Investigating the evolution and function of Wnt ligands

Wnt genes encode secreted glycoproteins which play an important role in development of all animals. Overall, thirteen subfamilies of Wnt ligands are present and all of them can already be found in the most basal metazoans. The importance of this large gene family and its evolutionary conservation intrigued me to analyse Wnts with a variety of different approaches. Starting with a broad evolutionary approach, the losses, conservation and duplication within the Wnt gene repertoire throughout the metazoan phylogeny were studied to understand the underlying evolutionary constrains which were fundamental to create this diverse Wnt landscape. I focussed on elucidating the Wnt gene losses and duplications in arthropods where I found support for the loss of Wnt2 and Wnt4 in all insects, loss of Wnt16 in all insets except Hemiptera and loss of Wnt8 and Wnt9 in Hymenoptera, while Chelicerata, such as spiders and scorpions have lost Wnt10. In horseshoe crabs, spiders and scorpions, duplications of Wnt7 and Wnt11 were observed. Taking some of the results from this broad evolutionary analysis, it would be interesting to understand on a finer scale how the expression or function of Wnt genes is conserved throughout more closely related species. Here, Lepidoptera became of certain interest due to their close relation toits sister groups Diptera and Coleoptera. The expression of Wnts is well known in Drosophila (Diptera) and Tribolium (Coleoptera) but relatively less is understood about Wnt gene expression in butterflies and moths (Lepidoptera). Showing the expression of Wnts in Lepidoptera and being able to compare these results with known patterns from closely related taxa could help to understand if also the function of Wnts could be conserved within phyla. Interestingly, it was possible to show that some Wnts genes (Wnt1, A and 10) have similar expression in all three analysed classes. This hints that in these closer related groups the function of Wnts could be conserved as well and therefore could also be able to influence the evolution of the ligands itself. In the third part of this thesis, the exact function of Wnts was even more narrowed down. For this purpose, Drosophila melanogaster was used and puzzlingly, even in a well-studied model organism such as Drosophila, the function of some of the Wnts is not fully understood. wingless for example is the most studied Wnt gene in Drosophila, while the role during development for Wnt6 and Wnt10 remains unclear. In the following analysis, the focus was on Wnt6 due to its high sequence similarity to wg, close genomic location and overlapping expression. Wnt6 is also highly conserved in all arthropods and additionally part of the conserved Wnt cluster (Wnt1-6-9-10). Hence, the function of Wnt6 during development was studied and also these results were linked to the question of how and why Wnt genes are conserved and why so many Wnt ligands are still present in many species. Previously, a potential role of Wnt6 during maxillary palp development was described which was used as a starting point for the functional analysis. Further, a new Wnt6 knockout line, using CRISPR/Cas9 was generated for comparison to a published knockout line. During the analysis a putative regulatory function of the first exon of Wnt6 was found, which might influence a crucial wg signal during palp development. Wnt6 itself might be involved in regulating the correct growth and pupariation signal during larval development. This analysis also added an additional components, including the regulation of Wnt ligands of the ancestral Wnt cluster to the potential evolutionary mechanisms. Taking all of these results together it was possible to highlight the large diversity of the Wnt landscape in arthropods and indicate clues about the underlying evolutionary mechanisms. Analysing the exact function of Wnt6 also revealed that the genomic location or the clustering of Wnts could play a role in constraining evolution on these genes due to regulatory region within the genes. Overall, this study contributes to increase our understanding of Wnt gene evolution as well as the function and regulation of Wnt ligands

Widespread retention of ohnologs in key developmental gene families following whole-genome duplication in arachnopulmonates

Whole-genome duplications (WGDs) have occurred multiple times during animal evolution, including in lineages leading to vertebrates, teleosts, horseshoe crabs, and arachnopulmonates. These dramatic events initially produce a wealth of new genetic material, generally followed by extensive gene loss. It appears, however, that developmental genes such as homeobox genes, signaling pathway components and microRNAs are frequently retained as duplicates (so-called ohnologs) following WGD. These not only provide the best evidence for WGD, but an opportunity to study its evolutionary consequences. Although these genes are well studied in the context of vertebrate WGD, similar comparisons across the extant arachnopulmonate orders are patchy. We sequenced embryonic transcriptomes from two spider species and two amblypygid species and surveyed three important gene families, Hox, Wnt, and frizzled, across these and 12 existing transcriptomic and genomic resources for chelicerates. We report extensive retention of putative ohnologs, further supporting the ancestral arachnopulmonate WGD. We also found evidence of consistent evolutionary trajectories in Hox and Wnt gene repertoires across three of the six arachnopulmonate orders, with interorder variation in the retention of specific paralogs. We identified variation between major clades in spiders and are better able to reconstruct the chronology of gene duplications and losses in spiders, amblypygids, and scorpions. These insights shed light on the evolution of the developmental toolkit in arachnopulmonates, highlight the importance of the comparative approach within lineages, and provide substantial new transcriptomic data for future study

Widespread retention of ohnologs in key developmental gene families following whole-genome duplication in arachnopulmonates

Whole-genome duplications (WGDs) have occurred multiple times during animal evolution, including in lineages leading to vertebrates, teleosts, horseshoe crabs, and arachnopulmonates. These dramatic events initially produce a wealth of new genetic material, generally followed by extensive gene loss. It appears, however, that developmental genes such as homeobox genes, signaling pathway components and microRNAs are frequently retained as duplicates (so-called ohnologs) following WGD. These not only provide the best evidence for WGD, but an opportunity to study its evolutionary consequences. Although these genes are well studied in the context of vertebrate WGD, similar comparisons across the extant arachnopulmonate orders are patchy. We sequenced embryonic transcriptomes from two spider species and two amblypygid species and surveyed three important gene families, Hox, Wnt, and frizzled, across these and 12 existing transcriptomic and genomic resources for chelicerates. We report extensive retention of putative ohnologs, further supporting the ancestral arachnopulmonate WGD. We also found evidence of consistent evolutionary trajectories in Hox and Wnt gene repertoires across three of the six arachnopulmonate orders, with interorder variation in the retention of specific paralogs. We identified variation between major clades in spiders and are better able to reconstruct the chronology of gene duplications and losses in spiders, amblypygids, and scorpions. These insights shed light on the evolution of the developmental toolkit in arachnopulmonates, highlight the importance of the comparative approach within lineages, and provide substantial new transcriptomic data for future study

The skeletal ontogeny of Astatotilapia burtoni – a direct-developing model system for the evolution and development of the teleost body plan

Abstract Background The experimental approach to the evolution and development of the vertebrate skeleton has to a large extent relied on “direct-developing” amniote model organisms, such as the mouse and the chicken. These organisms can however only be partially informative where it concerns secondarily lost features or anatomical novelties not present in their lineages. The widely used anamniotes Xenopus and zebrafish are “indirect-developing” organisms that proceed through an extended time as free-living larvae, before adopting many aspects of their adult morphology, complicating experiments at these stages, and increasing the risk for lethal pleiotropic effects using genetic strategies. Results Here, we provide a detailed description of the development of the osteology of the African mouthbrooding cichlid Astatotilapia burtoni, primarily focusing on the trunk (spinal column, ribs and epicentrals) and the appendicular skeleton (pectoral, pelvic, dorsal, anal, caudal fins and scales), and to a lesser extent on the cranium. We show that this species has an extremely “direct” mode of development, attains an adult body plan within 2 weeks after fertilization while living off its yolk supply only, and does not pass through a prolonged larval period. Conclusions As husbandry of this species is easy, generation time is short, and the species is amenable to genetic targeting strategies through microinjection, we suggest that the use of this direct-developing cichlid will provide a valuable model system for the study of the vertebrate body plan, particularly where it concerns the evolution and development of fish or teleost specific traits. Based on our results we comment on the development of the homocercal caudal fin, on shared ontogenetic patterns between pectoral and pelvic girdles, and on the evolution of fin spines as novelty in acanthomorph fishes. We discuss the differences between “direct” and “indirect” developing actinopterygians using a comparison between zebrafish and A. burtoni development

Wnt Gene Expression During Early Embryogenesis in the Nymphalid Butterfly Bicyclus anynana

Wnt signaling pathways are involved in many important cellular processes including proliferation and differentiation. Wnt ligands are released by source cells and signal to target cells by binding to the Frizzled receptor family and triggering changes in downstream target gene expression. Wnt signaling appeared at the base of metazoans and there was an early expansion in the repertoire of Wnt ligands to the 13 known subfamilies. However, little is known about functionality of these ligands in many animal lineages. Understanding the roles of these important signaling molecules in a wider range of animals is crucial to understand the regulation and evolution of cell fate during development and how this can lead to diversification. Here, we analyzed the Wnt repertoire among lepidopterans, where the embryological functionality of these ligands is understudied compared to other insect orders. To be able to explore Wnt gene roles during butterfly embryogenesis we first established a staging system for the butterfly model, Bicyclus anynana, and assayed the expression pattern of all eight lepidopteran Wnt genes during early butterfly development. We detected expression of Wnt1, Wnt10, and WntA in several expression domains, such as segmental stripes as well as expression of Wnt7 in the nervous system and Wnt11 in several head structures. Overall, our study provides, a basis for future research into butterfly embryogenesis and much needed new insights into the potential roles of Wnt genes in specifying cell fate in these animals as well as how this compares to other animals

Widespread retention of ohnologs in key developmental gene families following whole-genome duplication in arachnopulmonates

Whole-genome duplications (WGDs) have occurred multiple times during animal evolution, including in lineages leading to vertebrates, teleosts, horseshoe crabs, and arachnopulmonates. These dramatic events initially produce a wealth of new genetic material, generally followed by extensive gene loss. It appears, however, that developmental genes such as homeobox genes, signaling pathway components and microRNAs are frequently retained as duplicates (so-called ohnologs) following WGD. These not only provide the best evidence for WGD, but an opportunity to study its evolutionary consequences. Although these genes are well studied in the context of vertebrate WGD, similar comparisons across the extant arachnopulmonate orders are patchy. We sequenced embryonic transcriptomes from two spider species and two amblypygid species and surveyed three important gene families, Hox, Wnt, and frizzled, across these and 12 existing transcriptomic and genomic resources for chelicerates. We report extensive retention of putative ohnologs, further supporting the ancestral arachnopulmonate WGD. We also found evidence of consistent evolutionary trajectories in Hox and Wnt gene repertoires across three of the six arachnopulmonate orders, with interorder variation in the retention of specific paralogs. We identified variation between major clades in spiders and are better able to reconstruct the chronology of gene duplications and losses in spiders, amblypygids, and scorpions. These insights shed light on the evolution of the developmental toolkit in arachnopulmonates, highlight the importance of the comparative approach within lineages, and provide substantial new transcriptomic data for future study

Co-occurrence of targetable mutations in Non-small cell lung cancer (NSCLC) patients harboring MAP2K1 mutations

Background: MAP2K1 mutations are rare in non-small cell lung cancer (NSCLC) and considered to be mutually exclusive from known driver mutations. Activation of the MEK1-cascade is considered pivotal in resistance to targeted therapy approaches, and MAP2K1 K57 N mutation could be linked to resistance in preclinical models. We set out this study to detect MAP2K1 mutations and potentially targetable co-mutations using a molecular multiplex approach. Methods: Between 2012 and 2018, we routinely analyzed 14.512 NSCLC patients with two next-generation sequencing (NGS) panels. In a subset of patients, fluorescence in-situ hybridization was performed to detect rearrangements or amplifications. We assessed clinical parameters and co-occurring mutations and compared treatment outcomes of different forms of systemic therapy. Results: We identified 66 (0.5%) patients with MAP2K1 mutations. Both adenocarcinoma (n = 62) and squamous cell carcinoma (n = 4) histology. The presence of the mutations was linked to smoking, and transversions were more common than transitions. K57 N was the most frequent MAP2K1 mutation (n = 25). Additional mutations were found in 57 patients (86.4%). Mutations of TP53 were detected in 33 patients, followed by KEAP1 mutations in 28.1%. 24 patients (36.4%) had either MAP2K1-only or a co-occurring aberration considered targetable, including EGFR mutations, a BRAF V600E mutation and ROS1 rearrangements. Outcome analyses revealed a trend toward benefit from pemetrexed treatment. Conclusion: Our analysis shows that MAP2K1-mutated NSCLC patients might frequently present with potentially targetable aberrations. Their role in providing resistance in these subtypes and the possible therapeutic opportunities justify further analyses of this rare NSCLC subgroup

K-ras Mutation Subtypes in NSCLC and Associated Co-occuring Mutations in Other Oncogenic Pathways

Introduction: Although KRAS mutations in NSCLC have been considered mutually exclusive driver mutations for a long time, there is now growing evidence that KRAS-mutated NSCLC represents a genetically heterogeneous subgroup. We sought to determine genetic heterogeneity with respect to cancer-related co-mutations and their correlation with different KRAS mutation subtypes. Methods: Diagnostic samples from 4507 patients with NSCLC were analyzed by next-generation sequencing by using a panel of 14 genes and, in a subset of patients, fluorescence in situ hybridization. Next-generation sequencing with an extended panel of 14 additional genes was performed in 101 patients. Molecular data were correlated with clinical data. Whole-exome sequencing was performed in two patients. Results: We identified 1078 patients with KRAS mutations, of whom 53.5% had at least one additional mutation. Different KRAS mutation subtypes showed different patterns of co-occurring mutations. Besides mutations in tumor protein p53 gene (TP53) (39.4%), serine/threonine kinase 11 gene (STK11) (19.8%), kelch like ECH associated protein 1 gene (KEAP1) (12.9%), and ATM serine/threonine kinase gene (ATM) (11.9%), as well as MNNG HOS Transforming gene (MET) amplifications (15.4%) and erb-b2 receptor tyrosine kinase 2 gene (ERBB2) amplifications (13.8%, exclusively in G12C), we found rare co-occurrence of targetable mutations in EGFR (1.2%) and BRAF (1.2%). Whole-exome sequencing of two patients with co-occurring phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA) mutation revealed clonality of mutated KRAS in one patient and subclonality in the second, suggesting different evolutionary backgrounds. Conclusion: KRAS-mutated NSCLC represents a genetically heterogeneous subgroup with a high frequency of co-occurring mutations in cancer-associated pathways, partly associated with distinct KRAS mutation subtypes. This diversity might have implications for understanding the variability of treatment outcome in KRAS-mutated NSCLC and for future trial design. (C) 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc