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1. Extensive loss of Wnt genes in Tardigrada

   https://doi.org/10.1186/s12862-021-01954-y  

   Chavarria, Raul A. ; Game, Mandy ; Arbelaez, Briana ; Ramnarine, Chloe ; Snow, Zachary K. ; Smith, Frank W. ( December 2021 , BMC Ecology and Evolution)

   Wnt genes code for ligands that activate signaling pathways during development in Metazoa. Through the canonical Wnt (cWnt) signaling pathway, these genes regulate important processes in bilaterian development, such as establishing the anteroposterior axis and posterior growth. In Arthropoda, Wnt ligands also regulate segment polarity, and outgrowth and patterning of developing appendages. Arthropods are part of a lineage called Panarthropoda that includes Onychophora and Tardigrada. Previous studies revealed potential roles of Wnt genes in regulating posterior growth, segment polarity, and growth and patterning of legs in Onychophora. Unlike most other panarthropods, tardigrades lack posterior growth, but retain segmentation and appendages. Here, we investigated Wnt genes in tardigrades to gain insight into potential roles that these genes play during development of the highly compact and miniaturized tardigrade body plan.

   We analyzed published genomes for two representatives of Tardigrada,Hypsibius exemplarisandRamazzottius varieornatus. We identified single orthologs ofWnt4,Wnt5,Wnt9,Wnt11, andWntA, as well as twoWnt16paralogs in both tardigrade genomes. We only found aWnt2ortholog inH. exemplaris. We could not identify orthologs ofWnt1,Wnt6,Wnt7,Wnt8, orWnt10. We identified most other components of cWnt signaling in both tardigrade genomes. However, we were unable to identify an ortholog ofarrow/Lrp5/6, a gene that codes for a Frizzled co-receptor of Wnt ligands. Additionally, we found that some other animals that have lost several Wnt genes and are secondarily miniaturized, like tardigrades, are also missing an ortholog ofarrow/Lrp5/6. We analyzed the embryonic expression patterns of Wnt genes inH. exemplarisduring developmental stages that span the establishment of the AP axis through segmentation and leg development. We detected expression of all Wnt genes inH. exemplarisbesides one of theWnt16paralogs. During embryo elongation, expression of several Wnt genes was restricted to the posterior pole or a region between the anterior and posterior poles. Wnt genes were expressed in distinct patterns during segmentation and development of legs inH. exemplaris, rather than in broadly overlapping patterns.

   Our results indicate that Wnt signaling has been highly modified in Tardigrada. While most components of cWnt signaling are conserved in tardigrades, we conclude that tardigrades have lostWnt1,Wnt6,Wnt7,Wnt8, andWnt10, along witharrow/Lrp5/6. Our expression data may indicate a conserved role of Wnt genes in specifying posterior identities during establishment of the AP axis. However, the loss of several Wnt genes and the distinct expression patterns of Wnt genes during segmentation and leg development may indicate that combinatorial interactions among Wnt genes are less important during tardigrade development compared to many other animals. Based on our results, and comparisons to previous studies, we speculate that the loss of several Wnt genes in Tardigrada may be related to a reduced number of cells and simplified development that accompanied miniaturization and anatomical simplification in this lineage.

    

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2. Frizzled2 receives WntA signaling during butterfly wing pattern formation

   https://doi.org/10.1242/dev.201868  

   Hanly, Joseph J. ; Loh, Ling S. ; Mazo-Vargas, Anyi ; Rivera-Miranda, Teomie S. ; Livraghi, Luca ; Tendolkar, Amruta ; Day, Christopher R. ; Liutikaite, Neringa ; Earls, Emily A. ; Corning, Olaf B. W. H. ; et al ( August 2023 , Development)

   Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. While the secreted ligand WntA was shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologues of the Frizzled-family of Wnt receptors. Here we show that CRISPR mosaic knock-outs of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss-of-function in multiple nymphalids. While WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity (PCP) in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning, and shed light on the functional diversity of insect Frizzled receptors.

    

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3. Genome sequence of Ophryocystis elektroscirrha, an apicomplexan parasite of monarch butterflies: cryptic diversity and response to host-sequestered plant chemicals

   https://doi.org/10.1186/s12864-023-09350-0  

   Mongue, Andrew J. ; Martin, Simon H. ; Manweiler, Rachel E. ; Scullion, Helena ; Koehn, Jordyn L. ; de Roode, Jacobus C. ; Walters, James R. ( May 2023 , BMC Genomics)

   Apicomplexa are ancient and diverse organisms which have been poorly characterized by modern genomics. To better understand the evolution and diversity of these single-celled eukaryotes, we sequenced the genome ofOphryocystis elektroscirrha, a parasite of monarch butterflies,Danaus plexippus. We contextualize our newly generated resources within apicomplexan genomics before answering longstanding questions specific to this host-parasite system. To start, the genome is miniscule, totaling only 9 million bases and containing fewer than 3,000 genes, half the gene content of two other sequenced invertebrate-infecting apicomplexans,Porospora giganteaandGregarina niphandrodes. We found thatO. elektroscirrhashares different orthologs with each sequenced relative, suggesting the true set of universally conserved apicomplexan genes is very small indeed. Next, we show that sequencing data from other potential host butterflies can be used to diagnose infection status as well as to study diversity of parasite sequences. We recovered a similarly sized parasite genome from another butterfly,Danaus chrysippus, that was highly diverged from theO. elektroscirrhareference, possibly representing a distinct species. Using these two new genomes, we investigated potential evolutionary response by parasites to toxic phytochemicals their hosts ingest and sequester. Monarch butterflies are well-known to tolerate toxic cardenolides thanks to changes in the sequence of their Type II ATPase sodium pumps. We show thatOphryocystiscompletely lacks Type II or Type 4 sodium pumps, and related proteins PMCA calcium pumps show extreme sequence divergence compared to other Apicomplexa, demonstrating new avenues of research opened by genome sequencing of non-model Apicomplexa.

    

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4. SEC1A is a major Arabidopsis Sec1/Munc18 gene in vesicle trafficking during pollen tube tip growth

   https://doi.org/10.1111/tpj.15742  

   Beuder, Steven ; Lara‐Mondragón, Cecilia ; Dorchak, Alexandria ; MacAlister, Cora A. ( April 2022 , The Plant Journal)

   Pollen tubes (PTs) grow by the targeted secretion of new cell wall material to their expanding tip region. Sec1/Munc18 (SM) proteins promote membrane fusion through regulation of the SNARE complex. We have previously shown that disruption of protein glycosylation in theArabidopsis thaliana hpat1 hpat3double mutant leads to PT growth defects that can be suppressed by reducing secretion. Here, we identified five point mutant alleles of the SM proteinSEC1Aashpat1/3suppressors. The suppressors increased seed set, reduced PT growth defects and reduced the rate of glycoprotein secretion. In the absence of thehpatmutations,sec1areduced pollen germination and PT elongation producing shorter and wider PTs. Consistent with a defect in membrane fusion,sec1aPTs accumulated secretory vesicles. Thoughsec1ahad significantly reduced male transmission, homozygoussec1aplants maintained full seed set, demonstrating thatSEC1Awas ultimately dispensable for pollen fertility. However, when combined with a mutation in anotherSEC1‐likeSMgene,keule, pollen fertility was totally abolished. Mutation insec1b, the final member of the Arabidopsis SEC1 clade, did not enhance thesec1aphenotype. Thus, SEC1A is the major SM protein promoting pollen germination and tube elongation, but in its absence KEULE can partially supply this activity. When we examined the expression of the SM protein family in other species for which pollen expression data were available, we found that at least one Sec1‐like protein was highly expressed in pollen samples, suggesting a conserved role in pollen fertility in other species.

    

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5. Engineering heterologous enzyme secretion in Yarrowia lipolytica

   https://doi.org/10.1186/s12934-022-01863-9  

   Wang, Weigao ; Blenner, Mark A. ( July 2022 , Microbial Cell Factories)

   Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species,Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion inY. lipolyticainclude codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme.

   By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and$$\alpha$$$\alpha$-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion.

   Overall, our combined strategies not only proved effective in improving the protein production inYarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.

    

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