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PubMed1994

dishevelled is required during wingless signaling to establish both cell polarity and cell identity.

The dishevelled gene of Drosophila is required to establish coherent arrays of polarized cells and is also required to establish segments in the embryo. Here, we show that loss of dishevelled function in clones, in double heterozygotes with wingless mutants and in flies bearing a weak dishevelled transgene leads to patterning defects which phenocopy defects observed in wingless mutants alone. Further, polarized cells in all body segments require dishevelled function to establish planar cell polarity, and some wingless alleles and dishevelled; wingless double heterozygotes exhibit bristle polarity defects identical to those seen in dishevelled alone. The requirement for dishevelled in establishing polarity in cell autonomous. The dishevelled gene encodes a novel intracellular protein that shares an amino acid motif with several other proteins that are found associated with cell junctions. Clonal analysis of dishevelled in leg discs provides a unique opportunity to test the hypothesis that the wingless dishevelled interaction species at least one of the circumferential positional values predicted by the polar coordinate model. We propose that dishevelled encodes an intracellular protein required to respond to a wingless signal and that this interaction is essential for establishing both cell polarity and cell identity.

Amino Acid Sequence↗
PubMed1994

The Serrate locus of Drosophila and its role in morphogenesis of the wing imaginal discs: control of cell proliferation.

The Drosophila gene Serrate encodes a transmembrane protein with 14 EGF-like repeats in its extracellular domain. Here we show that loss-of-function mutations in this gene lead to larval lethality. Homozygous mutant larvae fail to differentiate the anterior spiracles, exhibit poorly developed mouth-hooks and show a severe reduction in the size of the wing and haltere primordia, which is not due to cell death. The few homozygous mutant escapers that pupariate develop into pharate adults that almost completely lack wings and halteres. Clonal analysis in the adult epidermis demonstrates a requirement for Serrate during wing and haltere development. Targeted ectopic expression of Serrate in the imaginal discs using the yeast transcriptional activator Gal4 results in regionally restricted induction of cell proliferation, e.g. the ventral tissues in the case of the wings and halteres. The results suggest that the wild-type function of Serrate is required for the control of position-specific cell proliferation during development of meso- and metathoracic dorsal discs, which in turn exerts a direct effect on morphogenesis.

Animals↗
PubMed1995

Suppressor of Hairless is required for signal reception during lateral inhibition in the Drosophila pupal notum.

Suppressor of Hairless (Su(H)) activity is zygotically required in larval imaginal discs for the singling out of adult sense organ precursor (SOP) cells: loss of Su(H) function results in too many proneural cluster cells adopting the SOP fate, while overexpression of the Su(H) protein prevents SOP specification. Su(H) null mutant alleles are recessive lethal at the late larval and early pupal stages. The development of Su(H) mutant cells in pupae was therefore studied in somatic clones. Clonal analysis first showed that Su(H) is required for the regular spacing of microchaete precursor cells, as clusters of mutant SOPs were detected at positions where singled out sense organ cells are normally found. Second, Su(H) mutant SOPs produced neuron-like cells, consistent with a late defect in Notch (N) signalling. Third, a careful cell-by-cell analysis of clone borders showed that Su(H) mutant cells may adopt the SOP fate even when directly adjacent to wild-type cells. Finally, quantitative clone border analysis indicates that the relative level of Su(H) gene dosage appears to bias the selection of the future SOP: cells with a higher level of Su(H) activity are more likely to adopt the epidermal fate. These results show that notum cells strictly require Su(H) activity for receiving the lateral inhibitory signal. Thus, the DNA-binding protein encoded by the Su(H) gene may act downstream of the N receptor to implement the epidermal, non-SOP fate.

Animals↗
PubMed1995

Notch regulates wingless expression and is not required for reception of the paracrine wingless signal during wing margin neurogenesis in Drosophila.

In the developing wing margin of Drosophila, wingless is normally expressed in a narrow stripe of cells adjacent to the proneural cells that form the sensory bristles of the margin. Previous work has shown that this wingless is required for the expression of the proneural achaete-scute complex genes and the subsequent formation of the sensory bristles along the margin; recently, it has been proposed that the proneural cells require the Notch protein to properly receive the wingless signal. We have used clonal analysis of a null allele of Notch to test this idea directly. We found that Notch was not required by prospective proneural margin cells for the expression of scute or the formation of sensory precursors, indicating Notch is not required for the reception of wingless signal. Loss of Notch from proneural cells produced cell-autonomous neurogenic phenotypes and precocious differentiation of sensory cells, as would be expected if Notch had a role in lateral inhibition within the proneural regions. However, loss of scute expression and of sensory precursors was observed if clones substantially included the normal region of wingless expression. These 'anti-proneural' phenotypes were associated with the loss of wingless expression; this loss may be partially or wholly responsible for the anti-proneural phenotype. Curiously, Notch- clones limited to the dorsal or ventral compartments could disrupt wingless expression and proneural development in the adjacent compartment. Analysis using the temperature-sensitive Notch allele indicated that the role of Notch in the regulation of wingless expression precedes the requirement for lateral inhibition in proneural cells. Furthermore, overexpression of wingless with a heat shock-wingless construct rescued the loss of sensory precursors associated with the early loss of Notch.

Animals↗
PubMed1996

Distinct spatial and temporal functions for PS integrins during Drosophila wing morphogenesis.

At the onset of pupariation in the Drosophila wing, the PS1 and PS2 integrins are expressed preferentially on the dorsal and ventral wing epithelia, respectively. Clonal analysis experiments have indicated that integrins are required to maintain the tight association of the wing surfaces. Surprisingly, we find that even in clones of cells lacking integrins the wing layers become apposed early in metamorphosis. However, following the normal period of wing separation, large integrin mutant clones do not become re-apposed in the pupa, and integrins are not organized in basal plaques in cells opposite a mutant clone. Paradoxically, our experiments indicate that at least one integrin function requires different integrins on the dorsal and ventral wing surfaces, however in some cases both alphaPS subunits can function to some degree on each wing surface. Finally, overexpression of an alphaPS subunit throughout the wing leads to a dominant wing blister phenotype, and the critical period for this phenotype is the beginning of pupariation. These data indicate that integrin requirements in wing morphogenesis can be separated into early (prepupal) and late (pupal) functions. The late function seems to reflect the traditional view of integrins as cell-matrix adhesion proteins. The early requirement, which probably requires dorsoventral segregation of PS1 and PS2, suggests functions for PS1 and PS2 in signaling events that regulate morphogenesis.

Animals↗
PubMed1996

The control of trichome spacing and number in Arabidopsis.

Arabidopsis trichomes are single-celled epidermal hairs that serve as a useful model for the study of plant cell differentiation. An examination of the distribution of trichomes early in their development revealed that developing trichomes occur adjacent to another trichome much less frequently than would be expected by chance. Clonal analysis of epidermal cell lineages ruled out a role for cell lineage in generating the observed minimum-distance spacing pattern. Taken together, these results are consistent with a role for lateral inhibition in the control of trichome development. We also report the identification of a new locus, Reduced Trichome Number (RTN), which affects the initiation of trichomes. This locus was initially detected by the reduced number of leaf trichomes on Landsberg erecta plants compared to that on Columbia plants. Quantitative Trait Locus mapping revealed that more than 73% of the variation in trichome number was due to a major locus near erecta on chromosome 2. The reduced number of trichomes conditioned by the Landsberg erecta allele of this locus appeared to be due to an early cessation of trichome initiation. The implications of these observations are discussed with regard to previously published models of trichome development.

Arabidopsis↗
PubMed1996

Alternatively spliced forms of the Drosophila alphaPS2 subunit of integrin are sufficient for viability and can replace the function of the alphaPS1 subunit of integrin in the retina.

The Drosophila inflated (if) gene encodes the alphaPS2 subunit of the PS family of integrins. The if transcript is spliced such that alphaPS2 is found in two alternative forms, alphaPS2(C) and alphaPS2(m8), which differ by 25 amino acid residues in a region shown to affect cation requirements and ligand specificity. In this study, we examine the functional significance of the protein isoforms of if by analyzing the ability of transgenes producing only one isoform to rescue developmental abnormalities associated with complete loss of PS2 integrin. We find that either form of alphaPS2 is sufficient to rescue if- animals to viability; however, the alphaPS2(C) form promotes higher survival of the organism. Furthermore, these studies suggest distinct roles for alphaPS2(C) and alphaPS2(m8) during development. When expressed in the developing wing, alphaPS2(m8) is more efficient at rescuing the if wing blister phenotype than is alphaPS2(C). Expression of alphaPS2(C) in the eye produces dominant disruption of photoreceptor organization. We have also examined the ability of alphaPS2 and alphaPS1 to maintain photoreceptor organization in the Drosophila retina. Clonal analysis of sectioned eyes suggests a requirement for alphaPS1, but not alphaPS2. However, ectopic expression of if(m8) or if(C) shows that either splice form Of alphaPS2 can functionally replace alphaPS1 and rescue the mew eye phenotype.

Alternative Splicing↗
PubMed1996

Mad acts downstream of Dpp receptors, revealing a differential requirement for dpp signaling in initiation and propagation of morphogenesis in the Drosophila eye.

Decapentaplegic (Dpp), a member of the TGF-beta family of cytokines, has been implicated in many patterning processes in Drosophila, including the initial steps of pattern formation in the developing eye. We show that the Mothers against dpp (Mad) gene is required for dpp signaling during eye development. Clonal analysis demonstrates a cell-autonomous function for Mad and genetic interactions indicate that Mad is an essential component of the signal transduction pathway downstream of the Dpp receptors in responding cells. Mad-mediated dpp signaling is absolutely required for the initiation of the morphogenetic furrow in the eye, but has only a minor role in its subsequent propagation across the eye disc. We also present evidence for the repression of wingless transcription by dpp signaling.

Alleles↗
PubMed1996

wingless signaling in the Drosophila eye and embryonic epidermis.

After the onset of pupation, sensory organ precursors, the progenitors of the interommatidial bristles, are selected in the developing Drosophila eye. We have found that wingless, when expressed ectopically in the eye via the sevenless promoter, blocks this process. Transgenic eyes have reduced expression of acheate, suggesting that wingless acts at the level of the proneural genes to block bristle development. This is in contrast to the wing, where wingless positively regulates acheate to promote bristle formation. The sevenless promoter is not active in the acheate-positive cells, indicating that the wingless is acting in a paracrine manner. Clonal analysis revealed a requirement for the genes porcupine, dishevelled and armadillo in mediating the wingless effect. Overexpression of zeste white-3 partially blocks the ability of wingless to inhibit bristle formation, consistent with the notion that wingless acts in opposition to zeste white-3. Thus the wingless signaling pathway in the eye appears to be very similar to that described in the embryo and wing. The Notch gene product has also been suggested to play a role in wingless signaling (J. P. Couso and A. M. Martinez Arias (1994) Cell 79, 259-72). Because Notch has many functions during eye development, including its role in inhibiting bristle formation through the neurogenic pathway, it is difficult to assess the relationship of Notch to wingless in the eye. However, we present evidence that wingless signaling still occurs normally in the complete absence of Notch protein in the embryonic epidermis. Thus, in the simplest model for wingless signalling, a direct role for Notch is unlikely.

Animals↗
PubMed1996

Evidence in the mouse for self-renewing stem cells in the formation of a segmented longitudinal structure, the myotome.

A novel method of clonal analysis has been used in the mouse to define the cellular events that lead to the formation of a segmented longitudinal structure, the myotome. Progenitor cells of the myotome were randomly marked during development by intragenic homologous recombination in transgenic mice expressing a reporter laacZ gene. 153 clones corresponding to 7829 cells, that is 20% of the myotomal population of one embryo, were obtained from 3000 E11.5 embryos. Their analysis leads to the hypothesis that, at E11.5, the 41 segments of the myotome have been mainly produced from a unique, spatially organised pool of self-renewing stem cells that accompanies the formation of the anterior-posterior axis.

Animals↗
PubMed1997

The allocation of early blastomeres to the ectoderm and endoderm is variable in the sea urchin embryo.

During sea urchin development, a tier-to-tier progression of cell signaling events is thought to segregate the early blastomeres to five different cell lineages by the 60-cell stage (E. H. Davidson, 1989, Development 105, 421-445). For example, the sixth equatorial cleavage produces two tiers of sister cells called 'veg1' and 'veg2,' which were projected by early studies to be allocated to the ectoderm and endoderm, respectively. Recent in vitro studies have proposed that the segregation of veg1 and veg2 cells to distinct fates involves signaling between the veg1 and veg2 tiers (O. Khaner and F. Wilt, 1991, Development 112, 881-890). However, fate-mapping studies on 60-cell stage embryos have not been performed with modern lineage tracers, and cell interactions between veg1 and veg2 cells have not been shown in vivo. Therefore, as an initial step towards examining how archenteron precursors are specified, a clonal analysis of veg1 and veg2 cells was performed using the lipophilic dye, DiI(C16), in the sea urchin species, Lytechinus variegatus. Both veg1 and veg2 descendants form archenteron tissues, revealing that the ectoderm and endoderm are not segregated at the sixth cleavage. Also, this division does not demarcate cell type boundaries within the endoderm, because both veg1 and veg2 descendants make an overlapping range of endodermal cell types. The allocation of veg1 cells to ectoderm and endoderm during cleavage is variable, as revealed by both the failure of veg1 descendants labeled at the eighth equatorial division to segregate predictably to either tissue and the large differences in the numbers of veg1 descendants that contribute to the ectoderm. Furthermore, DiI-labeled mesomeres of 32-cell stage embryos also contribute to the endoderm at a low frequency. These results show that the prospective archenteron is produced by a larger population of cleavage-stage blastomeres than believed previously. The segregation of veg1 cells to the ectoderm and endoderm occurs relatively late during development and is unpredictable, indicating that later cell position is more important than the early cleavage pattern in determining ectodermal and archenteron cell fates.

Animals↗
PubMed1998

Complete dissipation of coherent clonal growth occurs before gastrulation in mouse epiblast.

Observations on chimaeric mice argue that there must be considerable dispersal and intermingling of the clonal descendants of epiblast founder cells at an early stage in the development of the tissue. However, it has not been established when or how this occurs. Here we have used a genetic marker that enables donor cells to be visualized in situ to examine the early postimplantation distribution of clones obtained by transplanting epiblast founder cells into host blastocysts. We have also determined the spatial relationship between sister cells in non-chimaeric postimplantation epiblast by ionophoretic injection of a fluorescent macromolecule. Both experimental approaches support the conclusion that breakdown of coherent clonal growth accompanies epithelialization of the epiblast and is essentially complete by the onset of gastrulation. Furthermore, the clonal analysis shows that descendants of different epiblast founder cells continue to intermingle extensively well into organogenesis. We suggest that this sustained intermingling of cells in the epithelial epiblast, which does not occur in the adjacent visceral endoderm, depends on cells losing contact with the basal lamina when they divide. These findings have implications both for patterning of the early amniote embryo and for the growth of tall columnar epithelia in general.

Animals↗
PubMed1998

A subset of notch functions during Drosophila eye development require Su(H) and the E(spl) gene complex.

The Notch signalling pathway is involved in many processes where cell fate is decided. Previous work showed that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signalling represses atonal expression during lateral specification. In this paper we investigate which known components of the Notch pathway are involved in each signalling process. Using clonal analysis we show that a ligand of Notch, Delta, is required along with Notch for both proneural enhancement and lateral specification, while the downstream components Suppressor-of-Hairless and Enhancer-of-Split are involved only in lateral specification. Our data point to a distinct signal transduction pathway during proneural enhancement by Notch. Using misexpression experiments we also show that particular Enhancer-of-split bHLH genes can differ greatly in their contribution to lateral specification.

Animals↗
PubMed1998

The roles of the homeobox genes aristaless and Distal-less in patterning the legs and wings of Drosophila.

In the leg and wing imaginal discs of Drosophila, the expression domains of the homeobox genes aristaless (al) and Distal-less (Dll) are defined by the secreted signaling molecules Wingless (Wg) and Decapentaplegic (Dpp). Here, the roles played by al and Dll in patterning the legs and wings have been investigated through loss of function studies. In the developing leg, al is expressed at the presumptive tip and a molecularly defined null allele of al reveals that its only function in patterning the leg appears to be to direct the growth and differentiation of the structures at the tip. In contrast, Dll has previously been shown to be required for the development of all of the leg more distal than the coxa. Dll protein can be detected in a central domain in leg discs throughout most of larval development, and in mature discs this domain corresponds to the distal-most region of the leg, the tarsus and the distal tibia. Clonal analysis reveals that late in development these are the only regions in which Dll function is required. However, earlier in development Dll is required in more proximal regions of the leg suggesting it is expressed at high levels in these cells early in development but not later. This reveals a correlation between a temporal requirement for Dll and position along the proximodistal axis; how this may relate to the generation of the P/D axis is discussed. Dll is required in the distal regions of the leg for the expression of tarsal-specific genes including al and bric-a-brac. Dll mutant cells in the leg sort out from wild-type cells suggesting one function of Dll here is to control adhesive properties of cells. Dll is also required for the normal development of the wing, primarily for the differentiation of the wing margin.

Alleles↗
PubMed1999

Somatic signaling mediated by fs(1)Yb is essential for germline stem cell maintenance during Drosophila oogenesis.

Drosophila oogenesis starts when a germline stem cell divides asymmetrically to generate a daughter germline stem cell and a cystoblast that will develop into a mature egg. We show that the fs(1)Yb gene is essential for the maintenance of germline stem cells during oogenesis. We delineate fs(1)Yb within a 6.4 kb genomic region by transgenic rescue experiments. fs(1)Yb encodes a 4.1 kb RNA that is present in the third instar larval, pupal and adult stages, consistent with its role in regulating germline stem cells during oogenesis. Germline clonal analysis shows that all fs(1)Yb mutations are soma-dependent. In the adult ovary, fs(1)Yb is specifically expressed in the terminal filament cells, suggesting that fs(1)Yb acts in these signaling cells to maintain germline stem cells. fs(1)Yb encodes a novel hydrophilic protein with no potential signal peptide or transmembrane domains, suggesting that this protein is not itself a signal but a key component of the signaling machinery for germline stem cell maintenance.

Animals↗
PubMed2000

Combined activities of Gurken and decapentaplegic specify dorsal chorion structures of the Drosophila egg.

During Drosophila oogenesis Gurken, associated with the oocyte nucleus, activates the Drosophila EGF receptor in the follicular epithelium. Gurken first specifies posterior follicle cells, which in turn signal back to the oocyte to induce the migration of the oocyte nucleus from a posterior to an anterior-dorsal position. Here, Gurken signals again to specify dorsal follicle cells, which give rise to dorsal chorion structures including the dorsal appendages. If Gurken signaling is delayed and starts after stage 6 of oogenesis the nucleus remains at the posterior pole of the oocyte. Eggs develop with a posterior ring of dorsal appendage material that is produced by main-body follicle cells expressing the gene Broad-Complex. They encircle terminal follicle cells expressing variable amounts of the TGFbeta homologue, decapentaplegic. By ectopically expressing decapentaplegic and clonal analysis with Mothers against dpp we show that Decapentaplegic signaling is required for Broad-Complex expression. Thus, the specification and positioning of dorsal appendages along the anterior-posterior axis depends on the intersection of both Gurken and Decapentaplegic signaling. This intersection also induces rhomboid expression and thereby initiates the positive feedback loop of EGF receptor activation, which positions the dorsal appendages along the dorsal-ventral egg axis.

Animals↗
PubMed2000

Different clonal dispersion in the rostral and caudal mouse central nervous system.

We have performed a systematic clonal analysis to describe the modes of growth, dispersion and production of cells during the development of the mouse neural system. We have used mice expressing a LaacZ reporter gene under the control of the neuron specific enolase promoter to randomly generate LacZ clones in the central nervous system (CNS). We present evidence for (1) a pool of CNS founder cells that is not regionalized, i.e. give descendants dispersed along the entire A-P axis, (2) an early separation between pools of precursors for the anterior and posterior CNS and (3) distinct modes of production of progenitors in these two domains. More specifically, cell growth and dispersion of the progenitors follow a relatively coherent pattern throughout the anterior CNS, a mode that leads to a progressive regionalization of cell fates. In contrast, cell growth of progenitors of the SC appears to involve self-renewing stem cells that progress caudally during regression of the mode. Therefore, at least part of the area surrounding the node is composed of precursors with self-renewing properties and the development of the trunk is dependent on pools of stem cells regressing from A to P. Taken together with our analysis of the cell growth changes associated with neuromere formation (Mathis, L., Sieur, J., Voiculescu, O., Charnay, P. and Nicolas, J. F. (1999) Development 126, 4095-4106), our results suggest that major transitions in CNS development correspond to changes in cell behavior and may provide a link between morphogenesis and genetic patterning mechanisms (i.e. formation of the body plan).

Animals↗
PubMed2001

Sequential specification of neurons and glia by developmentally regulated extracellular factors.

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.

Animals↗