Scaling of internal organs during Drosophila embryonic development

Many species show a diverse range of sizes; for example, domestic dogs have large variation in body mass. Yet, the internal structure of the organism remains similar, i.e., the system scales to organism size. Drosophila melanogaster has been a powerful model system for exploring scaling mechanisms. In the early embryo, gene expression boundaries scale very precisely to embryo length. Later in development, the adult wings grow with remarkable symmetry and scale well with animal size. Yet, our knowledge of whether internal organs initially scale to embryo size remains largely unknown. Here, we utilize artificially small Drosophila embryos to explore how three critical internal organs—the heart, hindgut, and ventral nerve cord (VNC)—adapt to changes in embryo morphology. We find that the heart scales precisely with embryo length. Intriguingly, reduction in cardiac cell length, rather than number, appears to be important in controlling heart length. The hindgut, which is the first chiral organ to form, displays scaling with embryo size under large-scale changes in the artificially smaller embryos but shows few hallmarks of scaling within wild-type size variation. Finally, the VNC only displays weak scaling behavior; even large changes in embryo geometry result in only small shifts in VNC length. This suggests that the VNC may have an intrinsic minimal length that is largely independent of embryo length. Overall, our work shows that internal organs can adapt to embryo size changes in Drosophila, but the extent to which they scale varies significantly between organs.     

Changes in galactosyltransferase activity in chick pectoral muscle during embryonic development.

The two major vertebrate galactosyltransferases have been investigated in developing chick muscle in ovo and in vitro, and in cultured chick fibroblasts. The two enzymes were UDP-galactose-N-acetylglucosamine galactosyltransferase (galactosyltransferase I) and UDP-galactose-N-acetylgalactosamine galactosyltransferase (galactosyltransferase II). Both activities fell during muscle development in ovo. Galactosyltransferase I activity was constant from day 7 to day 16, after which it declined 5-fold, whereas galactosyltransferase II activity fell markedly from day 9 to 13 and 16 to 20, displaying an overall 8-fold decrease. In primary muscle cultures, galactosyltransferase I activity fell slightly during 7 days in culture, whereas galactosyltransferase II increased 2-fold during the same period. No significant change in activity of either galactosyltransferase was observed during intercellular recognition and fusion. Analysis of muscle cultures treated with cytosine arabinoside and of fibroblast cultures revealed that the majority of galactosyltransferase I activity in primary muscle cultures is associated with fibroblasts, whereas the majority of galactosyltransferase II activity is muscle-associated. The addition of 5-bromodeoxyuridine to primary muscle cultures resulted in a 3-fold rise in activities of both transferases.     

Expression pattern of DMAP-85 during Drosophila embryonic development

Microtubule-associated proteins (MAPs) play major regulatory roles on the organization and integrity of the cytoskeletal network. Previously, we identified DMAP-85, a Drosophila MAP that promotes tubulin polymerization in vitro. In this work, we examine the distribution of DMAP-85 and its association pattern with microtubules at embryonic stages. Immunoblots revealed that DMAP-85 was present throughout embryogenesis, but it was most abundant in stages 6-9. Immunofluorescence studies showed that DMAP-85 was associated with sub-populations of stable microtubules during embryo cellularization, and after gastrulation with interphase microtubule arrays. At late embryonic stages, it was preferentially found in the ventral nerve cord, co-localizing with axonal microtubules. These observations are in agreement with previous reports on DMAP-85 functions, suggesting that DMAP-85 might be required for the stabilization and organization of cytoplasmic microtubules during embryonic development.     

Glia development in the embryonic CNS of Drosophila.

Glial cells are pivotal players during the development and function of complex nervous systems. In Drosophila, recent genetic analyses have revealed several genes that control differentiation and function of CNS glial cells and their interactions with neurons can be studied in detail at the CNS midline, where it is essential for the correct establishment of the commissural axon pattern.     

Histones and histone acetylation during the embryonic development of Drosophila hydei

Histones and histone acetylation have been investigated during three stages of Drosophila hydei embryogenesis--early gastrula, late gastrula and organogenesis. No essential changes in the electrophoretic pattern of the histones have been revealed during the stages examined. However, we established an enhanced level of [14C]acetate incorporation at the time of extensive gene activation during gastrulation as well as some quantitative differences in the pattern of acetylation during gastrula and organogenesis. We consider most of them to be related to chromatin assembly during the stage of gastrulation and suggest that the correlation between histone acetylation and gene activity during Drosophila embryogenesis concerns histone H3 acetylation. The involvement of both acetylation and deacetylation in the steady-state acetylation level has been examined as well. We have found that the higher acetyltransferase activity is responsible for the enhanced level of acetate incorporation during gastrulation.     

Thymidine kinase activity in cardiac muscle during embryonic and postnatal development

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a K(m) value for thymidine of 1.6mum. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.     

Signals regulating muscle formation in the limb during embryonic development

Classically, somites have been the preparation of choice for the study of muscle development, while the limb bud is the preferred model of axis formation. Nevertheless, the limb bud offers some experimental advantages for muscle studies. This review describes the successive events involved in limb muscle formation during embryonic development, the properties of the key marker molecules and resumes our current knowledge of the signalling pathways involved.     

Arabidopsis VILLIN2 and VILLIN3 act redundantly in sclerenchyma development via bundling of actin filaments

The organization of the actin cytoskeleton has been implicated in sclerenchyma development. However, the molecular mechanisms linking the actin cytoskeleton to this process remain poorly understood. In particular, there have been no studies showing that direct genetic manipulation of the actin cytoskeleton affects sclerenchyma development. Villins belong to the villin/gelsolin/fragmin superfamily and are versatile actin-modifying proteins. Several recent studies have implicated villins in tip growth of single cells, but how villins act in multicellular plant development remains largely unknown. Here, we found that two closely related villin isovariants from Arabidopsis, VLN2 and VLN3, act redundantly in sclerenchyma development. Detailed analysis of cross-sections from inflorescence stems of vln2 vln3 double mutant plants revealed a reduction in stem size and in the number of vascular bundles; however, no defects in synthesis of the secondary cell wall were detected. Surprisingly, the vln2 vln3 double mutation did not affect cell elongation of inter-fascicular fibers. Biochemical analyses showed that recombinant VLN2 was able to cap, sever and bundle actin filaments, similar to VLN3. Consistent with these biochemical activities, loss of function of VLN2 and VLN3 resulted in a decrease in the amount of F-actin and actin bundles in plant cells. Collectively, our findings demonstrate that VLN2 and VLN3 act redundantly in sclerenchyma development via bundling of actin filaments.     

Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.

In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning.     

Dynamic expression of the cell adhesion molecule fasciclin I during embryonic development in Drosophila.

A number of different cell surface glycoproteins expressed in the central nervous system (CNS) have been identified in insects and shown to mediate cell adhesion in tissue culture systems. The fasciclin I protein is expressed on a subset of CNS axon pathways in both grasshopper and Drosophila. It consists of four homologous 150-amino acid domains which are unrelated to other sequences in the current databases, and is tethered to the cell surface by a glycosyl-phosphatidylinositol linkage. In this paper we examine in detail the expression of fasciclin I mRNA and protein during Drosophila embryonic development. We find that fasciclin I is expressed in several distinct patterns at different stages of development. In blastoderm embryos it is briefly localized in a graded pattern. During the germ band extended period its expression evolves through two distinct phases. Fasciclin I mRNA and protein are initially localized in a 14-stripe pattern which corresponds to segmentally repeated patches of neuroepithelial cells and neuroblasts. Expression then becomes confined to CNS and peripheral sensory (PNS) neurons. Fasciclin I is expressed on all PNS neurons, and this expression is stably maintained for several hours. In the CNS, fasciclin I is initially expressed on all commissural axons, but then becomes restricted to specific axon bundles. The early commissural expression pattern is not observed in grasshopper embryos, but the later bundle-specific pattern is very similar to that seen in grasshopper. The existence of an initial phase of expression on all commissural bundles helps to explain the loss-of-commissures phenotype of embryos lacking expression of both fasciclin I and of the D-abl tyrosine kinase. Fasciclin I is also expressed in several nonneural tissues in the embryo.     

Modeling the precision and robustness of Hunchback border during Drosophila embryonic development

During anterior-posterior axis specification in the Drosophila embryo, the Hunchback (Hb) protein forms a sharp boundary at the mid-point of the embryo with great positional precision. While Bicoid (Bcd) is a known upstream regulator for hb expression, there is evidence to suggest that Hb effectively filters out “noisy” data received from varied Bcd gradients. We use mathematical models to explore simple regulatory networks which filter out such noise to produce a precise Hb boundary. We find that in addition to Bcd and Hb, at least one freely evolving protein is necessary. An automated search yields a number of examples of three-protein networks exhibiting the desired precision. In all such networks, Hb diffuses much slower than the third protein. In addition, the action of Hb on the third protein is the opposite of the action of the third protein on hb (i.e. if Hb activates the third protein, then the third protein inhibits hb expression, and vice versa). Most of the discovered systems satisfy the known biological properties, that Bcd activates hb, and that Hb activates its own expression. We find that all network topologies satisfying these constraints arise among the networks exhibiting the desired precision. Investigating the dynamics of these networks, we find that under a general class of non-uniform initial conditions, Bcd can be eliminated from the system and the spatiotemporal evolution of these two proteins alone is sufficient to recapture the dynamics. We hypothesize that Bcd is needed only to spatially disturb the gradient of the third protein, and then becomes unnecessary in the further evolution of the Hb border. This provides a possible explanation as to why the Hb dynamics are robust under perturbations of the Bcd gradient. Under this hypothesis, other proteins would be able to assume the role of Bcd in our simulations (possibly in the case of evolutionary divergences or a redundancy in the process), with the only constraint that they act to positively regulate hb.     

mRNA usage during Drosophila melanogaster embryonic development. Analysis of nine cloned DNA segments

A set of nine phage lambda clones containing inserts from Drosophila melanogaster which are complementary to cDNA made from oocyte poly(A)+ RNA were selected from a larger group. These cloned elements code for a range of middle abundant RNA sequences which show no appreciable change in abundance during Drosophila embryogenesis. Seven of the nine clones are complementary to two oocyte RNAs, one to three RNAs and one to four RNAs. This study describes the changes that occur in these RNAs during embryonic development in the polysomal and non-polysomal fraction, and in the poly(A)+ RNA and poly(A)- RNA fraction. In all nine of these clones, greater than 70% of the complementary RNA is found in the polysomal region of a sucrose gradient. This proportion increases somewhat during development. Specific changes have been found during development in the proportion of RNA that is poly(A)+. Depending to the cloned sequence, this proportion may increase, decrease, or remain unchanged. For those clones that show a change, most of this change occurs between 8 and 19 h of development. Our data suggest, furthermore, the presence of a class of non-adenylated RNA being utilized during embryogenesis.     

Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development.

The Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. We now demonstrate that sugarless and sulfateless mutant embryos have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. Moreover, both Heartless- and Breathless-dependent MAPK activation is significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. We also find that overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization.     

Tissue distribution of PEBBLE RNA and pebble protein during Drosophila embryonic development

pebble (pbl) is required for cytokinesis during postblastoderm mitoses (Hime, G., Saint, R., 1992. Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila. Development 114, 165–171; Lehner, C.F., 1992. The pebble gene is required for cytokinesis in Drosophila. J. Cell Sci. 103, 1021–1030) and encodes a putative guanine nucleotide exchange factor (RhoGEF) for Rho1 GTPase (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301–2314). Mutations in pbl result in the absence of a contractile ring leading to a failure of cytokinesis and formation of polyploid multinucleate cells. Analysis of the subcellular distribution of PBL demonstrated that during mitosis, PBL accumulates at the cleavage furrow at the anaphase to telophase transition when assembly of a contractile ring is initiated (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301–2314). In addition, levels of PBL protein cycle during each round of cell division with the highest levels of PBL found in telophase and interphase nuclei. Here, we report the expression pattern of pbl during embryonic development. We show that PEBBLE RNA and PBL protein have a similar tissue distribution and are expressed in a highly dynamic pattern throughout embryogenesis. We show that PBL is strongly enriched in dividing nuclei in syncytial embryos and in pole cells as well as in nuclei of dividing cells in postblastoderm embryos. Our expression data correlate well with the phenotypes observed in pole cells and, particularly, with the absence of cytokinesis after cellular blastoderm formation in pbl mutants.     

Interspecies comparison of a gene pair with partially redundant function: the rst and kirre genes in D. virilis and D. melanogaster

Abstract The D. melanogaster rst and kirre genes encode two highly related immunoglobulin-like cell adhesion molecules that function redundantly during embryonic muscle development. The two genes appear to be derived from a common ancestor by gene duplication. Gene duplications have been proposed to be of major evolutionary significance since duplicated redundant sequences can accumulate mutations without detrimental effects for the organism and leave the duplicated genes free to assume novel functions. To address the issue of conservation of the duplicated sequences and their putative redundancy, as well as to identify putative functional divergence of the paralogs during drosophilid evolution, we performed an interspecies comparison of the rst and kirre genes from D. virilis and D. melanogaster. The D. virilis genome contains orthologues of both rst and kirre and hence the duplication took place before the split of the two lineages and has subsequently been conserved. However, whilst the Rst orthologues show a high degree of sequence similarity, this similarity is lower in Kirre orthologues. Especially the intracellular domains of D. virilis and D. melanogaster Kirre sequences are highly divergent: the D. virilis kirre gene lacks the 3′-most exon present in D. melanogaster, which contains motifs conserved between kirre and rst in D. melanogaster. Hence, while each of the two genes is highly conserved at the level of its exon-intron organization, the selection forces acting on the rst and kirre coding sequences are different. These findings are discussed in the light of general evolutionary mechanisms.     

Expression of Drosophila Ten-a,a dimeric receptor during embryonic development

Ten-a and Ten-m are the two Drosophila members of the newly discovered Ten-m family of dimeric type II transmembrane proteins. Here, we report complete cDNA cloning and protein expression patterns of Ten-a. The Ten-a protein, a dimeric receptor of about 500 kDa is mainly expressed on axons of the embryonic central nervous system and on muscle attachment sites.