bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell commitment and cell interactions in the ectoderm of Drosophila melanogaster
Authors: Stüttem Isabella , Campos-Ortega José A.
Published: April 2021

The separation of neural from epidermal progenitor cells in the ventral neuroectoderm of Drosophila is thought to be mediated by cellular interactions. In order to verify the occurrence of regulatory signals and to test the neurogenic capabilities of cells from various regions of the ectoderm, we have carried out homotopic and heterotopic transplantations of single ectodermal cells. We found that cells from any of the tested regions, with the exception of the proctodeal anlage, are capable of developing as neuroblasts following their transplantation into the ventral neuroectoderm. These neurogenic capabilities are gradually distributed. Cells from the procephalic and ventral neurogenic regions exhibit maximal capabilities, as shown by their behavior in heterotopic transplantations. However, the two neurogenic regions differ from each other in that no epidermalising signals can be demonstrated to occur within the procephalic neuroectoderm, whereas such signals are strong within the ventral neuroectoderm; in addition, neuralising signals from neighbouring cells seem to be necessary for neuroectodermal cells to develop as neuroblasts. Other ectodermal regions whose cells exhibit weaker neurogenic capabilities are, in decreasing order of capability, the dorsal epidermal anlage, the anterolateral region of the procephalic lobe, comprising the anlage of the pharynx, and the anterior pole of the embryo, corresponding to the anlagen of the stomodeum and ectodermal anterior midgut. We assume that, during development in situ, the neurogenic capabilities of all these cells are suppressed by inhibitory signals, which are released upon heterotopic transplantation into the neuroectoderm. A community effect which prevents groups of dorsal epidermal cells from taking on a neural fate upon their transplantation into the ventral neuroectoderm, is shown. Finally, we hypothesize that the lack of neurogenic capability in the cells from the proctodeal anlage is due to the absence of products of the proneural genes.
The involvement of retinoic acid in the development of the vertebrate central nervous system
Authors: Maden Malcolm , Holder Nigel
Published: April 2021

We discuss here both previously published data and our current experiments which suggest that the vitamin A derivative, retinoic acid (RA), may play a role in the development of the vertebrate central nervous system (CNS). This evidence comes from the following: both an excess and a deficiency of vitamin A causes embryonic defects of the CNS; RA has been detected endogenously in the CNS; RA stimulates neurite outgrowth; the retinoic acid receptors have been detected with interesting distributions in the CNS; the binding protein for retinol, namely cellular retinol binding protein (CRBP) is found in the radial glia of the ventral floor plate; the binding protein for RA, namely, cellular retinoic acid binding protein (CRABP) is found in particular sets of axons in the developing spinal cord, in particular rhombomeres in the developing hindbrain and in the neural crest. Some hypotheses for the possible role of RA in various aspects of CNS development are discussed.
Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila
Authors: Hime Gary , Saint Robert
Published: April 2021

Mutations at the pebble locus of Drosophila melanogaster result in embryonic lethality. Examination of homo-zygous mutant embryos at the end of embryogenesis revealed the presence of fewer and larger cells which contained enlarged nuclei. Characterization of the embryonic cell cycles using DAPI, propidium iodide, anti-tubulin and anti-spectrin staining showed that the first thirteen rapid syncytial nuclear divisions proceeded normally in pebble mutant embryos. Following cellular-ization, the postblastoderm nuclear divisions occurred (mitoses 14, 15 and 16), but cytokinesis was never observed. Multinucleate cells and duplicate mitotic figures were seen within single cells at the time of the cycle 15 mitoses. We conclude that zygotic expression of the pebble gene is required for cytokinesis following cellularization during Drosophila embryogenesis. We postulate that developmental regulation of zygotic transcription of the pebble gene is a consequence of the transition from syncytial to cellular mitoses during cycle 14 of embryogenesis.
Transforming Growth Factor- β1, β2 and - β3 in cartilage and bone cells during endochondral ossification in the chick
Authors: Thorp Barry H. , Anderson Irene , Jakowlew Sonia B.
Published: April 2021

The localization of TGF- β1, - β2 and - β3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF- β isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF- β antibodies. TGF- βs in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-β3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF- β isoforms supports the hypothesis that TGF- β has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF- β may be a marker of chondrocyte differentiation. TGF- β localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF- β in the induction of synthesis of extracellular matrix.
The formation of commissures in the Drosophila CNS depends on the midline cells and on the Notch gene
Authors: Menne Thomas V. , Klämbt Christian
Published: April 2021

The commissures of the Drosophila central nervous system (CNS) are formed in close relation to the ventral midline cells, a morphologically distinct set of cells located at the midline of the developing CNS. To analyze the function of these cells during commissure formation, we looked for mutations that result in the absence of commissures. One example of a gene that can give rise to such a phenotype is the neurogenic gene Notch. Here we show that mutant Notch embryos are devoid of commissural connections and have an abnormal midline. The midline cells of the embryonic Drosophila CNS are specified during the blastoderm stage about two hours before the first neuroblasts start to delaminate from the neurogenic region. To analyze Notch function for commissure development further, we took advantage of the Notchts1 allele. Temperature-shift experiments demonstrated that the lack of commissures in mutant Notch embryos results from defects in the anlage of the CNS midline cells. Here maternal as well as zygotic Notch function are required for the correct activation of the gene single-minded, since mutant Notch embryos derived from germ-line clones lack most of the single-minded-positive midline cells.
Functions of the cytoplasmic domain of the βPS integrin subunit during Drosophila development
Authors: Grinblat Yevgenya , Zusman Susan , Yee Gene , Hynes Richard O. , Kafatos Fotis C.
Published: April 2021

Integrins constitute a family of membrane-spanning, heterodimeric proteins that mediate adhesive interactions between cells and surrounding extracellular matrices (or other cells) and participate in signal transduction. We are interested in assessing integrin functions in the context of developing Drosophila melanogaster. This report, using mutants of the βPS subunit encoded by the myospheroid (mys) locus, analyzes the relationships between integrin protein structure and developmental functions in an intact organism.

As a first step in this analysis, we demonstrated the ability of a fragment of wild-type mys genomic DNA, introduced into the germ line in a P-element vector P[my+], to rescue phenotypes attributed to lack of (or defects in) the endogenous βPS during several discrete morphogenetic events. We then produced in vitro a series of modifications of the wild-type P[mys+] transposon, which encode βPS derivatives with mutations within the small and highly conserved cytoplasmic domain. In vivo analysis of these mutant transposons led to the following conclusions. The cytoplasmic tail of βPS is essential for all developmental functions of the protein that were assayed.An intron at a conserved position in the DNA sequence encoding the cytoplasmic tail is thought to participate in important alternative splicing events in vertebrate β integrin subunit genes, but is not required for the developmental functions of the mys gene assayed here.Phosphorylation on two conserved tyrosines found in the C terminus of the βPS cytoplasmic tail is not necessary for the tested developmental functions.Four highly conserved amino acid residues found in the N-terminal portion of the cytoplasmic tail are important but not critical for the developmental functions of βPS; furthermore, the efficiencies with which these mutant proteins function during different morphogenetic processes vary greatly, strongly suggesting that the cytoplasmic interactions involving PS integrins are developmentally modulated.
diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene
Authors: Castrillon Diego H. , Wasserman Steven A.
Published: April 2021

We show that the Drosophila gene diaphanous is required for cytokinesis. Males homozygous for the dia1 mutation are sterile due to a defect in cytokinesis in the germline. Females trans-heterozygous for dia1 and a deficiency are sterile and lay eggs with defective eggshells; failure of cytokinesis is observed in the follicle cell layer. Null alleles are lethal. Death occurs at the onset of pupation due to the absence of imaginal discs. Mitotic figures in larval neuroblasts were found to be polyploid, apparently due to a defect in cytokinesis. The predicted 123×103Mr protein contains two domains shared by the formin proteins, encoded by the limb deformity gene in the mouse. These formin homology domains, which we have termed FH1 and FH2, are also found in Bni1p, the product of a Saccharomyces cerevisiae gene required for normal cytokinesis in diploid yeast cells.
Ectopic induction and reorganization of Wnt-1 expression in quail/chick chimeras
Authors: Bally-Cuif Laure , Wassef Marion
Published: April 2021

When grafted ectopically into the diencephalon of a chick host embryo, a portion of met-mesencephalon straddling the met-mesencephalic constriction has the capacity to induce En-2 expression in the surrounding host tissue. Subsequently, tectal and cerebellar structures, composed of both host and grafted cells, are reconstructed in this ectopic location at the expense of the host diencephalon. Previous experiments indicated that the induction of En-2 was correlated with Wnt-1 expression within the graft. The aim of the present study was: (i) to determine whether Wnt-1 expression was spatially regulated within the graft, (ii) to investigate whether host Wnt-1-expressing cells were also involved in the ectopic met-mesencephalic development and, if so, (iii) to localize these Wnt-1-positive domains in relation to the patterning of the ectopically developing met-mesencephalic territory. We studied the expression profile of Wnt-1, in relation with that of other positional markers, in quail/chick chimeras where various portions of met-mesencephalon had been grafted into the diencephalon. We found that Wnt-1 expression was reorganized within the graft, and that it was also induced in the host in contact with the graft. Moreover, these ectopic expressions of Wnt-1, in both the grafted and the surrounding host tissues, were organized in concert to form a continuous positive line at the host/graft junction, the location of which depended on the precise origin of the graft. Finally, we found that this line was frequently located at the limit between territories expressing different positional markers. We propose that Wnt-1 expression is turned on at the junction between domains of different phenotypes, and may be used as a border to stabilize these adjacent differently committed territories.
A developmental analysis of oligodendroglial integrins in primary cells: changes in αv-associated β subunits during differentiation
Authors: Milner Richard , ffrench-Constant Charles
Published: April 2021

We have examined the expression of integrins on primary oligodendroglial cells during the differentiation of the pro- liferative oligodendrocyte precursor (O-2A progenitor) cell to the postmitotic oligodendrocyte. Cells of the oligoden- drocyte lineage expressed a limited repertoire of integrins: α6β1 and αv integrins including αvβ1, αvβ3 and αvβ5, as well as a potentially novel integrin αvβ80kDa. Integrin expression was developmentally regulated; during differen- tiation αvβ1 was reduced and αvβ5 upregulated. These results suggest that laminin and vitronectin are important extracellular matrix ligands for oligodendrocytes, and provide a rational explanation for previous observations that RGD peptides inhibit the expression of myelin-specific genes. They also suggest a simple model by which switching of integrin β subunits might regulate differentiation. As chimeric β1 integrins with a β5 cytoplasmic domain support proliferation less well than normal β1 integrins (Pasqualini and Hemler (1994), J. Cell Biol. 125, 447-460) the switch from αvβ1 to αvβ5 might play a key instructive role in the cessation of proliferation and subsequent differentiation.
Spatiotemporal dynamics of intracellular [Ca2+]i oscillations during the growth and meiotic maturation of mouse oocytes
Authors: Carroll John , Swann Karl , Whittingham David , Whitaker Michael
Published: April 2021

Calcium oscillations occur during meiotic maturation of mouse oocytes. They also trigger activation at fertilization. We have monitored [Ca2+]i in oocytes at different stages of growth and maturation to examine how the calcium release mechanisms alter during oogenesis. Spontaneous calcium oscillations occur every 2-3 minutes in the majority of fully grown (but immature) mouse oocytes released from antral follicles and resuming meiosis. The oscillations last for 2-4 hours after release from the follicle and take the form of global synchronous [Ca2+]i increases throughout the cell. Rapid image acquisition or cooling the bath temperature from 28°C to 16°C did not reveal any wave-like spatial heterogeneity in the [Ca2+]i signal. Calcium appears to reach highest levels in the germinal vesicle but this apparent difference of [Ca2+] in nucleus and cytoplasm is an artifact of dye loading. Smaller, growing immature oocytes are less competent: about 40% are able to resume meiosis and a similar proportion of these oocytes show spontaneous calcium oscillations. [Ca2+]i transients are not seen in oocytes that do not resume meiosis spontaneously in vitro. Nonetheless, these oocytes are capable of [Ca2+]i oscillations since they show them in response to the addition of carbachol or thimerosal. To examine how the properties of calcium release change during meiotic maturation, a calcium-releasing factor from sperm was microinjected into fully grown immature and mature oocytes. The sperm-factor-induced oscillations were about two-fold larger and longer in mature oocytes compared to immature oocytes. Calcium waves travelling at 40-60 µm/second were generated in mature oocytes, but not in immature oocytes. In some mature oocytes, successive calcium waves had different sites of origin. The modifications in the size and spatial organization of calcium transients during oocyte maturation may be a necessary prerequisite for normal fertilization.
Quantitative RT-PCR assays show Xist RNA levels are low in mouse female adult tissue, embryos and embryoid bodies
Authors: Buzin Carolyn H. , Mann Jeffrey R. , Singer-Sam Judith
Published: April 2021

We have investigated expression of the Xist gene in mouse female adult kidney, embryos and embryonic stem (ES) cells undergoing in vitro differentiation as embryoid bodies. Using the quantitative RT-PCR single nucleotide primer extension (SNuPE) assay, we found that the amount of Xist RNA in adult kidney of three mouse strains was less than ∼2000 transcripts per cell, with only modest differences between strains carrying different Xce alleles. Female embryos 7.5 days post coitum had the same number of Xist transcripts per cell as isogenic adult tissue. Using quantitative oligonucleotide hybridization assays after RT-PCR, we investigated Xist expression in ES lines heterozygous at the Pgk-1 and Xist loci. We found that, while in most (XX) ES lines Xist RNA levels increased during embryoid body formation, the levels seen were less than 10% those found in adult female kidney. In addition, we found that the allelic ratio of Xist transcripts from reciprocal (XX) ES cell lines differentiating in vitro was identical to that of isogenic 10.5 to 11.5 day female embryos. These latter results suggest that there is no pattern of preferential paternal imprinting during days 1 to 9 of in vitro differentiation of ES cells. However, the influence of the Xce locus on the randomness of X-inactivation in embyros seems to operate also in ES cell lines. Our overall conclusion is that the low levels of Xist RNA in female kidney, embryos and differentiating (XX) ES cells are compatible only with models that do not require Xist RNA to cover the entire inactive X chromosome.
Hypoblastic tissue and fibroblast growth factor induce blood tissue (haemoglobin) in the early chick embryo
Authors: Gordon-Thomson C. , Fabian B. C.
Published: April 2021

We have investigated the temporal and the causal basis of blood tissue specification in the chick embryo. Earlier workers have shown that the prospective blood-forming area is specified in a horseshoe-shaped area at the posterior side of the embryo. We found that cultured explants from the posterior marginal zone at stages XI to XIII (consisting of the posterior marginal zone and part of Koller’s sickle) have a high propensity to form haemoglobin (Hb), which could be inhibited at stage XI by adding antibody against basic fibroblast growth factor (bFGF) to the neutral culture medium; this treatment had no effect from stage XII onwards. The same result was found when whole embryos were cultured with an antiserum raised against bFGF, or with heparin.

In another series of experiments, we found that cultured pieces from the inner-core of stage XIII epiblasts (with or without hypoblast tissue) were able to form Hb, whereas inner-core pieces from the pre-hypoblast stages, namely stages X and XI, did not form Hb. The capacity to form Hb, however, could be conferred upon the inner-core pieces from stage X epiblasts if bFGF at a concentration of 75-150 ng/ml was added to the culture medium. Furthermore, and most pertinently, the capacity to form Hb could be conferred on stage X inner-core pieces when they were co-cultured with hypoblast from a stage XIII embryo in a sandwich explant. Thus the inductive role of the hypoblast appears to be mediated via bFGF. This conclusion was confirmed by our finding that the antibody to bFGF inhibited the formation of Hb in these hypoblast sandwich explants.

We propose that the erythropoietic cell lineage, i.e. blood tissue, is determined in the chick embryo before stage XII by a bFGF-type of inductive signal from the emerging hypoblast adjacent to Koller’s sickle, in the area encompassing the posterior marginal zone. The morphogenetic changes that take place in the epiblast and hypoblast between stages X and XIII are considered in the discussion, especially with respect to how the posterior horseshoe of blood-forming tissue arises in response to the inductive signal.
Integrin switching regulates normal trophoblast invasion
Authors: Damsky Caroline H. , Librach Clifford , Lim Kee-Hak , Fitzgerald Marilyn L. , McMaster Michael T. , Janatpour Mary , Zhou Yan , Logan Susan K. , Fisher Susan J.
Published: April 2021

Cells invade extracellular matrices in a regulated manner at specific times and places during normal development. A dramatic example is trophoblast invasion of the uterine wall. Previous studies have shown that differentiation of trophoblasts to an invasive phenotype is accompanied by temporally and spatially regulated switching of their integrin repertoire. In the first trimester human placenta, α6 integrins are restricted to cytotrophoblast (CTB) stem cells and downregulated in invasive CTBs, whereas α5β1 and α1β1 integrins are upregulated in differentiating and invasive CTBs. The goal of the present study was to determine whether these changes have functional consequences for CTB invasiveness. Using an in vitro invasion model, we determined first that aggregates of invading first trimester CTBs in vitro undergo the same pattern of integrin switching as was observed in situ, thereby validating the utility of the model. We then showed that antibody perturbation of interactions involving laminin or collagen type IV and their integrin α1/β1 receptor inhibited invasion by CTBs, whereas perturbing interactions between fibronectin and the α5/β1 fibronectin receptor accelerated invasion. Finally, we report that later gestation CTBs, which display greatly decreased invasive capacity, are also unable to upregulate α1β1 complexes, providing further evidence that this integrin is critical for CTB invasion. This gestational regulation is transcriptional. These data indicate that integrin switching observed during differentiation in situ has significant functional consequences for CTB invasion. The data suggest further that differentiating CTBs upregulate counterbalancing invasion-accelerating and invasion-restraining adhesion mechanisms. We propose that this contributes to regulating the depth of CTB invasion during normal implantation.
Alternative cell fate choice induced by low-level expression of a regulator of protein phosphatase 2A in the Drosophila peripheral nervous system
Authors: Shiomi Kensuke , Takeichi Masatoshi , Nishida Yasuyoshi , Nishi Yoshimi , Uemura Tadashi
Published: April 2021

The Drosophila gene twins encodes the regulatory B subunit of type 2A protein phosphatase. Here we report that its partial loss-of-function mutations caused abnormal morphogenesis in the adult peripheral nervous system. In wild-type flies, the mechanoreceptor, one major class of sensory organs, is composed of four specialized cells (one neuron and three accessory cells) that are derived from a single precursor cell. The hypomorphic twins mutations did not block division of this precursor, but most likely altered cell fate in this lineage to produce only accessory cells that form sensory structures. Stepwise reductions of twins protein enhanced this transformation. In these mutants, another regulatory subunit, A, and the catalytic subunit, C, of the phosphatase were expressed at normal levels. Therefore, the modulation of the phosphatase activity by the B subunit appears to be crucial for specification of neural cell identity.
The Caenorhabditis elegans MYOD homologue HLH-1 is essential for proper muscle function and complete morphogenesis
Authors: Chen Lihsia , Krause Michael , Sepanski Michael , Fire Andrew
Published: April 2021

A family of muscle-specific helix-loop-helix transcription factors (myoD, myogenin, myf-5 and MRF4) has been implicated in the control of vertebrate skeletal myogenesis. Searches for homologues of this family in Caenorhabditis elegans identified a single family member, hlh-1, which is expressed in striated muscles and their clonal precursors. We have isolated a null allele of hlh-1 following chemical mutagenesis. Animals homozygous for the null mutation produce contractile body-wall muscles, although muscle contractions are weak and coordination is defective. In addition to the evident muscle defects, mutant animals fail to complete embryonic elongation and die as larvae or young adults. Ultrastructural analysis of the mutant muscle reveals an apparently normal local lattice of thick and thin filaments, with more global defects in sarcomere organization and muscle cell placement. Mosaic studies using the point mutation and an extrachromosomal transgene indicate that the requirement for hlh-1 is fully zygotic, with no maternal hlh-1 requirement for either muscle development or viability.
The Caenorhabditis elegans gene lin-26 is required to specify the fates of hypodermal cells and encodes a presumptive zinc-finger transcription factor
Authors: Labouesse Michel , Sookhareea Satish , Horvitz H. Robert
Published: April 2021

The mutation lin-26(n156) prevents vulva formation in C. elegans by transforming the vulval precursor cells into neurons or neuroblasts. We have isolated and characterized three new lin-26 alleles, which result in embryonic lethality. These mutations cause a few other hypodermal cells to express a neural fate and most hypodermal cells to degenerate. lin-26 encodes a presumptive zinc-finger transcription factor. Our data indicate that lin-26 is required for cells to acquire the hypodermal fate.
Cranial paraxial mesoderm: regionalisation of cell fate and impact on craniofacial development in mouse embryos
Authors: Trainor Paul A. , Tan Seong-Seng , Tam Patrick P. L.
Published: April 2021

A combination of micromanipulative cell grafting and flu-orescent cell labelling techniques were used to examine the developmental fate of the cranial paraxial mesoderm of the 8.5-day early-somite-stage mouse embryo. Mesodermal cells isolated from seven regions of the cranial mesoderm, identified on the basis of their topographical association with specific brain segments were assessed for their contribution to craniofacial morphogenesis during 48 hours of in vitro development. The results demonstrate extensive cell mixing between adjacent but not alternate groups of mesodermal cells and a strict cranial-to-caudal distribution of the paraxial mesoderm to craniofacial structures. A two-segment periodicity similar to the origins of the branchial motor neurons and the distribution of the rhombencephalic neural crest cells was observed as the paraxial mesoderm migrates during formation of the first three branchial arches. The paraxial mesoderm colonises the mesenchymal core of the branchial arches, consistent with the location of the muscle plates. A dorsoventral regionalisation of cell fate similar to that of the somitic mesoderm is also found. This suggests evolution has conserved the fate of the murine cranial paraxial mesoderm as a multiprogenitor population which displays a predominantly myogenic fate. Heterotopic transplantation of cells to different regions of the cranial mesoderm revealed no discernible restriction in cell potency in the craniocaudal axis, reflecting considerable plasticity in the developmental fate of the cranial mesoderm at least at the time of experimentation. The distribution of the different groups of cranial mesoderm matches closely with that of the cranial neural crest cells suggesting the two cell populations may share a common segmental origin and similar destination.
Dorsoventral axis formation in Drosophila depends on the correct dosage of the gene gurken
Authors: Neuman-Silberberg F. S. , Schupbach T.
Published: April 2021

The Drosophila gene gurken participates in a signaling process that occurs between the germ line and the somatic cells (follicle cells) of the ovary. This process is required for correct patterning of the dorsoventral axis of both the egg and the embryo. gurken produces a spatially localized transcript which encodes a TGF-α-like molecule (Neuman-Sil-berberg and Schupbach, Cell 75, 165-174,1993). Mutations in gurken cause a ventralized phenotype in egg and embryo. To determine whether the gurken gene product plays an instructive role in dorsoventral patterning, we constructed females containing extra copies of a gurken transgene. Such females produce dorsalized eggs and embryos, which is expected if gurken acts as a limiting factor in the dorsoventral patterning process. In addition, the expression pattern of the gene rhomboid in the follicle cells is altered in ovaries of females containing extra copies of gurken. Our results indicate that changing gurken dosage in otherwise wild-type ovaries is sufficient to alter the number of somatic follicle cells directed to the dorsal fate. Therefore the gurken-torpedo signaling process plays an instructive role in oogenesis. It induces dorsal cell fates in the follicle cell epithelium and it controls the production of maternal components that will direct the embryonic dorsoventral pattern after fertilization.
Embryonic fat-cell lineage in Drosophila melanogaster
Authors: Hoshizaki Deborah Keiko , Blackburn Thomas , Price Catherine , Ghosh Mita , Miles Kathy , Ragucci Mark , Sweis Rami
Published: April 2021

The Drosophila adipose tissue, or fat body, and the body-wall muscle are two major tissues derived from the mesoderm. Although much is known about the lineage of muscle cells, little is known about the development of the fat body. Using known genes and an enhancer trap (29D), we have begun to trace the lineage of the cells comprising the fat body.

The genes Adh (alcohol dehydrogenase) and DCg1 (type IV collagen) code for gene products involved in fat-cell metabolism and therefore serve as terminal fat-cell differentiation markers. The expression of these genes was used to identify the fat body at stage 17 and to identify the start of terminal fat-cell differentiation at stage 15. We found that the steroid-hormone receptor gene, svp (seven-up), was expressed transiently within the fat-cell lineage from stages 12 to 14. We suggest that stage 12 marks the beginning of early fat-cell differentiation and that the svp-positive cells within the mesoderm are early precursor fat cells. To confirm the identity of these cells and to establish the role of svp in the developing fat cell, we examined svp mutant embryos for alterations in the expression of the two terminal fat-cell differentiation markers, Adh and DCg1. Loss of svp function resulted in the loss of Adh transcript and a reduction of DCg1 expression specifically in the fat body. Thus, svp plays a role in fat-body-specific expression of at least two terminal fat-cell differentiation genes. In contrast to svp, we found no evidence that the steroid receptor HNF-4(D) gene was expressed in the fat body nor that it was involved in the development of this tissue.

Using an enhancer-trap line (29D), we further traced the fat-cell lineage to nine bilateral clusters of cells within the mesoderm at germ-band extension. We suggest these 29D-positive cells represent the progenitor fat cells. In stage-12 embryos, the 29D-positive cell clusters can be identified within the mesoderm internal to nautilus-expressing cells. These data suggest that the precursor fat cells may be derived from the inner mesoderm, or spanchnopleura. Embryos deficient for the DNA region surrounding the site of the 29D enhancer trap lack most, if not all, of the cells in the fat-cell lineage. These embryos exhibit the loss of svp-positive precursor fat cells and concomitant loss of fat-body-specific expression of Adh and DCg1. The muscle-cell lineage, however, appears unaffected in embryos lacking the region containing the 29D enhancer trap. Both the nautilus-expressing cells, which are progenitors to body-wall muscle, and the visceral mesoderm were present in these embryos. These data suggest the presence of a gene(s) near the site of the 29D enhancer trap that is important in fat-cell determination. It is likely this Drosophila gene is associated with the 29D enhancer.
Fertile offspring derived from mammalian eggs lacking either animal or vegetal poles
Authors: Zernicka-Goetz Magdalena
Published: April 2021

In all animals so far tested, removing either pole of the undivided egg prevents normal development: embryos may arrest early, lack organs, or the adults may be sterile. These experiments have shown that spatial patterning of the egg is of utmost importance for subsequent development. However, the significance of spatial patterning in mammalian eggs is still controversial. To test the importance of egg polarity in the mouse a substantial amount of material either from the animal (polar body-associated) or the vegetal (opposite) pole of the fertilised egg was removed. One pole of the egg was cut away manually with a glass needle and the eggs were allowed to develop in vitro. Both kinds of surgical operation permit the development of blastocysts, which, after transfer to the uteri of pseudo-pregnant foster mothers, can produce viable offspring. Furthermore, these develop into fertile adult mice. I conclude that mouse eggs have no essential components that are localised uniquely to the animal or the vegetal pole and, therefore, do not rely for their axial development on maternal determinants that are so localised in the fertilised egg. Thus the mammalian egg appears to be very unusual in the animal kingdom in that it establishes the embryonic axes after the zygote has begun development.
Direct activation of Sex-lethal transcription by the Drosophila Runt protein
Authors: Kramer Sunita G. , Jinks Timothy M. , Schedl Paul , Gergen J. Peter
Published: April 2021

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. Here we demonstrate that runt also functions to activate transcription. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. Using an in vivo transient assay, we demonstrate that Runt’s DNA-binding activity is essential for Sex-lethal activation in vivo. These experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. Moreover, we demonstrate that a small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt- dependent transcriptional activation in vivo.
hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen
Authors: Lawrence Peter A. , Casal José , Struhl Gary
Published: April 2021

Like the Drosophila embryo, the abdomen of the adult consists of alternating anterior (A) and posterior (P) compartments. However the wing is made by only part of one A and part of one P compartment. The abdomen therefore offers an opportunity to compare two compartment borders (A/P is within the segment and P/A intervenes between two segments), and ask if they act differently in pattern formation. In the embryo, abdomen and wing P compartment cells express the selector gene engrailed and secrete Hedgehog protein whilst A compartment cells need the patched and smoothened genes in order to respond to Hedgehog. We made clones of cells with altered activities of the engrailed, patched and smoothened genes. Our results confirm (1) that the state of engrailed, whether ‘off’ or ‘on’, determines whether a cell is of A or P type and (2) that Hedgehog signalling, coming from the adjacent P compartments across both A/P and P/A boundaries, organises the pattern of all the A cells.

We have uncovered four new aspects of compartments and engrailed in the abdomen. First, we show that engrailed acts in the A compartment: Hedgehog leaves the P cells and crosses the A/P boundary where it induces engrailed in a narrow band of A cells. engrailed causes these cells to form a special type of cuticle. No similar effect occurs when Hedgehog crosses the P/A border. Second, we look at the polarity changes induced by the clones, and build a working hypothesis that polarity is organised, in both compartments, by molecule(s) emanating from the A/P but not the P/A boundaries. Third, we show that both the A and P compartments are each divided into anterior and posterior subdomains. This additional stratification makes the A/P and the P/A boundaries fundamentally distinct from each other. Finally, we find that when engrailed is removed from P cells (of, say, segment A5) they transform not into A cells of the same segment, but into A cells of the same parasegment (segment A6).
Developmental expression of Pax1/9 genes in urochordate and hemichordate gills: insight into function and evolution of the pharyngeal epithelium
Authors: Ogasawara Michio , Wada Hiroshi , Peters Heiko , Satoh Nori
Published: April 2021

The epithelium of the pharynx contributes to the formation of gills in hemichordates, urochordates, cephalochordates and primitive vertebrates, and is therefore a key structure for understanding developmental mechanisms underlying the establishment of chordate body plans. Pax1- and Pax9-related genes encode transcription factors which are expressed in the pharyngeal region of cephalochordates as well as in the vertebrate pharyngeal pouch epithelium that forms the thymus and parathyroid glands. To explore the molecular basis underlying the occurrence and modifications of the pharyngeal epithelium during evolution, we isolated cDNA clones for Pax1- and Pax9-related genes of urochordates (HrPax1/9 of Halocynthia roretzi and CiPax1/9 of Ciona intestinalis) and a hemichordate (PfPax1/9 of Ptychodera flava) from gill cDNA libraries. Each gene is present as a single copy per haploid genome. All of the cDNAs encode typical paired domains and octapeptides but not a homeodomain, as is also true of other Pax1- and Pax9-related genes. Molecular phylogenetic analysis based on comparison of the paired domain amino-acid sequences suggests that HrPax1/9, CiPax1/9 and PfPax1/9 belong to the Pax1/9 subfamily, and that they are descendants of a single precursor of Pax1/Pax9. Screening of HrPax1/9 cDNA clones yielded six different types of transcripts which were generated by alternative splicing. Northern blot, RT-PCR/Southern and in situ hybridization analyses revealed that HrPax1/9, CiPax1/9 and PfPax1/9 are not expressed during early embryogenesis but are expressed in the epithelia of differentiating gills, suggesting that these genes encode gill-specific transcription factors. The Pax1/9 genes therefore might provide the first developmental genetic corroboration of hypotheses of organ-level homology that unifies hemichordates, urochordates and cephalochordates.
Patterning of Drosophila leg sensory organs through combinatorial signaling by Hedgehog, Decapentaplegic and Wingless
Authors: Hays Rebecca , Buchanan Kathryn T. , Neff Carrie , Orenic Teresa V.
Published: April 2021

During development, global patterning events initiate signal transduction cascades which gradually establish an array of individual cell fates. Many of the genes which pattern Drosophila are expressed throughout development and specify diverse cell types by creating unique local environments which establish the expression of locally acting genes. This process is exemplified by the patterning of leg microchaete rows. hairy (h) is expressed in a spatially restricted manner in the leg imaginal disc and functions to position adult leg bristle rows by negatively regulating the proneural gene achaete, which specifies sensory cell fates. While much is known about the events that partition the leg imaginal disc and about sensory cell differentiation, the mechanisms that refine early patterning events to the level of individual cell fate specification are not well understood. We have investigated the regulation of h expression along the dorsal/ventral (D/V) axis of the leg adjacent to the anterior/posterior (A/P) compartment boundary and have found that it requires input from both D/V and A/P patterning mechanisms. Expression of the D/V axis h stripe (D/V-h) is controlled by dorsal- and ventral-specific enhancer elements which are targets of Decapentaplegic (Dpp) and Wingless (Wg) signaling, respectively, but which are also dependent on Hedgehog (Hh) signaling for activation. D/V-h expression is lost in smoothened mutant clones and is specifically activated by exogenously supplied Cubitus interruptus (Ci). D/V-h expression is also lost in clones deficient for Dpp and Wg signaling, but ectopic activation of D/V-h by Dpp and Wg is limited to the A/P compartment boundary where endogenous levels of full- length Ci are high. We propose that D/V-h expression is regulated in a non-linear pathway in which Ci plays a dual role. In addition to serving as an upstream activator of Dpp and Wg, Ci acts combinatorially with them to activate D/V- h expression.
Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila
Authors: Conley Catharine A. , Silburn Ross , Singer Matthew A. , Ralston Amy , Rohwer-Nutter Dan , Olson David J. , Gelbart William , Blair Seth S.
Published: April 2021

The BMP-like signaling mediated by the ligands Dpp and Gbb is required to reinforce the development of most veins in the Drosophila wing. However, the formation of the cross veins is especially sensitive to reductions in BMP-like signaling. We show here that the formation of the definitive cross veins occurs after the initial specification of the longitudinal veins in a process that requires localized BMP-like activity. Since Dpp and Gbb levels are not detectably higher in the early phases of cross vein development, other factors apparently account for this localized activity. Our evidence suggests that the product of the crossveinless 2 gene is a novel member of the BMP-like signaling pathway required to potentiate Gbb of Dpp signaling in the cross veins. crossveinless 2 is expressed at higher levels in the developing cross veins and is necessary for local BMP-like activity. The Crossveinless 2 protein contains a putative signal or transmembrane sequence, and a partial Von Willebrand Factor D domain similar to those known to regulate the formation of intramolecular and intermolecular bonds. It also contains five cysteine-rich domains, similar to the cysteine-rich domains found in Chordin, Short Gastrulation and Procollagen that are known to bind BMP-like ligands. These features strongly suggest that Crossveinless 2 acts extracelluarly or in the secretory pathway to directly potentiate Dpp or Gbb signaling.
Characterization of a novel subset of cardiac cells and their progenitors in theDrosophilaembryo
Authors: Ward Ellen J. , Skeath James B.
Published: April 2021

The Drosophila heart is a simple organ composed of two major cell types: cardioblasts, which form the simple contractile tube of the heart, and pericardial cells, which flank the cardioblasts. A complete understanding of Drosophila heart development requires the identification of all cell types that comprise the heart and the elucidation of the cellular and genetic mechanisms that regulate the development of these cells. Here, we report the identification of a new population of heart cells: the Odd skipped-positive pericardial cells (Odd-pericardial cells). We have used descriptive, lineage tracing and genetic assays to clarify the cellular and genetic mechanisms that control the development of Odd-pericardial cells. Odd skipped marks a population of four pericardial cells per hemisegment that are distinct from previously identified heart cells. We demonstrate that within a hemisegment, Odd-pericardial cells develop from three heart progenitors and that these heart progenitors arise in multiple anteroposterior locations within the dorsal mesoderm. Two of these progenitors divide asymmetrically such that each produces a two-cell mixed-lineage clone of one Odd-pericardial cell and one cardioblast. The third progenitor divides symmetrically to produce two Odd-pericardial cells. All remaining cardioblasts in a hemisegment arise from two cardioblast progenitors each of which produces two cardioblasts. Furthermore, we demonstrate that numb and sanpodo mediate the asymmetric divisions of the two mixed-lineage heart progenitors noted above.
The conditional medaka mutation eyeless uncouples patterning and morphogenesis of the eye
Authors: Winkler Sylke , Loosli Felix , Henrich Thorsten , Wakamatsu Yuko , Wittbrodt Joachim
Published: April 2021

In early vertebrate eye development, the retinal anlage is specified in the anterior neuroectoderm. During neurulation, the optic vesicles evaginate from the lateral wall of the prosencephalon. Here we describe the temperature-sensitive mutation eyeless in the Japanese medakafish. Marker gene analysis indicates that, whereas, specification of two retinal primordia and proximodistal patterning takes place in the mutant embryo, optic vesicle evagination does not occur and subsequent differentiation of the retinal primordia is not observed. The mutation eyeless thus uncouples patterning and morphogenesis at early steps of retinal development. Temperature-shift experiments indicate a requirement for eyeless activity prior to optic vesicle evagination. Cell transplantation shows that eyeless acts cell autonomously.
Drosophila puckered regulates Fos/Jun levels during follicle cell morphogenesis
Authors: Dobens Leonard L. , Martín-Blanco Enrique , Martínez-Arias Alfonso , Kafatos Fotis C. , Raftery Laurel A.
Published: April 2021

puckered (puc) encodes a VH1-like phosphatase that down- regulates Jun kinase (JNK) activity during dorsal closure of the Drosophila embryo. We report a role for puc in follicle cell morphogenesis during oogenesis. puc mRNA accumulates preferentially in the centripetally migrating follicle cells and cells of the elongating dorsal appendages. Proper levels of Puc activity in the follicle cells are critical for the production of a normal egg: either reduced or increased Puc activity result in incomplete nurse cell dumping and aberrant dorsal appendages. Phenotypes associated with puc mutant follicle cells include altered DE- cadherin expression in the follicle cells and a failure of nurse cell dumping to coordinate with dorsal appendage elongation, leading to the formation of cup-shaped egg chambers. The JNK pathway target A251-lacZ showed cell- type-specific differences in its regulation by puc and by the small GTPase DRac1. puc mutant cells displayed region- specific ectopic expression of the A251-lacZ enhancer trap whereas overexpression of a transgene encoding Puc was sufficient to suppress lacZ expression in a cell autonomous fashion. Strikingly, decreased or increased puc function leads to a corresponding increase or decrease, respectively, of Fos and Jun protein levels. Taken together, these data indicate that puc modulates gene expression responses by antagonizing a Rho GTPase signal transduction pathway that stabilizes the AP-1 transcription factor. Consistent with this, overexpression of a dominant negative DRac1 resulted in lower levels of Fos/Jun.