Immunoneutralization of insulin partially modifies dorsoventral patterning in developing frog embryos

Determination of anteroposterior and dorsoventral axes is an important early event in the development of vertebrates involving extensive cellular interactions including inductive events. Recently we showed that insulin plays an essential role in prepancreatic development of the frogMicrohyla ornata. In the present study we have investigated the effects of immunoneutralization of endogenous insulin on the process of pattern formation. Treatment of neurulating embryos with antiserum to insulin caused abnormal pattern formation. The defects included loss of normal architecture of the neural tube, reduction in the size of the neural tube and, most conspicuously, rotation of the dorsoventral axis of the neural tube, notochord and adjoining mesodermal elements. The effects could be alleviated partially by pretreatment of embryos with exogenous insulin. This supports our belief that insulin plays an important role in induction and pattern formation of the amphibian nervous system. In addition, using 2-deoxy-α-D-glucose, an inhibitor of glucose metabolism, it is shown that the stimulatory effects of exogenous insulin on developing frog embryos are, at least partially, through the glucose metabolism pathway. Preliminary results of this study were presented at the National Symposium on Genes and Human Environment, held at Hyderabad, February 1994 and DAE Symposium on Stress and Adaptive Responses in Biological Systems, held at Vadodara, March 1994.     

Cloning and expression pattern of a mouse homologue of drosophila sprouty in the mouse embryo.

Signaling molecules belonging to the Fibroblast growth factor (Fgf) family are necessary for directing bud outgrowth during tracheal development in Drosophila and lung development in mouse. A potential inhibitor of the Fgf signaling pathway, called Sprouty, has been identified in Drosophila. We have identified three potential mouse homologues of sprouty. One of them, called Sprouty4, exhibits a very restricted expression pattern. At 8.0 dpc (days post coitum) Sprouty4 is strongly expressed in the primitive streak region. At 9. 5 and 10.5 dpc, Sprouty4 is expressed in the nasal placode, the maxillary and mandibular processes, the otic vesicule, the second branchial arch, in the progress region of the limb buds and the presomitic mesoderm. Sprouty4 expression is also detected in the lateral region of the somites. In the developing lung, Sprouty4 is expressed broadly in the distal mesenchyme.     

Calcium mediates dorsoventral patterning of mesoderm in Xenopus.

Calcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that alpha1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity.     

Altered mitotic domains reveal fate map changes in Drosophila embryos mutant for zygotic dorsoventral patterning genes.

The spatial and temporal pattern of mitoses during the fourteenth nuclear cycle in a Drosophila embryo reflects differences in cell identities. We have analysed the domains of mitotic division in zygotic mutants that exhibit defects in larval cuticular pattern along the dorsoventral axis. This is a powerful means of fate mapping mutant embryos, as the altered position of mitotic domains in the dorsoventral pattern mutants correlate with their late cuticular phenotypes. In the mutants twist and snail, which fail to differentiate the ventrally derived mesoderm, mitoses specific to the mesoderm are absent. The lateral mesectodermal domain shows a partial ventral shift in twist mutants but a proportion of ventral cells do not behave characteristically, suggesting that twist has a positive role in the establishment of the mesoderm. In contrast, snail is required to repress mesectodermal fates in cells of the presumptive mesoderm. In the absence of both genes, the mesodermal and the mesectodermal anlage are deleted. Mutations at five loci delete specific pattern elements in the dorsal half of the embryo and cause partial ventralization. Mutations in the genes zerknüllt and shrew affect cell division only in the dorsalmost cells corresponding to the amnioserosa, while the genes tolloid, screw and decapentaplegic (dpp) affect divisions in both the prospective amnioserosa and the dorsal epidermis. We demonstrate that in each of these mutants dorsally placed mitotic domains are absent and this effect is correlated with an expansion and dorsal shift in the position of more ventral domains. The loss of activity in each of the five genes results in qualitatively similar alterations in the mitotic pattern; mutations with stronger ventralizing phenotypes affect increasingly greater subsets of the dorsal cells. Double mutant analysis indicates that these genes act in a concerted manner to specify dorsal fates. The correlation between phenotypic strength and the progressive loss of dorsal pattern elements in the ventralized mutants, suggests that one of these gene products, perhaps dpp, may provide positional information in a graded manner.     

The maternal NF-kappaB/dorsal gradient of Tribolium castaneum: dynamics of early dorsoventral patterning in a short-germ beetle

In the long-germ insect Drosophila melanogaster dorsoventral polarity is induced by localized Toll-receptor activation which leads to the formation of a nuclear gradient of the rel/ NF-kappaB protein Dorsal. Peak levels of nuclear Dorsal are found in a ventral stripe spanning the entire length of the blastoderm embryo allowing all segments and their dorsoventral subdivisions to be synchronously specified before gastrulation. We show that a nuclear Dorsal protein gradient of similar anteroposterior extension exists in the short-germ beetle, Tribolium castaneum, which forms most segments from a posterior growth zone after gastrulation. In contrast to Drosophila, (i) nuclear accumulation is first uniform and then becomes progressively restricted to a narrow ventral stripe, (ii) gradient refinement is accompanied by changes in the zygotic expression of the Tribolium Toll-receptor suggesting feedback regulation and, (iii) the gradient only transiently overlaps with the expression of a potential target, the Tribolium twist homolog, and does not repress Tribolium decapentaplegic. No nuclear Dorsal is seen in the cells of the growth zone of Tribolium embryos, indicating that here dorsoventral patterning occurs by a different mechanism. However, Dorsal is up-regulated and transiently forms a nuclear gradient in the serosa, a protective extraembryonic cell layer ultimately covering the whole embryo.     

Extracellular modulation of BMP activity in patterning the dorsoventral axis

Signaling via bone morphogenetic proteins (BMPs) regulates a vast array of diverse biological processes in the developing embryo and in postembryonic life. Many insights into BMP signaling derive from studies of the BMP signaling gradients that pattern cell fates along the embryonic dorsal-ventral (DV) axis of both vertebrates and invertebrates. This review examines recent developments in the field of DV patterning by BMP signaling, focusing on extracellular modulation as a key mechanism in the formation of BMP signaling gradients in Drosophila, Xenopus, and zebrafish.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

The mechanisms of dorsoventral patterning in the vertebrate neural tube

We describe the essential features of and the molecules involved in dorsoventral (DV) patterning in the neural tube. The neural tube is, from its very outset, patterned in this axis as there is a roof plate, floor plate, and differing numbers and types of neuroblasts. These neuroblasts develop into different types of neurons which express a different range of marker genes. Early embryological experiments identified the notochord and the somites as being responsible for the DV patterning of the neural tube and we now know that 4 signaling molecules are involved and are generated by these surrounding structures. Fibroblast growth factors (FGFs) are produced by the caudal mesoderm and must be down-regulated before neural differentiation can occur. Retinoic acid (RA) is produced by the paraxial mesoderm and is an inducer of neural differentiation and patterning and is responsible for down-regulating FGF. Sonic hedgehog (Shh) is produced by the notochord and floor plate and is responsible for inducing ventral neural cell types in a concentration-dependent manner. Bone morphogenetic proteins (BMPs) are produced by the roof plate and are responsible for inducing dorsal neural cell types in a concentration-dependent manner. Subsequently, RA is used twice more. Once from the somites for motor neuron differentiation and secondly RA is used to define the motor neuron subtypes, but in the latter case it is generated within the neural tube from differentiating motor neurons rather than from outside. These 4 signaling molecules also interact with each other, generally in a repressive fashion, and DV patterning shows how complex these interactions can be.     

Regional differences in BMP-dependence of dorsoventral patterning in the leech Helobdella

In the leech Helobdella, the ectoderm exhibits a high degree of morphological homonomy between body segments, but pattern elements in lateral ectoderm arise via distinct cell lineages in the segments of the rostral and midbody regions. In each of the four rostral segments, a complete set of ventrolateral (O fate) and dorsolateral (P fate) ectodermal pattern elements arises from a single founder cell, op. In the 28 midbody and caudal segments, however, there are two initially indeterminate o/p founder cells; the more dorsal of these is induced to adopt the P fate by BMP5-8 emanating from the dorsalmost ectoderm, while the more ventral cell assumes the O fate. Previous work has suggested that the dorsoventral patterning of O and P fates differs in the rostral region, but the role of BMP signaling in those segments has not been investigated. We show here that suppression of dorsal BMP5-8 signaling (which effects a P-to-O fate change in the midbody) has no effect on the patterning of O and P fates in the rostral region. Furthermore, ectopic expression of BMP5-8 in the ventral ectoderm (which induces an O-to-P fate change in the midbody) has no effect in the rostral region. Finally, expression of a dominant-negative BMP receptor (which induces a P-to-O fate change in the midbody) fails to affect O/P patterning in the rostral region. Thus, the rostral segments appear to use some mechanism other than BMP signaling to pattern O and P cell fates along the dorsoventral axis. From a mechanistic standpoint, the OP lineage of the rostral segments and the O-P equivalence group of the midbody and caudal segments constitute distinct developmental modules that rely to differing degrees on positional cues from surrounding ectoderm in order to specify homonomous cell fates.     

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods

Segmentation, i.e. the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. Here we present the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods.     

Domain mapping of tube, a protein essential for dorsoventral patterning of the Drosophila embryo.

The tube protein plays an essential role in the signal transduction pathway that establishes dorsoventral polarity in the Drosophila melanogaster embryo. Characterization of each of four tube mutants revealed a substitution or insertion in the amino-terminal half of the protein. This portion of the tube protein is also evolutionarily conserved, as demonstrated by isolation and sequencing of the Drosophila virilis tube gene. Moreover, RNA microinjection assays and germline transformation experiments demonstrated that the amino-terminal domain alone provides substantial levels of gene function: constructs encoding only the amino-terminal domain restore dorsoventral polarity to embryos lacking any maternal tube function. In the carboxyterminal domain, sequence conservation is concentrated in the five octapeptide repeats. Although the repeat-containing domain by itself provides no rescue of the tube maternal effect phenotype, it is necessary for wild-type levels of tube activity. This domain is thus likely to play an ancillary role in axis formation.     

Abnormal anteroposterior and dorsoventral patterning of the limb bud in the absence of retinoids.

We describe here how the early limb bud of the quail embryo develops in the absence of retinoids, including retinoic acid. Retinoid-deficient embryos develop to about stage 20/21, thus allowing patterns of early gene activity in the limb bud to be readily examined. Genes representing different aspects of limb polarity were analysed. Concerning the anteroposterior axis, Hoxb-8 was up-regulated and its border was shifted anteriorly whereas shh and the mesodermal expression of bmp-2 were down-regulated in the absence of retinoids. Concerning the apical ectodermal genes, fgf-4 was down-regulated whereas fgf-8 and the ectodermal domain of bmp-2 were unaffected. Genes involved in dorsoventral polarity were all disrupted. Wnt-7a, normally confined to the dorsal ectoderm, was ectopically expressed in the ventral ectoderm and the corresponding dorsal mesodermal gene Lmx-1 spread into the ventral mesoderm. En-1 was partially or completely absent from the ventral ectoderm. These dorsoventral patterns of expression resemble those seen in En-1 knockout mouse limb buds. Overall, the patterns of gene expression are also similar to the Japanese limbless mutant. These experiments demonstrate that the retinoid-deficient embryo is a valuable tool for dissecting pathways of gene activity in the limb bud and reveal for the first time a role for retinoic acid in the organisation of the dorsoventral axis.     

Drosophila-raf acts to elaborate dorsoventral pattern in the ectoderm of developing embryos.

In the early Drosophila embryo the activity of the EGF-receptor (Egfr) is required to instruct cells to adopt a ventral neuroectodermal fate. Using a gain-of-function mutation we showed that D-raf acts to transmit this and other late-acting embryonic Egfr signals. A novel role for D-raf was also identified in lateral cell development using partial loss-of-function D-raf mutations. Thus, we provide evidence that zygotic D-raf acts to specify cell fates in two distinct pathways that generate dorsoventral pattern within the ectoderm. These functional requirements for D-raf activity occur subsequent to its maternal role in organizing the anterioposterior axis. The consequences of eliminating key D-raf regulatory domains and specific serine residues in the transmission of Egfr and lateral epidermal signals were also addressed here.     

Microtubules and specification of the dorsoventral axis in frog embryos

The body plan of the frog is set-up by a rearrangement of the egg cytoplasm shortly after fertilization. Microtubules play several roles in this critical developmental event.