Tyrosine kinase inhibition produces specific alterations in axon guidance in the grasshopper embryo

Tyrosine kinase signaling pathways are essential for process outgrowth and guidance during nervous system development. We have examined the roles of tyrosine kinase activity in programming growth cone guidance decisions in an intact nervous system in which neurons can be individually identified. We applied the tyrosine kinase inhibitors herbimycin A and genistein to whole 40% grasshopper embryos placed in medium, or injected the inhibitors into intact grasshopper eggs. Both inhibitors caused interneuronal axons that normally would grow along the longitudinal connectives to instead leave the central nervous system (CNS) within the segmental nerve root and grow out toward the body wall muscles. In addition, herbimycin A produced pathfinding errors in which many longitudinal axons crossed the CNS midline. To study how this drug affected guidance decisions made by individual growth cones, we dye-filled the pCC interneuron, which normally extends an axon anteriorly along the ipsilateral longitudinal connective. In the presence of herbimycin A, the pCC growth cone was redirected across the anterior commissure. These phenotypes suggest that tyrosine kinase inhibition blocks a signaling mechanism that repels the growth cones of longitudinal connective neurons and prevents them from crossing the midline

Genetics of fat storage in flies and worms: what went wrong?

Body weight and fat storage are strongly influenced by an individual’s genetic makeup. In humans, genetic polymorphisms have been identified that have effects on body mass index (BMI) and fat content (Meyre et al., 2009; Speliotes et al., 2010; Choquet and Meyre, 2011a), and studies of monogenic rodent models of obesity have defined a variety of genes and signaling pathways that control fat storage and metabolism (Barsh and Schwartz, 2002). However, many other genes that regulate these processes undoubtedly remain to be discovered. Although forward genetic screens in the mouse have the potential to identify new obesity genes, such screens are expensive and lengthy endeavors

The cell surface receptor Tartan is a potential in vivo substrate for the receptor tyrosine phosphatase Ptp52F

Receptor-linked protein-tyrosine phosphatases (RPTPs) are essential regulators of axon guidance and synaptogenesis in Drosophila, but the signaling pathways in which they function are poorly defined. We identified the cell surface receptor Tartan (Trn) as a candidate substrate for the neuronal RPTP Ptp52F by using a modified two-hybrid screen with a substrate-trapping mutant of Ptp52F as "bait." Trn can bind to the Ptp52F substrate-trapping mutant in transfected Drosophila S2 cells if v-Src kinase, which phosphorylates Trn, is also expressed. Coexpression of wild-type Ptp52F causes dephosphorylation of v-Src-phosphorylated Trn. To examine the specificity of the interaction in vitro, we incubated Ptp52F-glutathione S-transferase (GST) fusion proteins with pervanadate-treated S2 cell lysates. Wild-type Ptp52F dephosphorylated Trn, as well as most other bands in the lysate. GST "pulldown" experiments demonstrated that the Ptp52F substrate-trapping mutant binds exclusively to phospho-Trn. Wild-type Ptp52F pulled down dephosphorylated Trn, suggesting that it forms a stable Ptp52F-Trn complex that persists after substrate dephosphorylation. To evaluate whether Trn and Ptp52F are part of the same pathway in vivo, we examined motor axon guidance in mutant embryos. trn and Ptp52F mutations produce identical phenotypes affecting the SNa motor nerve. The genes also display dosage-dependent interactions, suggesting that Ptp52F regulates Trn signaling in SNa motor neurons

Building a ladder to Hershey Heaven

When Alfred Hershey, one of the founders of molecular biology, was asked to describe his idea of scientific happiness, he said that it would be “to have one experiment that works, and keep doing it all the time”. By this he meant that it would be ideal to be able to conduct every experiment using the same tools and methods, and yet always generate new and interesting data (see Creager, 2001). However, molecular geneticists have not yet reached this “Hershey Heaven”. Today, when researchers want to discover more about a protein in an animal – for example, which tissues and cell types express the protein – they usually have to rely on antibodies that bind to the protein of interest. Unfortunately, good antibodies do not exist for most proteins, and it is time-consuming and expensive to generate and characterize new antibodies

Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways

Regulation of CNS and motor axon guidance in Drosophila by the receptor tyrosine phosphatase DPTP52F

Receptor-linked protein tyrosine phosphatases (RPTPs) regulate axon guidance and synaptogenesis in Drosophila embryos and larvae. We describe DPTP52F, the sixth RPTP to be discovered in Drosophila. Our genomic analysis indicates that there are likely to be no additional RPTPs encoded in the fly genome. Five of the six Drosophila RPTPs have C. elegans counterparts, and three of the six are also orthologous to human RPTP subfamilies. DPTP52F, however, has no clear orthologs in other organisms. The DPTP52F extracellular domain contains five fibronectin type III repeats and it has a single phosphatase domain. DPTP52F is selectively expressed in the CNS of late embryos, as are DPTP10D, DLAR, DPTP69D and DPTP99A. To define developmental roles of DPTP52F, we used RNA interference (RNAi)-induced phenotypes as a guide to identify Ptp52F alleles among a collection of EMS-induced lethal mutations. Ptp52F single mutant embryos have axon guidance phenotypes that affect CNS longitudinal tracts. This phenotype is suppressed in Dlar Ptp52F double mutants, indicating that DPTP52F and DLAR interact competitively in regulating CNS axon guidance decisions. Ptp52F single mutations also cause motor axon phenotypes that selectively affect the SNa nerve. DPTP52F, DPTP10D and DPTP69D have partially redundant roles in regulation of guidance decisions made by axons within the ISN and ISNb motor nerves

Redundancy and compensation in axon guidance: genetic analysis of the Drosophila Ptp10D/Ptp4E receptor tyrosine phosphatase subfamily

Background: Drosophila has six receptor protein tyrosine phosphatases (RPTPs), five of which are expressed primarily in neurons. Mutations in all five affect axon guidance, either alone or in combination. Highly penetrant CNS and motor axon guidance alterations are usually observed only when specific combinations of two or more RPTPs are removed. Here, we examine the sixth RPTP, Ptp4E, which is broadly expressed. Results: Ptp4E and Ptp10D are closely related Type III RPTPs. Non-drosophilid insect species have only one Type III RPTP, which is closest to Ptp10D. We found that Ptp4E mutants are viable and fertile. We then examined Ptp4E Ptp10D double mutants. These die before the larval stage, and have a mild CNS phenotype in which the outer longitudinal 1D4 bundle is frayed. Ptp10D Ptp69D double mutants have a strong CNS phenotype in which 1D4 axons abnormally cross the midline and the outer and middle longitudinal bundles are fused to the inner bundle. To examine if Ptp4E also exhibits synthetic phenotypes in combination with Ptp69D, we made Ptp4E Ptp69D double mutants and Ptp4E Ptp10D Ptp69D triple mutants. No phenotype was observed in the double mutant. The triple mutant phenotype differs from the Ptp10D Ptp69D phenotype in two ways. First, the longitudinal tracts appear more normal than in the double mutant; two or three bundles are observed, although they are disorganized and fused. Second, axons labelled by the SemaIIB-tMyc marker often cross in the wrong commissure. We also examined motor axon guidance, and found that no phenotypes are observed in any Ptp4E double mutant combination. However, triple mutants in which Ptp4E Ptp10D was combined with Ptp69D or Ptp52F exhibited stronger phenotypes than the corresponding Ptp10D double mutants. Conclusions: Type III RPTPs are required for viability in Drosophila, since Ptp4E Ptp10D double mutants die before the larval stage. Unlike Ptp10D, Ptp4E appears to be a relatively minor player in the control of axon guidance. Strong phenotypes are only observed in triple mutants in which both Type III RPTPs are eliminated together with Ptp69D or Ptp52F. Our results allow us to construct a complete genetic interaction matrix for all six of the RPTPs

R3 receptor tyrosine phosphatases: Conserved regulators of receptor tyrosine kinase signaling and tubular organ development

R3 receptor tyrosine phosphatases (RPTPs) are characterized by extracellular domains composed solely of long chains of fibronectin type III repeats, and by the presence of a single phosphatase domain. There are five proteins in mammals with this structure, two in Drosophila and one in Caenorhabditis elegans. R3 RPTPs are selective regulators of receptor tyrosine kinase (RTK) signaling, and a number of different RTKs have been shown to be direct targets for their phosphatase activities. Genetic studies in both invertebrate model systems and in mammals have shown that R3 RPTPs are essential for tubular organ development. They also have important functions during nervous system development. R3 RPTPs are likely to be tumor suppressors in a number of types of cancer

Visualization of binding patterns for five Leucine-rich repeat proteins in the Drosophila embryo

Leucine-rich repeat (LRR) domain-containing proteins play central roles in organizing neural connectivity. The LRR is a protein-recognition motif and proteins with extracellular LRR (eLRR) domains mediate intercellular communication and cell adhesion, which in turn regulate neuronal processes such as axon guidance, target selection, synapse formation and stabilization of connections (de Wit et al. 2011). The LRR-domain containing Slits and their Robo receptors are one of the best characterized examples of ligand-receptor pairs that regulate midline crossing and axon guidance in both Drosophila and vertebrates (Brose et al. 1999; Dickson and Gilestro 2006). There are 66 eLRR proteins in Drosophila, many of which are expressed in the nervous system and exhibit strikingly specific expression patterns, often labeling distinct subpopulations of neurons (Lauren et al. 2003; Dolan et al. 2007). The binding partners and functions of many of these eLRR proteins remain unknown. We have previously described a novel method to identify ligands and/or binding partners for extracellular proteins (Fox and Zinn 2005; Lee et al. 2013; Ozkan et al. 2013). This method involves using fusion proteins containing the extracellular domain (ECD) of a protein fused to a pentamerization domain (COMP), followed by human placental alkaline phosphatase (AP). These AP fusion proteins are used to stain live-dissected stage 16 Drosophila embryos. The resulting staining patterns can be used as a template to identify expression patterns of the binding partners of the AP fusion protein. Using this technique, we have identified ligands for the receptor tyrosine phosphatases Ptp10D, Lar and Ptp69D (Bali et al. 2019; Fox and Zinn 2005; Lee et al. 2013). Here, we describe novel binding patterns for 5 eLRR proteins using their respective AP fusion proteins. Tartan (trn) and Capricious (caps) are two closely-related eLRR proteins with known functions in embryonic motor axon guidance and the innervation of antennal lobe glomeruli by olfactory sensory axons (Kurusu et al. 2008; Hong et al. 2009). Studies of trn and caps single and double mutants suggest that the two genetically interact and may function through a common binding partner (Milan et al. 2005; Kurusu et al. 2008). Tartan may be a substrate for the receptor tyrosine phosphatase Ptp52F (Bugga et al. 2009). We stained wild-type live-dissected stage 16 Drosophila embryos with trn-AP and caps-AP fusion proteins separately, and found distinct as well as overlapping staining patterns for both fusion proteins. Both trn-AP and caps-AP bind to longitudinal axons in the ventral nerve cord (VNC), with stronger binding seen in one particular axon bundle close to the midline (arrows, a1’ and b2’). Both also show binding to muscles (arrows, a2’ and b3’), indicating that they interact with a binding partner expressed on the surface of muscles. trn-AP shows binding to a subset of sensory neurons (arrow, a3’), which caps-AP does not. In addition, caps-AP binds to the transverse nerve, which emanates from the midline and is located on the dorsal side of the VNC (arrow, b1’). Fish-lips (Fili) is an eLRR with roles in the regulation of apoptosis (Adachi-Yamada et al. 2005) and olfactory receptor neuron (ORN) targeting in the antennal lobe (Xie et al. 2019). It is expressed at moderately-high levels during embryonic stages 12 – 17 and during 24 – 48 hours after puparium formation (modENCODE Temporal Expression Data, FlyBase). These developmental stages correspond to peak synaptogenesis times, implying a developmental role of Fili in regulating synaptogenesis. Thus, identification of binding partners of Fili is crucial to understand its roles in CNS development. Staining of wild-type stage 16 embryos with Fili-AP fusion protein shows a restricted binding pattern in the CNS, indicating a similar restricted expression pattern of its binding partners. It binds to a set of dorsal midline neurons (arrow, c1’) and a subset of longitudinal axons in the VNC (arrow, c2’). A subset of midline cells, putatively glial cells are also labeled with Fili-AP. Strong binding is seen to the transverse nerve in the VNC (c1) and in the periphery (arrow, c3’), while no labeling is seen to the SNa in the same focal plane (arrow, c3). Reduced ocelli (rdo) is a gene that regulates ocelli development (Caldwell et al. 2007) and encodes an eLRR protein of unknown function. Caldwell et al. 2007 showed a broad expression pattern of the encoded protein in the adult nervous system. We performed staining of wild-type stage 16 embryos with rdo-AP fusion protein and found a very strong binding signal in the longitudinal and commissural axons of the VNC (arrow, d1’). This binding was limited to the VNC, and no binding was observed to the muscles (data not shown), indicating that the eLRR encoded by rdo interacts with neuronal-specific ligands. We also observed binding in a subset of midline glial cells in the VNC (arrow, d2’). 2mit is another gene encoding a putative eLRR and is expressed in the developing nervous system. It has a putative role in regulating short-term memory (Baggio et al. 2013). No other information is known about this eLRR. We stained wild-type stage 16 embryos with 2mit-AP fusion protein and saw a wide pattern of binding by this fusion protein, unlike the other restricted patterns observed above. Both longitudinal, commissural as well as exiting motor axons in the VNC are labeled by 2mit-AP (arrows, e1’). Moreover, we observed a pan-cellular pattern of labeling in the periphery as well as in the VNC, where 2-mit-AP binding signal is seen on the surface of cells, resulting in a cell-membrane staining pattern (arrow, e2’). This implies that the eLRR encoded by 2mit is capable of interacting with ligands expressed on neuronal as well as non-neuronal cell types. These binding patterns provide clues to the expression patterns of proteins that these eLRRs might interact with to regulate various developmental processes