Removal of pomt1 in zebrafish leads to loss of α-dystroglycan glycosylation and dystroglycanopathy phenotypes.

Biallelic mutations in Protein O-mannosyltransferase 1 (POMT1) are among the most common causes of a severe group of congenital muscular dystrophies (CMDs) known as dystroglycanopathies. POMT1 is a glycosyltransferase responsible for the attachment of a functional glycan mediating interactions between the transmembrane glycoprotein dystroglycan and its binding partners in the extracellular matrix (ECM). Disruptions in these cell-ECM interactions lead to multiple developmental defects causing brain and eye malformations in addition to CMD. Removing Pomt1 in the mouse leads to early embryonic death due to the essential role of dystroglycan during placental formation in rodents. Here, we characterized and validated a model of pomt1 loss of function in the zebrafish showing that developmental defects found in individuals affected by dystroglycanopathies can be recapitulated in the fish. We also discovered that pomt1 mRNA provided by the mother in the oocyte supports dystroglycan glycosylation during the first few weeks of development. Muscle disease, retinal synapse formation deficits, and axon guidance defects can only be uncovered during the first week post fertilization by generating knock-out embryos from knock-out mothers. Conversely, maternal pomt1 from heterozygous mothers was sufficient to sustain muscle, eye, and brain development only leading to loss of photoreceptor synapses at 30 days post fertilization. Our findings show that it is important to define the contribution of maternal mRNA while developing zebrafish models of dystroglycanopathies and that offspring generated from heterozygous and knock-out mothers can be used to differentiate the role of dystroglycan glycosylation in tissue formation and maintenance.

Duplicated zebrafish (Danio rerio) inositol phosphatases inpp5ka and inpp5kb diverged in expression pattern and function.

One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland and retina indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P2. Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish.

Digging behavior discrimination test to probe burrowing and exploratory digging in male and female mice.

Digging behavior is often used to test motor function and repetitive behaviors in mice. Different digging paradigms have been developed for behaviors related to anxiety and compulsion in mouse lines generated to recapitulate genetic mutations leading to psychiatric and neurological disorders. However, the interpretation of these tests has been confounded by the difficulty of determining the motivation behind digging in mice. Digging is a naturalistic mouse behavior that can be focused toward different goals, that is foraging for food, burrowing for shelter, burying objects, or even for recreation as has been shown for dogs, ferrets, and human children. However, the interpretation of results from current testing protocols assumes the motivation behind the behavior often concluding that increased digging is a repetitive or compulsive behavior. We asked whether providing a choice between different types of digging activities would increase sensitivity to assess digging motivation. Here, we present a test to distinguish between burrowing and exploratory digging in mice. We found that mice prefer burrowing when the option is available. When food restriction was used to promote a switch from burrowing to exploration, males readily switched from burrowing to digging outside, while females did not. In addition, when we tested a model of intellectual disability and autism spectrum disorder that had shown inconsistent results in the marble burying test, the Cc2d1a conditional knockout mouse, we found greatly reduced burrowing only in males. Our findings indicate that digging is a nuanced motivated behavior and suggest that male and female rodents may perform it differently.

Anticancer Activity and In Vitro to In Vivo Mechanistic Recapitulation of Novel Ruthenium-Based Metallodrugs in the Zebrafish Model.

Ruthenium is popular as a metal core for chemotherapeutics, due to versatile molecular coordination. Because new metallodrugs are synthesized at high rates, our studies included assays in zebrafish to expedite the initial evaluation as anticancer agents. Here we evaluated novel metallodrugs (PMC79 and LCR134), and cisplatin, a widely used platinum-based chemotherapeutic. We hypothesized that this model could characterize anticancer properties and recapitulate previous in vitro results in vivo. Our findings suggest anticancer properties of PMC79 and LCR134 were similar with less toxicity than cisplatin. Exposures from 24 to 72 h at or below the LOAELs of PMC79 and LCR134 (3.9 µM and 13.5 µm, respectively), impaired blood vessel development and tailfin regeneration. Blood vessel examination through live imaging of larvae revealed distinct regional antiangiogenic impacts. The significant decrease in gene expression of the VEGF-HIF pathway and beta-actin could explain the morphological effects observed in the whole organism following exposure. Tailfin amputation in larvae exposed to PMC79 or LCR134 inhibited tissue regrowth and cell division, but did not impact normal cell proliferation unlike cisplatin. This suggests Ru drugs may be more selective in targeting cancerous cells than cisplatin. Additionally, in vitro mechanisms were confirmed. PMC79 disrupted cytoskeleton formation in larvae and P-glycoprotein transporters in vivo was inhibited at low doses which could limit off-target effects of chemotherapeutics. Our results demonstrate the value for using the zebrafish in metallodrug research to evaluate mechanisms and off-target effects. In light of the findings reported in this article, future investigation of PMC79 and LCR134 are warranted in higher vertebrate models.