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Heavy metals are both integral parts of cells and environmental toxicants, and their deregulation is associated with severe cellular dysfunction and various diseases. Here we show that the Hippo pathway plays a critical role in regulating heavy metal homeostasis. Hippo signalling deficiency promotes the transcription of heavy metal response genes and protects cells from heavy metal-induced toxicity, a process independent of its classic downstream effectors YAP and TAZ. Mechanistically, the Hippo pathway kinase LATS phosphorylates and inhibits MTF1, an essential transcription factor in the heavy metal response, resulting in the loss of heavy metal response gene transcription and cellular protection. Moreover, LATS activity is inhibited following heavy metal treatment, where accumulated zinc directly binds and inhibits LATS. Together, our study reveals an interplay between the Hippo pathway and heavy metals, providing insights into this growth-related pathway in tissue homeostasis and stress response., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovo D dominant female-sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini-white (w + mW.hs ), or rosy (ry + ) and neomycin (neo R ) transgenes are in common use. Parallel ovo D lines were developed using w + mW.hs FRT insertions on the X and chromosomes 2R and 3L, as well as ry + , neo R FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry + , neo R FRT background, are not amenable to germline clonal analysis without labor-intensive recombination onto chromosome arms containing a w + mW.hs FRT. Here we report the creation of a new ovo D line for the ry + , neo R FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal-effect phenotypes of novel brother of tout-velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages., (© 2016 Wiley Periodicals, Inc.)

The N-terminal head domain of kinesin heavy chain (Khc) is well known for generating force for transport along microtubules in cytoplasmic organization processes during metazoan development, but the functions of the C-terminal tail are not clear. To address this, we studied the effects of tail mutations on mitochondria transport, determinant mRNA localization and cytoplasmic streaming in Drosophila. Our results show that two biochemically defined elements of the tail - the ATP-independent microtubule-binding sequence and the IAK autoinhibitory motif - are essential for development and viability. Both elements have positive functions in the axonal transport of mitochondria and determinant mRNA localization in oocytes, processes that are accomplished by biased saltatory movement of individual cargoes. Surprisingly, there were no indications that the IAK autoinhibitory motif acts as a general downregulator of Kinesin-1 in those processes. Time-lapse imaging indicated that neither tail region is needed for fast cytoplasmic streaming in oocytes, which is a non-saltatory bulk transport process driven solely by Kinesin-1. Thus, the Khc tail is not constitutively required for Kinesin-1 activation, force transduction or linkage to cargo. It might instead be crucial for more subtle elements of motor control and coordination in the stop-and-go movements of biased saltatory transport.

Mutations in the Drosophila variable nurse cells (vnc) gene result in female sterility and oogenesis defects, including egg chambers with too many or too few nurse cells. We show that vnc corresponds to Arrest Defective1 (Ard1) and encodes the catalytic subunit of NatA, the major N-terminal acetyl-transferase complex. While N-terminal acetylation is one of the most prevalent covalent protein modifications in eukaryotes, analysis of its role in development has been challenging since mutants that compromise NatA activity have not been described in any multicellular animal. Our data show that reduced ARD1 levels result in pleiotropic oogenesis defects including abnormal cyst encapsulation, desynchronized cystocyte division, disrupted nurse cell chromosome dispersion, and abnormal chorion patterning, consistent with the wide range of predicted NatA substrates. Furthermore, we find that loss of Ard1 affects cell survival/proliferation and is lethal for the animal, providing the first demonstration that this modification is essential in higher eukaryotes., (© 2010 Wiley-Liss, Inc.)

The formation of the ventral furrow during Drosophila gastrulation is driven by coordinated apical constriction. Cell-cell adhesion is thought to regulate apical constriction, but the mechanisms are poorly understood. DE-cadherin, an epithelial classic cadherin, has in its membrane-proximal extracellular region a suite of domains absent from vertebrate/urochordate classic cadherins. We constructed DEΔP, a DE-cadherin derivative that lacks the membrane-proximal half of the extracellular region but retains the entire cytoplasmic domain and still exhibits strong cell-cell binding ability. The extracellular region of DEΔP consists of only cadherin repeats, mimicking vertebrate/urochordate classic cadherins. In animals lacking DE-cadherin, DEΔP organized adherens junction assembly and functioned fully in many cadherin-dependent processes, including oogenesis. Embryos in which DE-cadherin was entirely replaced by DEΔP established the blastoderm epithelium but failed to form a ventral furrow. Apical constrictions were initiated relatively normally but subsequently decelerated. These were then followed by catastrophic disruption of the junctional network. Our results suggest that although the membrane-proximal half of the DE-cadherin extracellular region is dispensable for many developmental events, it is essential for efficient and robust apical constriction during ventral furrow formation., (© 2010 The Authors. Journal compilation © 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)

To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (12C6 and 13C6). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.

Morphogen gradients play fundamental roles in patterning and cell specification during development by eliciting differential transcriptional responses in target cells. In Drosophila, Decapentaplegic (Dpp), the BMP2/4 homolog, downregulates transcription of the nuclear repressor brinker (brk) in a concentration-dependent manner to generate an inverse graded distribution. Both Dpp and Brk are crucial for directing Dpp target gene expression in defined domains and the consequent execution of distinct developmental programs. Thus, determining the mechanism by which the brk promoter interprets the Dpp activity gradient is essential for understanding both Dpp-dependent patterning and how graded signaling activity can generate different responses through transcriptional repression. We have uncovered key features of the brk promoter that suggest it uses a complex enhancer logic not represented in current models. First, we find that the regulatory region contains multiple compact modules that can independently drive brk-like expression patterns. Second, each module contains binding sites for the Schnurri/Mad/Medea (SMM) complex, which mediates Dpp-dependent repression, linked to regions that direct activation. Third, the SMM repression complex acts through a distance-dependent mechanism that probably uses the canonical co-repressor C-terminal Binding Protein (CtBP). Finally, our data suggest that inputs from multiple regulatory modules are integrated to generate the final pattern. This unusual promoter organization may be necessary for brk to respond to the Dpp gradient in a precise and robust fashion.

Heparan sulfate proteoglycans (HSPGs) are extracellular macromolecules found on virtually every cell type in eumetazoans. HSPGs are composed of a core protein covalently linked to glycosaminoglycan (GAG) sugar chains that bind and modulate the signaling efficiency of many ligands, including Hedgehog (Hh), Wingless (Wg) and Bone morphogenetic proteins (BMPs). Here, we show that, in Drosophila, loss of HSPGs differentially affects embryonic Hh, Wg and BMP signaling. We find that a stage-specific block to GAG synthesis prevents HSPG expression during establishment of the BMP activity gradient that is crucial for dorsal embryonic patterning. Subsequently, GAG synthesis is initiated coincident with the onset of Hh and Wg signaling which require HSPGs. This temporal regulation is achieved by the translational control of HSPG synthetic enzymes through internal ribosome entry sites (IRESs). IRES-like features are conserved in GAG enzyme transcripts from diverse organisms, suggesting that this represents a novel evolutionarily conserved mechanism for regulating GAG synthesis and modulating growth factor activity.

Bone Morphogenetic Proteins (Bmps) are secreted growth factors that play crucial roles in animal development across the phylogenetic spectrum. Bmp signaling results in the phosphorylation and nuclear translocation of Smads, downstream signal transducers that bind DNA. In Drosophila, the zinc finger protein Schnurri (Shn) plays a key role in signaling by the Bmp2/Bmp4 homolog Decapentaplegic (Dpp), by forming a Shn/Smad repression complex on defined promoter elements in the brinker (brk) gene. Brk is a transcriptional repressor that downregulates Dpp target genes. Thus, brk inhibition by Shn results in the upregulation of Dpp-responsive genes. We present evidence that vertebrate Shn homologs can also mediate Bmp responsiveness through a mechanism similar to Drosophila Shn. We find that a Bmp response element (BRE) from the Xenopus Vent2 promoter drives Dpp-dependent expression in Drosophila. However, in sharp contrast to its activating role in vertebrates, the frog BRE mediates repression in Drosophila. Remarkably, despite these opposite transcriptional polarities, sequence changes that abolish cis-element activity in Drosophila also affect BRE function in Xenopus. These similar cis requirements reflect conservation of trans-acting factors, as human Shn1 (hShn1; HIVEP1) can interact with Smad1/Smad4 and assemble an hShn1/Smad complex on the BRE. Furthermore, both Shn and hShn1 activate the BRE in Xenopus embryos, and both repress brk and rescue embryonic patterning defects in shn mutants. Our results suggest that vertebrate Shn proteins function in Bmp signal transduction, and that Shn proteins recruit coactivators and co-repressors in a context-dependent manner, rather than acting as dedicated activators or repressors.

To understand the actions of morphogens, it is crucial to determine how they elicit different transcriptional responses in different cell types. Here, we identify a BMP-responsive enhancer of Msx2, an immediate early target of bone morphogenetic protein (BMP) signaling. We show that the BMP-responsive region of Msx2 consists of a core element, required generally for BMP-dependent expression, and ancillary elements that mediate signaling in diverse developmental settings. Analysis of the core element identified two classes of functional sites: GCCG sequences related to the consensus binding site of Mad/Smad-related BMP signal transducers; and a single TTAATT sequence, matching the consensus site for Antennapedia superclass homeodomain proteins. Chromatin immunoprecipitation and mutagenesis experiments indicate that the GCCG sites are direct targets of BMP restricted Smads. Intriguingly, however, these sites are not sufficient for BMP responsiveness in mouse embryos; the TTAATT sequence is also required. DNA sequence comparisons reveal this element is highly conserved in Msx2 promoters from mammalian orders but is not detectable in other vertebrates or non-vertebrates. Despite this lack of conservation outside mammals, the Msx2 BMP-responsive element serves as an accurate readout of Dpp signaling in a distantly related bilaterian - Drosophila. Strikingly, in Drosophila embryos, as in mice, both TTAATT and GCCG sequences are required for Dpp responsiveness, showing that a common cis-regulatory apparatus can mediate the transcriptional activation of BMP-regulated genes in widely divergent bilaterians.

Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. We have isolated mutations in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Our phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, we find that mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. Our results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv.

Background: Motor proteins of the minus end-directed cytoplasmic dynein and plus end-directed kinesin families provide the principal means for microtubule-based transport in eukaryotic cells. Despite their opposing polarity, these two classes of motors may cooperate in vivo. In Drosophila circumstantial evidence suggests that dynein acts in the localization of determinants and signaling factors during oogenesis. However, the pleiotropic requirement for dynein throughout development has made it difficult to establish its specific role., Results: We analyzed dynein function in the oocyte by disrupting motor activity through temporally restricted expression of the dynactin subunit, dynamitin. Our results indicate that dynein is required for several processes that impact patterning; such processes include localization of bicoid (bcd) and gurken (grk) mRNAs and anchoring of the oocyte nucleus to the cell cortex. Surprisingly, dynein function is sensitive to reduction in kinesin levels, and germ line clones lacking kinesin show defects in dorsal follicle cell fate, grk mRNA localization, and nuclear attachment that are similar to those resulting from the loss of dynein. Significantly, dynein and dynactin localization is perturbed in these animals. Conversely, kinesin localization also depends on dynein activity., Conclusions: We demonstrate that dynein is required for nuclear anchoring and localization of cellular determinants during oogenesis. Strikingly, mutations in the kinesin motor also disrupt these processes and perturb dynein and dynactin localization. These results indicate that the activity of the two motors is interdependent and suggest a model in which kinesin affects patterning indirectly through its role in the localization and recycling of dynein.

In Drosophila, a BMP-related ligand Decapentaplegic (Dpp) is essential for cell fate specification during embryogenesis and in imaginal disc development. Dpp signaling culminates in the phosphorylation and nuclear translocation of Mothers against dpp (Mad), a receptor-specific Smad that can bind DNA and regulate the transcription of Dpp-responsive genes. Genetic analysis has implicated Schnurri (Shn), a zinc finger protein that shares homology with mammalian transcription factors, in the Dpp signal transduction pathway. However, a direct role for Shn in regulating the transcriptional response to Dpp has not been demonstrated. In this study we show that Shn acts as a DNA-binding Mad cofactor in the nuclear response to Dpp. Shn can bind DNA in a sequence-specific manner and recognizes sites within a well-characterized Dpp-responsive promoter element, the B enhancer of the Ultrabithorax (Ubx) gene. The Shn-binding sites are relevant for in vivo expression, since mutations in these sites affect the ability of the enhancer to respond to Dpp. Furthermore we find that Shn and Mad can interact directly through discrete domains. To examine the relative contribution of the two proteins in the regulation of endogenous Dpp target genes we developed a cell culture assay and show that Shn and Mad act synergistically to induce transcription. Our results suggest that cooperative interactions between these two transcription factors could play an important role in the regulation of Dpp target genes. This is the first evidence that Dpp/BMP signaling in flies requires the direct interaction of Mad with a partner transcription factor., (Copyright 2000 Academic Press.)

The BMP-related ligand Decapentaplegic (Dpp) has a well-characterized role in pattern formation during Drosophila embryogenesis and in larval development. Previous work has shown that transcription of Dpp-responsive genes requires the activity of the BMP-specific Smad, Mothers against dpp (Mad). In this study we investigated the role of the zinc finger transcription factor Schnurri (Shn) in mediating the nuclear response to Dpp during adult patterning. Using clonal analysis, we show that wing imaginal disc cells mutant for shn fail to transcribe the genes spalt, optomotor blind, vestigial, and Dad, that are known to be induced by dpp signaling. shn clones also ectopically express brinker, a gene that is downregulated in response to dpp, thus implicating Shn in both activation and repression of Dpp target genes. We demonstrate that loss of shn activity affects anterior-posterior patterning and cell proliferation in the wing blade, in a manner that reflects the graded requirement for Dpp in these processes. Furthermore, we find that shn is expressed in the pupal wing and plays a distinct role in mediating dpp-dependent vein differentiation at this stage. The absence of shn activity results in defects that are similar in nature and severity to those caused by elimination of Mad, suggesting that Shn has an essential role in dpp signal transduction in the developing wing. Our data are consistent with a model in which Shn acts as a cofactor for Mad., (Copyright 2000 Academic Press.)

The 3231-nucleotide-pair (ntp) sequence of one end of one of the two linear mitochondrial (mt) DNA molecules of Hydra attenuata (phylum Cnidaria, class Hydrozoa, order Anthomedusae) has been determined. This segment contains complete genes for tRNA(f-Met), l-rRNA, tRNA(Trp), subunit 2 of cytochrome c oxidase (COII), subunit 8 of ATP synthetase (ATPase8), and the 5' 136 ntp of ATPase6. These genes are arranged in the order given and are transcribed from the same strand of the molecule. As in two other cnidarians, the hexacorallian anthozoan Metridium senile and the octocorallian anthozoan Sarcophyton glaucum, the mt-genetic code of H. attenuata is near standard. The only modification appears to be that TGA specifies tryptophan rather than termination. Also as in M. senile and S. glaucum, the encoded H. attenuata mt-tRNA(f-Met) has primary and secondary structural features resembling those of Escherichia coli initiator tRNA(t-Met). As the encoded mt-tRNA(Trp) cannot be folded into a totally orthodox secondary structure, two alternative forms are suggested. The encoded H. attenuata mt-l-rRNA is 1738 nt, which is 451 nt shorter than the M. senile mt-l-rRNA. Comparisons of secondary structure models of these two mt-l-rRNAs indicate that most of the size difference results from loss of nucleotides in the H. attenuata molecule at a minimum of 46 locations, which includes elimination of six distinct helical elements.

Nuclear movement is critical for several developmental processes in eukaryotes. Drosophila oogenesis provides a paradigmatic example in which localization of the nucleus generates a source of cellular asymmetry that is used in patterning both the anterior-posterior and the dorsal-ventral axes of the oocyte. In this study we show that mutations in the Drosophila Lissencephaly1 (DLis1) gene result in partial ventralization of the eggshell. DLis1 mutations affect the localization of gurken mRNA and protein in the oocyte. These defects are correlated with incorrect positioning of the oocyte nucleus, suggesting that DLis1 is required for nuclear migration. DLis1 shows significant sequence conservation across the evolutionary spectrum. Fungal cognates of DLis1 are involved in nuclear migration while homologs in humans and mice are implicated in neuronal migration. DLis1 shows genetic interactions with the Glued and Dynein heavy chain subunits of the dynein/dynactin complex, supporting the idea that the Lis1 family of proteins plays a role in microtubule motor-based nuclear motility., (Copyright 2000 Academic Press.)

Decapentaplegic (dpp), a TGF beta-related ligand, plays a key role in Drosophila development. Although dpp receptors have been isolated, the downstream components of the signaling pathway remain to be identified. We have cloned the schnurri (shn) gene and show that it encodes a putative zinc finger transcription factor homologous to the human major histocompatibility complex-binding proteins 1 and 2. Mutations in shn affect multiple events that require dpp signaling as well as the transcription of dpp-responsive genes. Genetic interactions and the strikingly similar phenotypes of mutations in shn and the dpp receptors encoded by thick veins and punt suggest that shn plays a downstream role in dpp signaling.

During embryogenesis Drosophila pole cells, like germ cells in many other vertebrates and invertebrates, actively migrate before assembling into the gonad. Five separate steps can be distinguished: an initial passive displacement of the germ cells by gastrulation movements, an amoeboid migratory phase during which the pole cells pass through the endoderm, migration on endoderm toward mesoderm, separation into two bilateral groups associated with the gonadal mesoderm precursors, followed by condensation into the gonad itself. We have analyzed gonad assembly in embryonic pattern mutants to determine whether distinct cues are required in this process. We show that the initiation of migration does not require the presence of the mesoderm, the eventual target of the germ cells. Rather, migration is triggered as a consequence of the differentiation of the endoderm. Examination of embryos mutant for maternal genes of the terminal group suggests that a primary role of the endoderm in this process is to allow the germ cell access to the interior of the embryo. In addition, we show that normal gonad assembly requires a region of the embryo that includes the posterior compartment of the fifth and the sixth abdominal segments.

Cell migration is a common feature of development. In order to understand more about the factors that control these movements we have embarked on further analysis of the migration of Drosophila germ cells. This process involves passage of the germ cells across the gut primordium and migration toward the mesoderm where the somatic gonad forms. We are particularly interested in the early phase of this migration when the germ cells interact with the amnioproctodeal invagination, the developing gut, before entering into association with the mesoderm. We will summarize the results of our and other studies of these events before describing a number of enhancer trap lines which show expression in the amnioproctodeal invagination during the early phase of germ cell migration. These reveal more about the complexity of this tissue and suggest this tissue is capable of guiding the early phase of germ cell migration.

The rel family of proteins can be defined as a group of proteins that share sequence homology over a 300 amino acid region termed the rel domain. The rel family comprises important regulatory proteins from a wide variety of species and includes the Drosophila morphogen dorsal, the mammalian transcription factor NF-kappa B, the avian oncogene v-rel, and the cellular proto-oncogene c-rel. Over the last two years it has become apparent that these proteins function as DNA-binding transcription factors, and that their activity is regulated at the level of subcellular localization.