The RNF220 domain nuclear factor Teyrha-Meyrha (Tey) regulates the migration and differentiation of specific visceral and somatic muscles in Drosophila.

Development of the visceral musculature of the Drosophila midgut encompasses a closely coordinated sequence of migration events of cells from the trunk and caudal visceral mesoderm that underlies the formation of the stereotypic orthogonal pattern of circular and longitudinal midgut muscles. Our study focuses on the last step of migration and morphogenesis of longitudinal visceral muscle precursors and shows that these multinucleated precursors utilize dynamic filopodial extensions to migrate in dorsal and ventral directions over the forming midgut tube. The establishment of maximal dorsoventral distances from one another, and anteroposterior alignments, lead to the equidistant coverage of the midgut with longitudinal muscle fibers. We identify Teyrha-Meyhra (Tey), a tissue-specific nuclear factor related to the RNF220 domain protein family, as a crucial regulator of this process of muscle migration and morphogenesis that is further required for proper differentiation of longitudinal visceral muscles. In addition, Tey is expressed in a single somatic muscle founder cell in each hemisegment, regulates the migration of this founder cell, and is required for proper pathfinding of its developing myotube to specific myotendinous attachment sites.

Simulation and in vivo experimentation predict AdamTS-A location of function during caudal visceral mesoderm migration in Drosophila.

BACKGROUND: Caudal visceral mesoderm (CVM) cells migrate as a loose collective along the trunk visceral mesoderm (TVM) and are surrounded by extracellular matrix (ECM). In this study, we examined how one extracellular protease, AdamTS-A, facilitates CVM migration. RESULTS: A comparison of mathematical simulation to experimental results suggests that location of AdamTS-A action in CVM cells is on the sides of the cell not in contact with the TVM, predominantly at the CVM-ECM interface. CVM migration from a top-down view showed CVM cells migrating along the outside of the TVM substrate in the absence of AdamTS-A. Moreover, overexpression of AdamTS-A resulted in similar, but milder, mis-migration of the CVM. These results contrast with the salivary gland where AdamTS-A is proposed to cleave connections at the trailing edge of migrating cells. Subcellular localization of GFP-tagged AdamTS-A suggests that this protease is not limited to functioning at the trailing edge of CVM cells. CONCLUSION: Using both in vivo experimentation and mathematical simulations, we demonstrated that AdamTS-A cleaves connections between CVM cells and the ECM on all sides not attached to the TVM. Clearly, AdamTS-A has a more expansive role around the entire cell in cleaving cell-ECM attachments in cells migrating as a loose collective.

The extracellular protease AdamTS-B inhibits vein formation in the Drosophila wing.

Vein patterning in the Drosophila wing provides a powerful tool to study regulation of various signaling pathways. Here we show that the ADAMTS extracellular protease AdamTS-B (CG4096) is expressed in the embryonic wing imaginal disc precursor cells and the wing imaginal disc, and functions to inhibit wing vein formation. Knock-down of AdamTS-B displayed posterior crossveins (PCVs) with either extra branches or deltas, or wider PCVs, and a wandering distal tip of the L5 longitudinal vein. Conversely, over-expression of AdamTS-B resulted in a complete absence of the PCV, an incomplete anterior crossvein, and missing distal end of the L5 longitudinal vein. We conclude that AdamTS-B inhibits wing vein formation through negative regulation of signaling pathways, possibly BMP as well as Egfr, displaying the complexity of roles for this family of extracellular proteases.

The secreted AdamTS-A metalloprotease is required for collective cell migration.

Members of the ADAMTS family of secreted metalloproteases play crucial roles in modulating the extracellular matrix (ECM) in development and disease. Here, we show that ADAMTS-A, the Drosophila ortholog of human ADAMTS 9 and ADAMTS 20, and of C. elegans GON-1, is required for cell migration during embryogenesis. AdamTS-A is expressed in multiple migratory cell types, including hemocytes, caudal visceral mesoderm (CVM), the visceral branch of the trachea (VBs) and the secretory portion of the salivary gland (SG). Loss of AdamTS-A causes defects in germ cell, CVM and VB migration and, depending on the tissue, AdamTS-A functions both autonomously and non-autonomously. In the highly polarized collective of the SG epithelium, loss of AdamTS-A causes apical surface irregularities and cell elongation defects. We provide evidence that ADAMTS-A is secreted into the SG lumen where it functions to release cells from the apical ECM, consistent with the defects observed in AdamTS-A mutant SGs. We show that loss of the apically localized protocadherin Cad99C rescues the SG defects, suggesting that Cad99C serves as a link between the SG apical membrane and the secreted apical ECM component(s) cleaved by ADAMTS-A. Our analysis of AdamTS-A function in the SG suggests a novel role for ADAMTS proteins in detaching cells from the apical ECM, facilitating tube elongation during collective cell migration.

Drosophila neuroligin 2 is required presynaptically and postsynaptically for proper synaptic differentiation and synaptic transmission.

Trans-synaptic adhesion between Neurexins (Nrxs) and Neuroligins (Nlgs) is thought to be required for proper synapse organization and modulation, and mutations in several human Nlgs have shown association with autism spectrum disorders. Here we report the generation and phenotypic characterization of Drosophila neuroligin 2 (dnlg2) mutants. Loss of dnlg2 results in reduced bouton numbers, aberrant presynaptic and postsynaptic development at neuromuscular junctions (NMJs), and impaired synaptic transmission. In dnlg2 mutants, the evoked responses are decreased in amplitude, whereas the total active zone (AZ) numbers at the NMJ are comparable to wild type, suggesting a decrease in the release probability. Ultrastructurally, the presynaptic AZ number per bouton area and the postsynaptic density area are both increased in dnlg2 mutants, whereas the subsynaptic reticulum is reduced in volume. We show that both presynaptic and postsynaptic expression of Dnlg2 is required to restore synaptic growth and function in dnlg2 mutants. Postsynaptic expression of Dnlg2 in dnlg2 mutants and wild type leads to reduced bouton growth whereas presynaptic and postsynaptic overexpression in wild-type animals results in synaptic overgrowth. Since Nlgs have been shown to bind to Nrxs, we created double mutants. These mutants are viable and display phenotypes that closely resemble those of dnlg2 and dnrx single mutants. Our results provide compelling evidence that Dnlg2 functions both presynaptically and postsynaptically together with Neurexin to determine the proper number of boutons as well as the number of AZs and size of synaptic densities during the development of NMJs.

The FGF8-related signals Pyramus and Thisbe promote pathfinding, substrate adhesion, and survival of migrating longitudinal gut muscle founder cells.

Fibroblast growth factors (FGFs) frequently fulfill prominent roles in the regulation of cell migration in various contexts. In Drosophila, the FGF8-like ligands Pyramus (Pyr) and Thisbe (Ths), which signal through their receptor Heartless (Htl), are known to regulate early mesodermal cell migration after gastrulation as well as glial cell migration during eye development. Herein, we show that Pyr and Ths also exert key roles during the long-distance migration of a specific sub-population of mesodermal cells that migrate from the caudal visceral mesoderm within stereotypic bilateral paths along the trunk visceral mesoderm toward the anterior. These cells constitute the founder myoblasts of the longitudinal midgut muscles. In a forward genetic screen for regulators of this morphogenetic process we identified loss of function alleles for pyr. We show that pyr and ths are expressed along the paths of migration in the trunk visceral mesoderm and endoderm and act largely redundantly to help guide the founder myoblasts reliably onto and along their substrate of migration. Ectopically-provided Pyr and Ths signals can efficiently re-rout the migrating cells, both in the presence and absence of endogenous signals. Our data indicate that the guidance functions of these FGFs must act in concert with other important attractive or adhesive activities of the trunk visceral mesoderm. Apart from their guidance functions, the Pyr and Ths signals play an obligatory role for the survival of the migrating cells. Without these signals, essentially all of these cells enter cell death and detach from the migration substrate during early migration. We present experiments that allowed us to dissect the roles of these FGFs as guidance cues versus trophic activities during the migration of the longitudinal visceral muscle founders.

HLH54F is required for the specification and migration of longitudinal gut muscle founders from the caudal mesoderm of Drosophila.

HLH54F, the Drosophila ortholog of the vertebrate basic helix-loop-helix domain-encoding genes capsulin and musculin, is expressed in the founder cells and developing muscle fibers of the longitudinal midgut muscles. These cells descend from the posterior-most portion of the mesoderm, termed the caudal visceral mesoderm (CVM), and migrate onto the trunk visceral mesoderm prior to undergoing myoblast fusion and muscle fiber formation. We show that HLH54F expression in the CVM is regulated by a combination of terminal patterning genes and snail. We generated HLH54F mutations and show that this gene is crucial for the specification, migration and survival of the CVM cells and the longitudinal midgut muscle founders. HLH54F mutant embryos, larvae, and adults lack all longitudinal midgut muscles, which causes defects in gut morphology and integrity. The function of HLH54F as a direct activator of gene expression is exemplified by our analysis of a CVM-specific enhancer from the Dorsocross locus, which requires combined inputs from HLH54F and Biniou in a feed-forward fashion. We conclude that HLH54F is the earliest specific regulator of CVM development and that it plays a pivotal role in all major aspects of development and differentiation of this largely twist-independent population of mesodermal cells.

Drosophila Tey represses transcription of the repulsive cue Toll and generates neuromuscular target specificity.

Little is known about the genetic program that generates synaptic specificity. Here we show that a putative transcription factor, Teyrha-Meyhra (Tey), controls target specificity, in part by repressing the expression of a repulsive cue, Toll. We focused on two neighboring muscles, M12 and M13, which are innervated by distinct motoneurons in Drosophila. We found that Toll, which encodes a transmembrane protein with leucine-rich repeats, was preferentially expressed in M13. In Toll mutants, motoneurons that normally innervate M12 (MN12s) formed smaller synapses on M12 and instead appeared to form ectopic nerve endings on M13. Conversely, ectopic expression of Toll in M12 inhibited synapse formation by MN12s. These results suggest that Toll functions in M13 to prevent synapse formation by MN12s. We identified Tey as a negative regulator of Toll expression in M12. In tey mutants, Toll was strongly upregulated in M12. Accordingly, synapse formation on M12 was inhibited. Conversely, ectopic expression of tey in M13 decreased the amount of Toll expression in M13 and changed the pattern of motor innervation to the one seen in Toll mutants. These results suggest that Tey determines target specificity by repressing the expression of Toll. These results reveal a mechanism for generating synaptic specificity that relies on the negative regulation of a repulsive target cue.

sli is required for proper morphology and migration of sensory neurons in the Drosophila PNS.

Neurons and glial cells coordinate with each other in many different aspects of nervous system development. Both types of cells are receiving multiple guidance cues to guide the neurons and glial cells to their proper final position. The lateral chordotonal organs (lch5) of the Drosophila peripheral nervous system (PNS) are composed of five sensory neurons surrounded by four different glial cells, scolopale cells, cap cells, attachment cells and ligament cells. During embryogenesis, the lch5 neurons go through a rotation and ventral migration to reach their final position in the lateral region of the abdomen. We show here that the extracellular ligand sli is required for the proper ventral migration and morphology of the lch5 neurons. We further show that mutations in the Sli receptors Robo and Robo2 also display similar defects as loss of sli, suggesting a role for Slit-Robo signaling in lch5 migration and positioning. Additionally, we demonstrate that the scolopale, cap and attachment cells follow the mis-migrated lch5 neurons in sli mutants, while the ventral stretching of the ligament cells seems to be independent of the lch5 neurons. This study sheds light on the role of Slit-Robo signaling in sensory neuron development.

The extracellular protease stl functions to inhibit migration of v'ch1 sensory neuron during Drosophila embryogenesis.

Proper migration of cells through the dense and complex extracellular matrix (ECM) requires constant restructuring of the ECM to allow cells to move forward in a smooth manner. This restructuring can occur through the action of extracellular enzymes. Among these extracellular enzymes is the ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin repeats) family of secreted extracellular proteases. Drosophila stl encodes an ADAMTS protease expressed in and around the peripheral nervous system (PNS) during embryogenesis. The absence of stl displayed one specific neuron, the v'ch1 sensory neuron, migrating to its target sooner than in wild type. During normal development, the v'ch1 sensory neuron migrates dorsally at the same time it is extending an axon ventrally toward the CNS. Surprisingly, in the absence of stl, the v'ch1 neuron migrated further dorsally as compared to the wild type at stage 15, but did not migrate past its correct target at stage 16, suggesting a novel role for this extracellular protease in inhibiting migration of this neuron past a certain point.

Hepatic glutamine metabolism.

Expression of high activities of both glutamine synthetase and glutaminase allows the liver to play a major role in the regulation of glutamine homeostasis. The liver shows net glutamine output in metabolic acidosis, in prolonged starvation and animals bearing tumors, net glutamine uptake in the postabsorptive state, on consuming high protein diets, and in uncontrolled diabetes or sepsis. Liver glutamine synthetase is expressed only in a small population of perivenous cells that allows it to salvage any ammonia not incorporated into urea in periportal cells. Hepatic glutaminase is a unique isozyme found only in periportal liver parenchymal cells where it provides glutamate and ammonia for the urea cycle. Control of hepatic glutamine metabolism occurs almost exclusively through changes in the activity of glutaminase, with no change in glutamine synthetase flux.