msh may play a conserved role in dorsoventral patterning of the neuroectoderm and mesoderm

Many of the mechanisms that govern the patterning of the Drosophila neuroectoderm and mesoderm are still unknown. Here we report the sequence, expression, and regulation of the homeobox gene msh, which is likely to play an important role in the early patterning events of these two tissue primordia. msh expression is first observed in late blastoderm embryos and occurs in longitudinal bands of cells that are fated to become lateral neuroectoderm. This expression is under the control of dorsoventral axis-determination genes and depends on dpp-mediated repression in the dorsal half of the embryo and on fib-(EGF-) mediated repression ventrally. The bands of msh expression define the cells that will form the lateral columns of proneural gene expression and give rise to the lateral row of SI neuroblasts. This suggests that msh may be one of the upstream regulators of the achaete-scute (AS-C) genes and may play a role that is analogous to that of the homeobox gene vnd/NK2 in the medial sector of the neuroectoderm. During neuroblast segregation, msh expression is maintained in a subset of neuroblasts, indicating that msh, like vnd/NK2, could function in both dorsoventral patterning of the neuroectoderm and neuroblast specification. The later phase of msh expression that occurs after the first wave of neuroblast segregation in defined ectodermal and mesodermal clusters of cells points to similar roles of msh in patterning and cell fate specification of the peripheral nervous system, dorsal musculature, and the fat body. A comparison of the expression patterns of the vertebrate homologs of msh, vnd/NK2, and AS-C genes reveals striking similarities in dorsoventral patterning of the Drosophila and vertebrate neuroectoderm and indicates that genetic circuitries in neural patterning are evolutionarily conserved.     

orb is required for anteroposterior and dorsoventral patterning during Drosophila oogenesis

We describe mutations in the orb gene, identified previously as an ovarian-specific member of a large family of RNA-binding proteins. Strong orb alleles arrest oogenesis prior to egg chamber formation, an early step of oogenesis, whereas females mutant for a maternal-effect lethal orb allele lay eggs with ventralized eggshell structures. Embryos that develop within these mutant eggs display posterior patterning defects and abnormal dorsoventral axis formation. Consistent with such embryonic phenotypes, orb is required for the asymmetric distribution of oskar and gurken mRNAs within the oocyte during the later stages of oogenesis. In addition, double heterozygous combinations of orb and grk or orb and top/DER alleles reveal that mutations in these genes interact genetically, suggesting that they participate in a common pathway. Orb protein, which is localized within the oocyte in wild-type females, is distributed ubiquitously in stage 8-10 orb mutant oocytes. These data will be discussed in the context of a model proposing that Orb is a component of the cellular machinery that delivers mRNA molecules to specific locations within the oocyte and that this function contributes to both D/V and A/P axis specification during oogenesis.     

Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos

Glycogen synthase kinase 3 (GSK-3) is homologous to the product of the Drosophila gene shaggy (zeste-white 3), which is required for signalling by wingless during Drosophila development. To test whether GSK-3 is also involved in vertebrate pattern formation, its role was investigated during early Xenopus development. It was found that dominant-negative GSK-3 mutants induced dorsal differentiation, whereas wild-type GSK-3 induced ventralization. These results indicate that GSK-3 is required for ventral differentiation, and suggest that dorsal differentiation may involve the suppression of GSK-3 activity by a wingless/wnt-related signal.     

Control of dorsoventral somite patterning by Wnt-1 and beta-catenin

In vertebrates, the dorsoventral patterning of somitic mesoderm is controlled by factors expressed in adjacent tissues. The ventral neural tube and the notochord function to promote the formation of the sclerotome, a ventral somite derivative, while the dorsal neural tube and the surface ectoderm have been shown to direct somite cells to a dorsal dermomyotomal fate. A number of signaling molecules are expressed in these inducing tissues during times of active cell fate specification, including members of the Hedgehog, Wnt, and BMP families. However, with the exception of the ventral determinant Sonic hedgehog (Shh), the functions of these signaling molecules with respect to dorsoventral somite patterning have not been determined. Here we investigate the role of Wnt-1, a candidate dorsalizing factor, in the regulation of sclerotome and dermomyotome formation. When ectopically expressed in the presomitic mesoderm of chick embryos in ovo, Wnt-1 differentially affects the expression of dorsal and ventral markers. Specifically, ectopic Wnt-1 is able to completely repress ventral (sclerotomal) markers and to enhance and expand the expression of dorsal (dermomyotomal) markers. However, Wnt-1 appears to be unable to convert all somitic mesoderm to a dermomyotomal fate. Delivery of an activated form of beta-catenin to somitic mesoderm mimics the effects of Wnt-1, demonstrating that Wnt-1 likely acts directly on somitic mesoderm, and not through adjacent tissues via an indirect signal relay mechanism. Taken together, our results support a model for somite patterning where sclerotome formation is controlled by the antagonistic activities of Shh and Wnt signaling pathways.     

Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus

The marginal zone is a ring of tissue that gives rise to a characteristic dorsoventral pattern of mesoderm in amphibian embryos. Bmp-4 is thought to play an important role in specifying ventral mesodermal fate. Here we show (1) that different doses of Bmp-4 are sufficient to pattern four distinct mesodermal cell types and to pattern gene expression in the early gastrula marginal zone into three domains, (2) that there is a graded requirement for a Bmp signal in mesodermal patterning, and (3) that Bmp-4 has long-range activity which can become graded in the marginal zone by the antagonizing action of noggin. The results argue that Bmp-4 acts as a morphogen in dorsoventral patterning of mesoderm.     

Conserved Sp?tzle/Toll signaling in dorsoventral patterning of Xenopus embryos

Three Cynomolgus monkeys (Macaca fascicularis) learned a flavour-visual conditional discrimination problem, in which one of two possible food items was presented at the beginning of each trial, and acted as an instruction cue to signal which of two visually distinct stimulus objects the animal must displace on that trial in order to obtain a further food reward. The task was learned first in light then in dark conditions. Following rhinal cortex ablation the animals were unable to use the flavour properties of the food items to guide visual choices, performing at close to chance levels. Postoperative performance on a food preference test showed that their problem in associating a flavour cue with a visual object in the conditional learning task also extended to aberrant choice of foods based on their visual appearance.     

Conservation of BMP signaling in zebrafish mesoderm patterning

To investigate the conservation of mechanisms for mesodermal patterning between zebrafish and Xenopus, we isolated two cDNA clones encoding bone morphogenetic protein (BMP)-related proteins from a zebrafish cDNA library. Based on their predicted amino acid sequences, these two clones were designated as zbmp-2 and zbmp-4. Whole-mount in situ hybridization analysis revealed that in gastrula embryo, both genes were localized in the ventral part of the embryo, consistent with the proposed function of Xenopus BMP-4 in ventral mesoderm specification. zbmp-4 expression, however, was also seen in the embryonic shield, the most dorsal mesodermal structure. To examine the ability of zbmp-2 to ventralize mesoderm, we injected synthetic mRNA into zebrafish embryos and found that overexpression of this gene eliminated dorsal structures including notochord at both morphological and molecular level. In contrast, expression of ventral marker gene eve1 was expanded to the dorsal side. These effects are analogous to the ventralization of embryos caused by ectopic xBMP-4 expression. Taken together, one may conclude that the developmental mechanisms for mesodermal patterning regulated by BMPs are evolutionarily conserved between amphibians and teleosts.     

Determination of the zebrafish forebrain: induction and patterning

We report an analysis of forebrain determination and patterning in the zebrafish Danio rerio. In order to study these events, we isolated zebrafish homologs of two neural markers, odd-paired-like (opl), which encodes a zinc finger protein, and fkh5, which encodes a forkhead domain protein. At mid-gastrula, expression of these genes defines a very early pattern in the presumptive neurectoderm, with opl later expressed in the telencephalon, and fkh5 in the diencephalon and more posterior neurectoderm. Using in vitro explant assays, we show that forebrain induction has occurred even earlier, by the onset of gastrulation (shield stage). Signaling from the early gastrula shield, previously shown to be an organizing center, is sufficient for activation of opl expression in vitro. In order to determine whether the organizer is required for opl regulation, we removed from late blastula stage embryos either the presumptive prechordal plate, marked by goosecoid (gsc) expression, or the entire organizer, marked by chordin (chd) expression. opl was correctly expressed after removal of the presumptive prechordal plate and consistently, opl was correctly expressed in one-eyed pinhead (oep) mutant embryos, where the prechordal plate fails to form. However, after removal of the entire organizer, no opl expression was observed, indicating that this region is crucial for forebrain induction. We further show that continued organizer function is required for forebrain induction, since beads of BMP4, which promotes ventral fates, also prevented opl expression when implanted during gastrulation. Our data show that forebrain specification begins early during gastrulation, and that a wide area of dorsal mesendoderm is required for its patterning.     

windbeutel, a gene required for dorsoventral patterning in Drosophila, encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum

The formation of the dorsoventral axis of the Drosophila embryo depends on cell-cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilization, when the egg has already been laid. We have performed a molecular analysis of windbeutel. We have found that windbeutel encodes a putative resident protein of the endoplasmic reticulum, and has homologs in rats and humans. The gene is expressed for a brief period of time in the follicle cells of the ovary, at around the time when the dorsoventral axis of the egg chamber is first established. We propose that Windbeutel is responsible for the folding and/or modification of a specific factor that is secreted from the follicle cells and participates in the activation of the ventralizing signal.     

A role for Xenopus Frizzled 8 in dorsal development

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors of the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photoaffinity labeling was progressively blocked by cGMP at concentrations higher than 10 microM, whereas cAMP or 5'-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, cIMP, and beta-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 microM, which compared favorably with an IC50 of 5 microM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.     

The role of the msh homeobox gene during Drosophila neurogenesis: implication for the dorsoventral specification of the neuroectoderm

Development of the Drosophila central nervous system begins with the delamination of neural and glial precursors, called neuroblasts, from the neuroectoderm. An early and important step in the generation of neural diversity is the specification of individual neuroblasts according to their position. In this study, we describe the genetic analysis of the msh gene which is likely to play a role in this process. The msh/Msx genes are one of the most highly conserved families of homeobox genes. During vertebrate spinal cord development, Msx genes (Msx1-3) are regionally expressed in the dorsal portion of the developing neuroectoderm. Similarly in Drosophila, msh is expressed in two longitudinal bands that correspond to the dorsal half of the neuroectoderm, and subsequently in many dorsal neuroblasts and their progeny. We showed that Drosophila msh loss-of-function mutations led to cell fate alterations of neuroblasts formed in the dorsal aspect of the neuroectoderm, including a possible dorsal-to-ventral fate switch. Conversely, ectopic expression of msh in the entire neuroectoderm severely disrupted the proper development of the midline and ventral neuroblasts. The results provide the first in vivo evidence for the role of the msh/Msx genes in neural development, and support the notion that they may perform phylogenetically conserved functions in the dorsoventral patterning of the neuroectoderm.     

A genetic framework for floral patterning

The initial steps of flower development involve two classes of consecutively acting regulatory genes. Meristem-identity genes, which act early to control the initiation of flowers, are expressed throughout the incipient floral primordium. Homeotic genes, which act later to specify the identity of individual floral organs, are expressed in distinct domains within the flower. The link between the two classes of genes has remained unknown so far. Here we show that the meristem-identity gene LEAFY has a role in controlling homeotic genes that is separable from its role in specifying floral fate. On the basis of our observation that LEAFY activates different homeotic genes through distinct mechanisms, we propose a genetic framework for the control of floral patterning.     

Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein

Determination of fate maps and cell lineage tracing have previously been carried out in the zebrafish embryo by following the progeny of individual cells injected with fluorescent dyes. We review the information obtained from these experiments and then present an approach to fate mapping and cell movement tracing, utilizing the activation of caged fluorescein-dextran. This method has several advantages over single-cell injections in that it is rapid, allows cells at all depths in the embryo to be marked, can be used to follow cells starting at any time during development, and allows an appreciation of the movements of cells located in a coherent group at the time of uncaging. We demonstrate that the approach is effective in providing additional and complementary information on prospective mesoderm and brain tissues studied previously. We also present, for the first time, a fate map of placodal tissues including the otic vesicle, lateral line, cranial ganglia, lens, and olfactory epithelium. The prospective placodal cells are oriented at the 50% epiboly stage on the ventral side of the embryo with anterior structures close to the animal pole, and posterior structures nearer to the germ ring.     

Delta-Notch signalling and the patterning of sensory cell differentiation in the zebrafish ear: evidence from the mind bomb mutant

Mechanosensory hair cells in the sensory patches of the vertebrate ear are interspersed among supporting cells, forming a fine-grained pattern of alternating cell types. Analogies with Drosophila mechanosensory bristle development suggest that this pattern could be generated through lateral inhibition mediated by Notch signalling. In the zebrafish ear rudiment, homologues of Notch are widely expressed, while the Delta homologues deltaA, deltaB and deltaD, coding for Notch ligands, are expressed in small numbers of cells in regions where hair cells are soon to differentiate. This suggests that the delta-expressing cells are nascent hair cells, in agreement with findings for Delta1 in the chick. According to the lateral inhibition hypothesis, the nascent hair cells, by expressing Delta protein, would inhibit their neighbours from becoming hair cells, forcing them to be supporting cells instead. The zebrafish mind bomb mutant has abnormalities in the central nervous system, somites, and elsewhere, diagnostic of a failure of Delta-Notch signalling: in the CNS, it shows a neurogenic phenotype accompanied by misregulated delta gene expression. Similar misregulation of delta ; genes is seen in the ear, along with misregulation of a Serrate homologue, serrateB, coding for an alternative Notch ligand. Most dramatically, the sensory patches in the mind bomb ear consist solely of hair cells, which are produced in great excess and prematurely; at 36 hours post fertilization, there are more than ten times as many as normal, while supporting cells are absent. A twofold increase is seen in the number of otic neurons also. The findings are strong evidence that lateral inhibition mediated by Delta-Notch signalling controls the pattern of sensory cell differentiation in the ear.