Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.     

Zygotically active genes that affect the spatial expression of the fushi tarazu segmentation gene during early Drosophila embryogenesis

The establishment of the segmental body pattern of Drosophila requires the coordinated functions of three classes of zygotically active genes early in development. We have examined the effects of mutations in these genes on the spatial expression of the fushi tarazu (ftz) pair-rule segmentation gene. Mutations in four gap loci and in three pair-rule loci dramatically affect the initial pattern of transverse stripes of ftz-containing nuclei. Five other pair-rule genes and several other loci that affect the larval cuticular pattern do not detectably affect ftz expression. No simple regulatory relationships can be deduced. Rather, expression of the ftz gene depends upon the interactions among the different segmentation genes active at each position along the anterior-posterior axis of the early embryo.     

Non-periodic cues generate seven ftz stripes in the Drosophila embryo

We have examined the expression pattern of the segmentation gene fushi tarazu (ftz) by in situ hybridization to whole mount embryos using digoxygenin labeled probes. This method has revealed previously undetected stages in the development of the ftz RNA pattern. The ftz stripes arise individually in a distinct, non-linear order along the anterior-posterior axis of the embryo. In addition, the stripes develop differentially along the dorsal-ventral axis; most stripes emerge on the ventral side and then gradually spread dorsally until they surround the entire circumference of the embryo. The order of appearance of ftz stripes is not inversely correlated with the order of appearance of hairy (h) stripes as would be expected if ftz stripes were generated by h repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, eve or runt, with normal expression patterns decaying only after cellularization. Thus, the so called primary pair-rule genes are involved in the refinement rather than establishment of the ftz stripes. The contribution of cis-acting regulatory elements to the ftz pattern was examined. The zebra and upstream elements interact to generate seven correctly positioned stripes at the end of cellularization. However, stripe establishement is not correctly mimicked by any ftz/lac fusion gene: stripes arise in an order drastically different from the endogenous ftz gene suggesting the existence of ftz regulatory elements outside the 10-kb region examined to date. These observations suggest that the ftz pattern is directed by at least two independent regulatory systems: first, stripe establishment is directed by regionally distributed factors that act differentially in individual stripes along both anterior-posterior and dorsal-ventral axes of the egg and, second, stripe refinement and maintenance are mediated by pair-rule gene products that interact with previously identified ftz regulatory elements. This multi-level regulation provides a back-up system that ensures the development of seven stripes in the blastoderm.     

Genetic control of cell fate in the termini of the Drosophila embryo.

Cell fates in the anterior and posterior termini of the Drosophila embryo are programmed by multiple zygotic genes that are regulated in response to a maternally encoded signal transduction pathway. These genes specify terminal as distinct from central cell fates, program pattern along the anteroposterior and dorsoventral axes of the termini, and also control endoderm specification and terminal morphogenetic movements. Here, we use a genetic interaction test to dissect the zygotic components of the terminal genetic hierarchy. We show that two genes, lines and empty spiracles, act downstream of tailless to repress central and promote terminal cell fates along the anteroposterior axis of the termini. Genes that control dorsoventral pattern in the termini and genes that program terminal morphogenesis act in distinct branches of the genetic hierarchy that are independent of tailless.     

cactus, a maternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophila embryos.

The dorsoventral pattern of the Drosophila embryo is mediated by a gradient of nuclear localization of the dorsal protein which acts as a morphogen. Establishment of the nuclear concentration gradient of dorsal protein requires the activities of the 10 maternal 'dorsal group' genes whose function results in the positive regulation of the nuclear uptake of the dorsal protein. Here we show that in contrast to the dorsal group genes, the maternal gene cactus acts as a negative regulator of the nuclear localization of the dorsal protein. While loss of function mutations of any of the dorsal group genes lead to dorsalized embryos, loss of cactus function results in a ventralization of the body pattern. Progressive loss of maternal cactus activity causes progressive loss of dorsal pattern elements accompanied by the expansion of ventrolateral and ventral anlagen. However, embryos still retain dorsoventral polarity, even if derived from germline clones using the strongest available, zygotic lethal cactus alleles. In contrast to the loss-of-function alleles, gain-of-function alleles of cactus cause a dorsalization of the embryonic pattern. Genetic studies indicate that they are not overproducers of normal activity, but rather synthesize products with altered function. Epistatic relationships of cactus with dorsal group genes were investigated by double mutant analysis. The dorsalized phenotype of the dorsal mutation is unchanged upon loss of cactus activity. This result implies that cactus acts via dorsal and has no independent morphogen function. In all other dorsal group mutant backgrounds, reduction of cactus function leads to embryos that express ventrolateral pattern elements and have increased nuclear uptake of the dorsal protein at all positions along the dorsoventral axis. Thus, the cactus gene product can prevent nuclear transport of dorsal protein in the absence of function of the dorsal group genes. Genetic and cytoplasmic transplantation studies suggest that the cactus product is evenly distributed along the dorsoventral axis. Thus the inhibitory function that cactus product exerts on the nuclear transport of the dorsal protein appears to be antagonized on the ventral side. We discuss models of how the action of the dorsal group genes might counteract the cactus function ventrally.     

A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo

The dorsoventral axis of the Drosophila embryo is determined by a morphogen gradient established by the action of 12 maternal-effect genes: the dorsal group genes and cactus. One of the dorsal group genes, dorsal (dl), encodes the putative morphogen. Although no overall asymmetry in the distribution of dorsal protein is observed, a gradient of nuclear concentration of dl protein is established during cleavage stages, with a maximum at the ventral side of the egg. At the dorsal side of the egg, the protein remains in the cytoplasm. Nuclear localization of the dl protein, and hence gradient formation, is blocked in dorsalizing alleles of all of the other dorsal group genes, while in ventralizing mutants nuclear localization extends to the dorsal side of the egg. A correlation between dl protein distribution and embryonic pattern in mutant embryos indicates that the nuclear concentration of the dl protein determines pattern along the dorsoventral axis.     

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.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

A screen for genes that interact with the Drosophila pair-rule segmentation gene fushi tarazu

The pair-rule gene fushi tarazu (ftz) of Drosophila is expressed at the blastoderm stage in seven stripes that serve to define the even-numbered parasegments. ftz encodes a DNA-binding homeodomain protein and is known to regulate genes of the segment polarity, homeotic, and pair-rule classes. Despite intensive analysis in a number of laboratories, how ftz is regulated and how it controls its targets are still poorly understood. To help understand these processes, we conducted a screen to identify dominant mutations that enhance the lethality of a ftz temperature-sensitive mutant. Twenty-six enhancers were isolated, which define 21 genes. All but one of the mutations recovered show a maternal effect in their interaction with ftz. Three of the enhancers proved to be alleles of the known ftz protein cofactor gene ftz-f1, demonstrating the efficacy of the screen. Four enhancers are alleles of Atrophin (Atro), the Drosophila homolog of the human gene responsible for the neurodegenerative disease dentatorubral-pallidoluysian atrophy. Embryos from Atro mutant germ-line mothers lack the even-numbered (ftz-dependent) engrailed stripes and show strong ftz-like segmentation defects. These defects likely result from a reduction in Even-skipped (Eve) repression ability, as Atro has been shown to function as a corepressor for Eve. In this study, we present evidence that Atro is also a member of the trithorax group (trxG) of Hox gene regulators. Atro appears to be particularly closely related in function to the trxG gene osa, which encodes a component of the brahma chromatin remodeling complex. One additional gene was identified that causes pair-rule segmentation defects in embryos from homozygous mutant germ-line mothers. The single allele of this gene, called bek, also causes nuclear abnormalities similar to those caused by alleles of the Trithorax-like gene, which encodes the GAGA factor.     

The polarity of the dorsoventral axis in the Drosophila embryo is defined by an extracellular signal

Twelve maternal effect loci are required for the production of Drosophila embryos with a correct dorsoventral axis. Analysis of mosaic females indicates that the expression of the genes nudel, pipe, and windbeutel is required in the somatic tissue, presumably in the follicle cells that surround the oocyte. Thus, information coming from outside the egg cell influences dorsoventral pattern formation during embryogenesis. In transplantation experiments, the perivitelline fluid from the compartment surrounding the embryo can restore dorsoventral pattern to embryos from females mutant for nudel, pipe, or windbeutel. The positioning of the transplanted pervitelline fluid also determines the polarity of the restored dorsoventral axis. We propose that the polarizing activity, normally present at the ventral side of the egg, is a ligand for the Toll receptor. Presumably, local activation of the Toll protein by the ligand initiates the formation of the nuclear concentration gradient of the dorsal protein, thereby determining dorsoventral pattern.     

The polarising role of cell adhesion molecules in early development

Polarising a cell or an embryo is a crucial and recurrent event during development, as it is important for cell differentiation and migration. Cells can become polarised along their apical-basal axis and also within the plane of the tissue layer to which they belong. The embryo develops three axes: the anteroposterior, the dorsoventral and the left-right axis. Recent work indicates instructive roles for cell adhesion molecules in establishing not only apical-basal polarity but also planar cell polarity and, surprisingly, in the generation of left-right asymmetry in vertebrates. Signalling cascades that regulate polarity formation seem to be conserved among different organisms, thereby raising the intriguing question of whether this also holds true for the cell adhesion molecules.     

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.     

Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression.

The Hox genes are a class of putative developmental control genes that are thought to be involved in the specification of positional identity along the anteroposterior axis of the vertebrate embryo. It is apparent from their expression pattern that their regulation is dependent upon positional information. In a previous analysis of the Hox-1.1 promoter in transgenic mice, we identified sequences that were sufficient to establish transgene expression in a specific region of the embryo. The construct used, however, did not contain enough regulatory sequences to reproduce all aspects of Hox-1.1 expression. In particular, neither a posterior boundary nor a restriction of expression to prevertebrae was achieved. Here we show correct regulation by Hox-1.1 sequences in transgenic mice and identify the elements responsible for different levels of control. Concomitant with the subdivision of mesodermal cells into different lineages during gastrulation and organogenesis, Hox-1.1 expression is restricted to successively smaller sets of cells. Distinct elements are required at different stages of development to execute this developmental programme. One position-responsive element (130 bp nontranslated leader) was shown to be crucial for the restriction of expression not only along the anteroposterior axis of the embryo, setting the posterior border, but also along the dorsoventral axis of the neural tube and to the lineage giving rise to the prevertebrae. Thus, Hox-1.1 expression is established in a specific region of the embryo and in a specific lineage of the mesoderm by restricting the activity of the promoter by the combined effect of several regulatory elements.     

Analysis of maternal effect mutant combinations elucidates regulation and function of the overlap of hunchback and Krüppel gene expression in the Drosophila blastoderm embryo

The metameric organisation of the Drosophila embryo is generated early during development, due to the action of maternal effect and zygotic segmentation and homeotic genes. The gap genes participate in the complex process of pattern formation by providing a link between the maternal and the zygotic gene activities. Under the influence of maternal gene products they become expressed in distinct domains along the anteroposterior axis of the embryo; negative interactions between neighboring gap genes are thought to be involved in establishing the expression domains. The gap gene activities in turn are required for the correct patterning of the pair-rule genes; little is known, however, about the underlying mechanisms. We have monitored the distribution of gap and pair-rule genes in wild-type embryos and in embryos in which the anteroposterior body pattern is greatly simplified due to combinations of maternal effect mutations (staufen exuperantia, vasa exuperantia, vasa exuperantia, bicoid oskar, bicoid oskar torsolike, vasa torso exuperantia). We show that the domains of protein distribution of the gap genes hunchback and Krüppel overlap in wild-type embryos. Based on the analysis of the maternal mutant combinations, we suggest an explanation of how this overlap is generated. Furthermore, our data show that different constellations of gap gene activities provide different input for the pair-rule genes, and thus strongly suggest that the overlap of hunchback and Krüppel in wild-type is functional in the formation of the patterns of pair-rule genes.     

Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.

The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.