Kristi A. Wharton

Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats.

The primary structure of the major embryonic Notch transcript is presented, as determined by sequence analysis of overlapping cDNA clones. The 10,148 bp sequence corresponding to this transcript possesses an 8109 bp open reading frame that potentially codes for a 2703 amino acid protein. We show that this polypeptide contains a repeated structure composed of 36 tandemly arranged 40 amino acid long repeats, which show homology to the epidermal growth factor and other proteins containing EGF-like repeats. Hydropathy plots suggest that the putative Notch protein may span the membrane. We relate these findings to the developmental action of Notch and speculate that the locus may be involved in a cell-cell interaction mechanism that is essential for the differentiation of the ectoderm into neural and epidermal precursors.

Opa: a novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster

The principal transcription product of Notch, a locus involved in the neurogenesis of D. melanogaster, is a developmentally regulated poly(A)+ RNA approximately 10.5 kb in length. Analysis of the structure of this RNA has revealed a 93 bp repeated sequence that is shared by many other developmentally regulated transcription units. Nucleotide sequence analysis of the repeat shows an unusual structure consisting predominantly of the triplets CAG and CAA, both of which can code for the amino acid Gln. We present evidence indicating that the Notch repeat is a member of a novel family of repetitive elements, which we term the opa family. Our data suggest that some of these elements may be not only transcribed but also translated. We compare opa with other known transcribed repeats and speculate on its functional significance.

A Comparative Study of Desktop, Fishtank, and Cave Systems for the Exploration of Volume Rendered Confocal Data Sets

We present a participant study that compares biological data exploration tasks using volume renderings of laser confocal microscopy data across three environments that vary in level of immersion: a desktop, fishtank, and cave system. For the tasks, data, and visualization approach used in our study, we found that subjects qualitatively preferred and quantitatively performed better in the cave compared with the fishtank and desktop. Subjects performed real-world biological data analysis tasks that emphasized understanding spatial relationships including characterizing the general features in a volume, identifying colocated features, and reporting geometric relationships such as whether clusters of cells were coplanar. After analyzing data in each environment, subjects were asked to choose which environment they wanted to analyze additional data sets in - subjects uniformly selected the cave environment.

Dual function of the Drosophila Alk1/Alk2 ortholog Saxophone shapes the Bmp activity gradient in the wing imaginal disc

Wing patterning in Drosophila requires a Bmp activity gradient created by two Bmp ligands, Gbb and Dpp, and two Bmp type I receptors, Sax and Tkv. Gbb provides long-range signaling, while Dpp signals preferentially to cells near its source along the anteroposterior (AP) boundary of the wing disc. How each receptor contributes to the signaling activity of each ligand is not well understood. Here, we show that while Tkv mediates signals from both Dpp and Gbb, Sax exhibits a novel function for a Bmp type I receptor: the ability to both promote and antagonize signaling. Given its high affinity for Gbb, this dual function of Sax impacts the function of Gbb in the Bmp activity gradient more profoundly than does Dpp. We propose that this dual function of Sax is dependent on its receptor partner. When complexed with Tkv, Sax facilitates Bmp signaling, but when alone, Sax fails to signal effectively and sequesters Gbb. Overall, our model proposes that the balance between antagonizing and promoting Bmp signaling varies across the wing pouch, modulating the level and effective range, and, thus, shaping the Bmp activity gradient. This previously unknown mechanism for modulating ligand availability and range raises important questions regarding the function of vertebrate Sax orthologs.

Twisted Perspective: New Insights into Extracellular Modulation of BMP Signaling during Development

TGF-␤-like ligands, the Bone Morphogenetic Proteins (BMPs), is the direct modulation of ligand activity by extracellular factors (reviewed in De Robertis and Sasai, 1996; Figure 1). In amphibians, opposing activities of BMP4 and its antagonist Chordin (Chd) are responsible for subdivision of the embryonic dorsal–ventral axis. A Brown University similar role for BMP4 and Chd orthologs has been impli-Providence, Rhode Island 02912 cated in dorsal–ventral patterning of the zebrafish embryo (Mullins, 1998). Thus, in the vertebrates, BMP activity specifies ventral fates in the early embryo, and is During the past several decades, considerable research antagonized by localized expression of Chd orthologs efforts have elucidated the central components of many dorsally. In Drosophila, the same relationship exists be-signal transduction pathways in animal systems. More tween the BMP homologs decapentaplegic (dpp) and recently attention has shifted to the importance of mod-screw (scw) and the Chd ortholog short gastrulation ulating intercellular signaling processes during develop-(sog), but the axis is inverted: dpp and scw specify dorsal ment, as it is clear that fine-tuned regulation of signaling fates in the embryo and are antagonized by sog, which systems in vivo results in subtle distinctions at the cellu-is expressed ventrally. In both vertebrates and inverte-lar level. Extracellular modulators have been a particular brates, the activity of Chd orthologs is antagonized by a focus of investigation, including proteins that affect re-family of metalloproteases including Drosophila Tolloid ceptor stability, ligand function, or ligand availability. In (Tld), Xenopus Xolloid (Xol), Human BMP-1, and their these latter types of regulation, proteins such as proteo-orthologs in other vertebrates. Biochemical studies on glycans, heparan sulfate modifying enzymes, and prote-the Xenopus, Drosophila, and mouse proteins have ases have been recognized as key players, as have demonstrated that Tld/Xol/BMP1 cleavage inactivates a diverse class of diffusible modulators that influence Sog/Chd and promotes BMP signaling (Marqué s et al., ligand activity by inhibiting or facilitating its function (see references contained within reviews by Capdevila Xol proteins function as BMP agonists. modulator that influences the availability of BMP ligands, on the Drosophila gene twisted gastrulation (tsg) and a family of the TGF-␤ superfamily of signaling molecules. its vertebrate orthologs reveal another level of complex-It appears that for TGF-␤ signaling in general a major ity in the extracellular modulation of BMP signaling dur-means of signal regulation is the modulation of ligand ing development. tsg was first identified in Drosophila as availability and as yet this system provides …

Gbb/BMP signaling is required to maintain energy homeostasis in Drosophila.

The coordination of animal growth and development requires adequate nutrients. During times of insufficient food, developmental progression is slowed and stored energy is utilized to ensure that cell and tissue survival are maintained. Here, we report our finding that the Gbb/BMP signaling pathway, known to play an important role in many developmental processes in both vertebrates and invertebrates, is critical in the Drosophila larval fat body for regulating energy homeostasis. Animals with mutations in the Drosophila BMP-5,7 orthologue, glass bottom boat (gbb), or in its signaling components, display phenotypes similar to nutrient-deprived and Tor mutant larvae. These phenotypes include a developmental delay with reduced overall growth, a transparent appearance, and altered total lipid, glucose and trehalose levels. We find that Gbb/BMP signaling is required in the larval fat body for maintaining proper metabolism, yet interestingly, following nutrient deprivation larvae in turn show a loss of BMP signaling in fat body cells indicating that Gbb/BMP signaling is a central player in homeostasis. Finally, despite strong phenotypic similarities between nutrient-compromised animals and gbb mutants, distinct differences are observed in the expression of a group of starvation responsive genes. Overall, our results implicate Gbb/BMP signaling as a new pathway critical for positive regulation of nutrient storage and energy homeostasis during development.

A Large Bioactive BMP Ligand with Distinct Signaling Properties Is Produced by Alternative Proconvertase Processing

Mutations in an evolutionarily conserved processing site in bone morphogenetic proteins are associated with developmental disorders. Ligand Size Matters Ligands in the bone morphogenetic protein (BMP) family are critical in development and have been implicated in various pathological processes. The mature BMPs are generated by processing of proproteins. Akiyama et al. identified an alternative processing site in Glass bottom boat (Gbb), the Drosophila ortholog of vertebrate BMP5, 6, and 7. Processing at this site generated a larger ligand that they called Gbb38 and that was more active over a longer range in the developing wing disc than was its smaller counterpart. Mutations in this conserved processing site in three different members of the human BMP family were associated with various human development disorders. Thus, differential processing of BMP ligands may contribute to cell- and tissue-specific responses to BMP signaling. Dimers of conventional transforming growth factor–β (TGF-β) and bone morphogenetic protein (BMP) ligands are composed of two 100– to 140–amino acid peptides that are produced through the proteolytic processing of a proprotein precursor by proconvertases, such as furin. We report the identification of an evolutionarily conserved furin processing site in the amino terminus (NS) of the Glass bottom boat (Gbb; the Drosophila ortholog of vertebrate BMP5, 6, and 7) proprotein that generates a 328–amino acid, active BMP ligand distinct from the conventional 130–amino acid ligand. Gbb38, the large ligand form of Gbb, exhibited greater signaling activity and a longer range than the shorter form Gbb15. The abundance of Gbb15 and Gbb38 varied among different tissues, raising the possibility that differential processing could account for tissue-specific behaviors of BMPs. In human populations, mutations that abolished the NS cleavage site in BMP4, BMP15, or anti-Müllerian hormone were associated with cleft lip with or without cleft palate (BMP4), premature ovarian failure (BMP15), and persistent Müllerian duct syndrome (anti-Müllerian hormone), suggesting the importance of NS processing during development. The identification of this large BMP ligand form and the functional differences between large and small ligands exemplifies the potential for differential proprotein processing to substantially affect BMP and TGF-β signaling output in different tissue and cellular contexts.

Hyperactive BMP signaling induced by ALK2(R206H) requires type II receptor function in a Drosophila model for classic fibrodysplasia ossificans progressiva.

Background: Fibrodysplasia Ossificans Progressiva (FOP) is an autosomal dominant disorder characterized by episodic deposition of heterotopic bone in place of soft connective tissue. All FOP‐associated mutations map to the BMP type I receptor, ALK2, with the ALK2R206H mutant form found in the vast majority of patients. The mechanism(s) regulating the expressivity of hyperactive ALK2R206H signaling throughout a patients life is not well understood. Results: In Drosophila, human ALK2R206H receptor induces hyperactive BMP signaling. As in vertebrates, elevated signaling associated with ALK2R206H in Drosophila is ligand‐independent. We found that a key determinant for ALK2R206H hyperactivity is a functional type II receptor. Furthermore, our results indicate that like its Drosophila ortholog, Saxophone (Sax), wild‐type ALK2 can antagonize, as well as promote, BMP signaling. Conclusions: The dual function of ALK2 is of particular interest given the heterozygous nature of FOP, as the normal interplay between such disparate behaviors could be shifted by the presence of ALK2R206H receptors. Our studies provide a compelling example for Drosophila as a model organism to study the molecular underpinnings of complex human syndromes such as FOP. Developmental Dynamics 241:200–214, 2012.

Crimpy Enables Discrimination of Presynaptic and Postsynaptic Pools of a BMP at the Drosophila Neuromuscular Junction

Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbbs proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal.

Functional Analysis of saxophone , the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPs) direct a myriad of developmental and adult homeostatic events through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like Kinase1 and -2 (ALK1/ACVRL1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp+. In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sax mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by saxP, a viable allele previously reported to be null, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVRl-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-β signaling.