We conclude that Hox gene expression within the vertebrate CNS is not confined exclusively to the segmented hindbrain and spinal cord, but rather that a presumptive fore/midbrain expression domain arose early in vertebrate origins and has been conserved for at least 400 million years.     

Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue

A novel gene, Xenopus Polycomblike 2 (XPcl2), which encodes a protein similar to Drosophila Polycomblike was cloned and characterized. Polycomblike belongs to the Polycomb group proteins, which maintain stable expression patterns for the clustered homeotic genes in the Drosophila embryo by forming multimeric complexes on chromatin. XPcl2 shows greater amino acid sequence homology to human and mouse M96 (hPcl2, mPcl2) than Xenopus Pcl1 (XPcl1), mouse Tctex3 (mPcl1) and human PHF1 (hPcl1), indicating that at least two types of Polycomblike genes are conserved between amphibians and mammals. XPcl2 mRNA is present both maternally and zygotically, and the temporal expression profile is distinct from XPcl1, another member of the Polycomblike family in Xenopus. XPcl2 is highly expressed in the anterior-dorsal region of Xenopus following the neurula stage in a manner similar to XPcl1. Overexpression of XPcl2 disturbs the development of the anterior central nervous system, eye and cement gland. In the XPcl2-overexpressing embryo, a hindbrain marker, Krox20, and a spinal cord marker, HoxB9, are expressed more posteriorly, suggesting an alteration in the anterior-posterior patterning of the neural tissue. In addition, XPcl2 represses Zic3- and noggin-induced anterior neural markers, but not neural crest markers in animal cap explants. These results indicate that XPcl2 regulates anterior neural tissue development and the anterior-posterior patterning of the neural tissue.     

Retinoic acid induces changes in the localization of homeobox proteins in the antero-posterior axis of Xenopus laevis embryos.

We have studied the localization of the proteins of Xeb1 and Xeb2, two homeobox (hbx)-containing genes that are expressed during the early development of Xenopus laevis. Both proteins are expressed in juxtaposed and partially overlapping domains along the antero-posterior axis of Xenopus laevis embryos, with clearly defined anterior boundaries. Xeb2 is predominantly expressed in the caudal region of the hindbrain, whereas the Xeb1 protein is located in the most rostral region of the spinal cord. Furthermore, both proteins are expressed in single cells dispersed in the lateral flanks of the embryo in positions that correlate with the expression domains in the neural tube. We suggest that these cells are migratory neural crest cells that have acquired positional information in the neural tube prior to migration. The Xeb2 protein was also detected in the most posterior branchial arches and the pronephros. In stage 45 embryos, nuclei of the IX-X cranial ganglia, the lung buds and cells spreading into the forelimb rudiment express the Xeb2 antigen. The Xeb1 protein was also detected in the lung buds and the forelimb rudiment. To examine the effect of retinoic acid on expression, gastrula embryos were treated with all-trans retinoic acid (RA). Increasing concentrations of RA caused progressive truncation of anterior structures. The most severely affected embryos lacked eyes, nasal pits, forebrain, midbrain and otic vesicles, and the anterior boundary of the hindbrain seemed to be displaced rostrally. This alteration correlates with a progressive displacement of the anterior boundary of the expression domain of Xeb2. On the other hand, 10(-6) M RA induces an ectopic site of Xeb1 expression at the anterior end of the central nervous system, located just anterior to the extended domain of Xeb2 whereas expression in the spinal cord remains unaffected.     

otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation.

We previously showed that otx2 regulates Xenopus cement gland formation in the ectoderm. Here, we show that otx2 is sufficient to direct anterior neural gene expression, and that its activity is required for cement gland and anterior neural determination. otx2 activity at midgastrula activates anterior and prevents expression of posterior and ventral gene expression in whole embryos and ectodermal explants. These data suggest that part of the mechanism by which otx2 promotes anterior determination involves repression of posterior and ventral fates. A dominant negative otx2-engrailed repressor fusion protein (otx2-En) ablates endogenous cement gland formation, and inhibits expression of the mid/hindbrain boundary marker engrailed-2. Ectoderm expressing otx2-En is not able to respond to signals from the mesoderm to form cement gland, and is impaired in its ability to form anterior neural tissue. These results compliment analyses in otx2 mutant mice, indicating a role for otx2 in the ectoderm during anterior neural patterning.     

Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, we found that Wnt signaling was sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allowed the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induced Dlx2 expression and inhibited the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts directed the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling resulted in the induction of dT specific markers, but only in tissues that expressed Irx3.     

Knockdown of the complete Hox paralogous group 1 leads to dramatic hindbrain and neural crest defects

The Hox paralogous group 1 (PG1) genes are the first and initially most anterior Hox genes expressed in the embryo. In Xenopus, the three PG1 genes, Hoxa1, Hoxb1 and Hoxd1, are expressed in a widely overlapping domain, which includes the region of the future hindbrain and its associated neural crest. We used morpholinos to achieve a complete knockdown of PG1 function. When Hoxa1, Hoxb1 and Hoxd1 are knocked down in combination, the hindbrain patterning phenotype is more severe than in the single or double knockdowns, indicating a degree of redundancy for these genes. In the triple PG1 knockdown embryos the hindbrain is reduced and lacks segmentation. The patterning of rhombomeres 2 to 7 is lost, with a concurrent posterior expansion of the rhombomere 1 marker, Gbx2. This effect could be via the downregulation of other Hox genes, as we show that PG1 function is necessary for the hindbrain expression of Hox genes from paralogous groups 2 to 4. Furthermore, in the absence of PG1 function, the cranial neural crest is correctly specified but does not migrate into the pharyngeal arches. Embryos with no active PG1 genes have defects in derivatives of the pharyngeal arches and, most strikingly, the gill cartilages are completely missing. These results show that the complete abrogation of PG1 function in Xenopus has a much wider scope of effect than would be predicted from the single and double PG1 knockouts in other organisms.