For a given gene, different mutations influence organismal phenotypes to varying degrees. However, the expressivity of these variants not only depends on the DNA lesion associated with the mutation, but also on factors including the genetic background and rearing environment. The degree to which these factors influence related alleles, genes, or pathways similarly, and whether similar developmental mechanisms underlie variation in the expressivity of a single allele across conditions and among alleles is poorly understood. Besides their fundamental biological significance, these questions have important implications for the interpretation of functional genetic analyses, for example, if these factors alter the ordering of allelic series or patterns of complementation. We examined the impact of genetic background and rearing environment for a series of mutations spanning the range of phenotypic effects for both the scalloped and vestigial genes, which influence wing development in Drosophila melanogaster. Genetic background and rearing environment influenced the phenotypic outcome of mutations, including intra-genic interactions, particularly for mutations of moderate expressivity. We examined whether cellular correlates (such as cell proliferation during development) of these phenotypic effects matched the observed phenotypic outcome. While cell proliferation decreased with mutations of increasingly severe effects, surprisingly it did not co-vary strongly with the degree of background dependence. We discuss these findings and propose a phenomenological model to aid in understanding the biology of genes, and how this influences our interpretation of allelic effects in genetic analysis.

The PI 3-kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3-phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34-Schwann cell knockout (Vps34SCKO) mice show severe hypomyelination in peripheral nerves. Vps34−/− Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34−/− Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34−/− Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic though endosomes. In Vps34SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to Nrg1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34SCKO mice.

Micronuclei are derived from missegregated chromosomes and frequently lose membrane integrity, leading to DNA damage, innate immune activation, and metastatic signaling. Here, we demonstrate that two characteristics of the trapped chromosome, length and gene density, are key contributors to micronuclei membrane stability and determine the timing of micronucleus rupture. We demonstrate that these results are not due to chromosome-specific differences in spindle position or initial protein recruitment during post-mitotic nuclear envelope assembly. Micronucleus size strongly correlates with lamin B1 levels and nuclear pore density in intact micronuclei, but, unexpectedly, lamin B1 levels do not completely predict nuclear lamina organization or membrane stability. Instead, small gene-dense micronuclei have decreased nuclear lamina gaps compared to large micronuclei, despite very low levels of lamin B1. Our data strongly suggest that nuclear envelope composition defects previously correlated with membrane rupture only partly explain membrane stability in micronuclei. We propose that an unknown factor linked to gene density has a separate function that inhibits the appearance of nuclear lamina gaps and delays membrane rupture until late in the cell cycle.

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42For a given gene, different mutations influence organismal phenotypes to varying 43 degrees. However, the expressivity of these variants not only depends on the DNA lesion 44 associated with the mutation, but also on factors including the genetic background and 45 rearing environment. The degree to which these factors influence related alleles, genes, or 46 pathways similarly, and whether similar developmental mechanisms underlie variation in the 47 expressivity of a single allele across conditions and variation across alleles is poorly 48 understood. Besides their fundamental biological significance, these questions have 49 important implications for the interpretation of functional genetic analyses, for example, if 50 these factors alter the ordering of allelic series or patterns of complementation. We 51 examined the impact of genetic background and rearing environment for a series of 52 mutations spanning the range of phenotypic effects for both the scalloped and vestigial 53 genes, which influence wing development in Drosophila melanogaster. Genetic background 54 and rearing environment influenced the phenotypic outcome of mutations, including intra-55 genic interactions, particularly for mutations of moderate expressivity. We examined whether 56 cellular correlates (such as cell proliferation during development) of these phenotypic effects 57 matched the observed phenotypic outcome. While cell proliferation decreased with 58 mutations of increasingly severe effects, surprisingly it did not co-vary strongly with the 59 degree of background dependence. We discuss these findings and propose a 60 phenomenological model to aid in understanding the biology of genes, and how this 61 influences our interpretation of allelic effects in genetic analysis. 62 63 Author Summary 64 Different mutations in a gene, or in genes with related functions, can have effects of 65 varying severity. Studying sets of mutations and analyzing how they interact are essential 66 components of a geneticist's toolkit. However, the effects caused by a mutation depend not 67 only on the mutation itself, but on additional genetic variation throughout an organism's 68 genome and on the environment that organism has experienced. Therefore, identifying how 69 the genomic and environmental context alter the expression of mutations is critical for 70 making reliable inferences about how genes function. Yet studies on this context 71 dependence have largely been limited to single mutations in single genes. We examined 72 how the genomic and environmental context influence the expression of multiple mutations 73 in two related genes affecting the fruit fly wing. Our results show that the genetic and 74 3 environmental context generally affect the expression of related mutations in similar ways. 75 However, the interactions between two different mutations in a single gene sometimes 76 depended strongly on context. In addition, cell proliferation in the developing wing and adult 77 wing size were not affected by the genetic and environmental context...

The form of Charcot–Marie-Tooth type 4B (CMT4B) disease caused by mutations in myotubularin-related 5 (MTMR5; also called SET Binding Factor 1; SBF1) shows a spectrum of axonal and demyelinating nerve phenotypes. This contrasts with the CMT4B subtypes caused by MTMR2 or MTMR13 (SBF2) mutations, which are characterized by myelin outfoldings and classic demyelination. Thus, it is unclear whether MTMR5 plays an analogous or distinct role from that of its homolog, MTMR13, in the peripheral nervous system (PNS). MTMR5 and MTMR13 are pseudophosphatases predicted to regulate endosomal trafficking by activating Rab GTPases and binding to the phosphoinositide 3-phosphatase MTMR2. In the mouse PNS, Mtmr2 was required to maintain wild type levels of Mtmr5 and Mtmr13, suggesting that these factors function in discrete protein complexes. Genetic elimination of both Mtmr5 and Mtmr13 in mice led to perinatal lethality, indicating that the two proteins have partially redundant functions during embryogenesis. Loss of Mtmr5 in mice did not cause CMT4B-like myelin outfoldings. However, adult Mtmr5−/− mouse nerves contained fewer myelinated axons than control nerves, likely as a result of axon radial sorting defects. Consistently, Mtmr5 levels were highest during axon radial sorting and fell sharply after postnatal day seven. Our findings suggest that Mtmr5 and Mtmr13 ensure proper axon radial sorting and Schwann cell myelination, respectively, perhaps through their direct interactions with Mtmr2. This study enhances our understanding of the non-redundant roles of the endosomal regulators MTMR5 and MTMR13 during normal peripheral nerve development and disease.

The form of Charcot-Marie-Tooth type 4B (CMT4B) disease caused by mutations in myotubularin-related 5 (MTMR5; also called SET Binding Factor 1; SBF1) shows a spectrum of axonal and demyelinating nerve phenotypes. This contrasts with the CMT4B subtypes caused by MTMR2 or MTMR13 (SBF2) mutations, which are characterized by myelin outfoldings and classic demyelination. Thus, it is unclear whether MTMR5 plays an analogous or distinct role from that of its homolog, MTMR13, in the peripheral nervous system (PNS). MTMR5 and MTMR13 are pseudophosphatases predicted to regulate endosomal trafficking by activating Rab GTPases and binding to the phosphoinositide 3-phosphatase MTMR2. In the mouse PNS, Mtmr2 was required to maintain wild type levels of Mtmr5 and Mtmr13, suggesting that these factors function in discrete protein complexes. Genetic elimination of both Mtmr5 and Mtmr13 in mice led to perinatal lethality, indicating that the two proteins have partially redundant functions during embryogenesis. Loss of Mtmr5 in mice did not cause CMT4B-like myelin outfoldings.However, adult Mtmr5 -/mouse nerves contained fewer myelinated axons than control nerves, likely as a result of axon radial sorting defects. Mtmr5 levels were highest during axon radial sorting, whereas Mtmr13 levels rose as myelin formed, and remained high through adulthood.Our findings suggest that Mtmr5 and Mtmr13 ensure proper axon radial sorting and Schwann cell myelination, respectively, perhaps through their direct interactions with Mtmr2. This study enhances our understanding of the non-redundant roles of the endosomal regulators MTMR5 and MTMR13 during normal peripheral nerve development and disease.During the development of the peripheral nervous system (PNS), Schwann cells associate with bundles of mixed diameter axons. In a process known as radial sorting, Schwann cells segregate large diameter axons (>1 μm) from smaller axons, which remain in bundles (1, 2). Once sorted into 1:1 associations with Schwann cells, large caliber axons are wrapped in a specialized, multilayer membrane sheath called myelin (3). Myelin facilitates rapid, saltatory nerve impulse conduction by clustering sodium channels at nodes of Ranvier, and by reducing axonal capacitance (4). Small caliber axons (<1 μm) remain unmyelinated and are supported by Schwann cells in Remak bundles (2). Both myelinating and Remak Schwann cells provide metabolic support to maintain axon integrity (3, 5-8). Both radial sorting and subsequent myelination require coordinated cytoskeleton reorganization, and receptor signaling from the adaxonal and abaxonal Schwann cell surfaces (1, 3). The precise control of downstream signaling is thought to be mediated in part by receptor trafficking through the endolysosomal pathway (9-11).Genomic studies have strongly suggested a link between abnormal endosomal trafficking and demyelinating Charcot-Marie-Tooth (CMT) disease (12). CMT is the most common inherited neurological disorder, affecting 1 in 2500 people (13). Axonal and demyelinating forms of CMT are d...

Identifying the genetic architecture of complex traits is of interest to many geneticists, including those interested in human disease, plant and animal breeding and evolutionary genetics. Despite advances in sequencing technologies and GWAS statistical methods improving our ability to identify variants with smaller effect sizes, many of these identified polymorphisms fail to be replicated in subsequent studies. In addition to sampling variation, this reflects the complexities introduced by factors including environmental variation, genetic background and differences in allele frequencies among populations. Using Drosophila melanogaster wing shape, we ask if we can replicate allelic effects of polymorphisms first identified in a GWAS (Pitchers et al. 2019) in three genes: dachsous (ds), extra-macrochaete (emc) and neuralized (neur), using artificial selection in the lab and bulk segregant mapping in natural populations. We demonstrate that shape changes associated with these genes is aligned with major axes of phenotypic and genetic variation in natural populations. Following 7 generations of artificial selection along ds and emc shape change vectors, we observe genetic differentiation of variants in ds and in genomic regions with other genes in the hippo signaling pathway, indicating available genetic diversity of a population summarized in G influences alleles captured by selection. Despite the success with artificial selection, bulk segregant analysis using natural populations did not detect these same variants, likely due to the contribution of environmental variation, low minor allele frequencies coupled with small effect sizes of the contributing variants.