Endoderm Patterning in Zebrafish: Pancreas Development: A Dissertation

The pancreas is located below the liver and adjacent to the small intestine where it connects to the duodenum. It consists of exocrine and endocrine components. The exocrine portion makes enzymes which are deposited in the duodenum to digest fats, proteins, and carbohydrates. Exocrine tissue also makes bicarbonates that neutralize stomach acids. The endocrine portion produces hormones such as insulin and glucagon which are released into the blood stream. These hormones regulate glucose transport into the body\u27s cells and are crucial for energy production. The pancreas is associated with diseases such as cancer, diabetes, Annular pancreas and Nesidioblastosis. Annular pancreas and Nesidioblastosis are congenital malformations associated with excess endocrine tissue of the pancreas and its structures. Understanding the development of the pancreas might lead to insight of these diseases. The pancreas arises from the endoderm. In zebrafish, Nodal signaling activates mix-type and gata genes that then function together to regulate sox32 expression which is necessary and sufficient to induce endoderm formation. Interestingly, sox32 is exclusive to zebrafish and works synergistically with pou5f1 to regulate its own expression and turn on sox17 expression. sox17is evolutionarily conserved from zebrafish to mouse and is necessary for endoderm formation. Signals from within the endoderm and the surrounding mesoderm specify regions in the endoderm to develop into the pancreas and other endodermal organs. Sonic hedgehog (shh) expression in the foregut establishes the anterior boundary of the pancreas primordium while cdx4 expression establishes the posterior boundary, but what regulates these factors is unclear. We determined that two Three Amino Acid Loop Extension (TALE) homeodomain transcription cofactors, Meis3 and Pbx4, regulate shh expression in the anterior endoderm. Disrupting either meis3 or pbx4 reduces shh expression in the anterior endoderm. As a result, anterior ectopic insulin expression occurs outside the normal pancreatic domain. Therefore, we discovered upstream regulatory factors of shhexpression in the anterior endoderm, which is necessary for patterning the endoderm and pancreas primordium. We performed an ENU (N-ethyl-N-nitrosurea) haploid screen to look for endocrine pancreas mutants and to find other factors involved in pancreas development and patterning. From the screen, we characterized two mutants. We identified an aldh1a2 mutant, aldh1a2um22, which blocks the production of Retinoic Acid (RA) from vitamin A. While RA is known to be necessary for differentiation of the pancreas and liver, we also found it to be necessary for intestine differentiation. Two other aldh family genes exist in the zebrafish genome, but our data suggests that aldh1a2is the only Aldh that functions in endoderm differentiation and it is maternally deposited. From the screen, we discovered a second mutant, 835.4, that spontaneously arose within the background. pou5f1 expression is normal in mutant embryos, but sox32 expression is reduced and sox17 expression is lost. Downstream endoderm genes of sox17 are also lost and as a result no endodermal organs develop. Rescue experiments indicate that the mutation is located between sox32 and sox17 in the endoderm pathway. We currently have not been successful at mapping this mutation and therefore are unable to rule out the possibility that it lies in the sox17 gene. However, our data suggest that the mutation occurs in a new gene that is necessary for sox17 expression, potentially working with sox32 and/or pou5f1

Maternal and Zygotic aldh1a2 Activity Is Required for Pancreas Development in Zebrafish

We have isolated and characterized a novel zebrafish pancreas mutant. Mutant embryos lack expression of isl1 and sst in the endocrine pancreas, but retain isl1 expression in the CNS. Non-endocrine endodermal gene expression is less affected in the mutant, with varying degrees of residual expression observed for pdx1, carbA, hhex, prox1, sid4, transferrin and ifabp. In addition, mutant embryos display a swollen pericardium and lack fin buds. Genetic mapping revealed a mutation resulting in a glycine to arginine change in the catalytic domain of the aldh1a2 gene, which is required for the production of retinoic acid from vitamin A. Comparison of our mutant (aldh1a2um22) to neckless (aldh1a2i26), a previously identified aldh1a2 mutant, revealed similarities in residual endodermal gene expression. In contrast, treatment with DEAB (diethylaminobenzaldehyde), a competitive reversible inhibitor of Aldh enzymes, produces a more severe phenotype with complete loss of endodermal gene expression, indicating that a source of Aldh activity persists in both mutants. We find that mRNA from the aldh1a2um22 mutant allele is inactive, indicating that it represents a null allele. Instead, the residual Aldh activity is likely due to maternal aldh1a2, since we find that translation-blocking, but not splice-blocking, aldh1a2 morpholinos produce a phenotype similar to DEAB treatment. We conclude that Aldh1a2 is the primary Aldh acting during pancreas development and that maternal Aldh1a2 activity persists in aldh1a2um22 and aldh1a2i26 mutant embryos

Potential Interactions Between Diatoms and Bacteria are Shaped by Trace Element Gradients in the Southern Ocean

The growth of diatoms in the Southern Ocean, especially the region surrounding the West Antarctic Peninsula, is frequently constrained by low dissolved iron and other trace metal concentrations. This challenge may be overcome by mutualisms between diatoms and co-occurring associated bacteria, in which diatoms produce organic carbon as a substrate for bacterial growth, and bacteria produce siderophores, metal-binding ligands that can supply diatoms with metals upon uptake as well as other useful secondary compounds for diatom growth like vitamins. To examine the relationships between diatoms and bacteria in the plankton (diatom) size class (\u3e 3 mu m), we sampled both bacterial and diatom community composition with accompanying environmental metadata across a naturally occurring concentration gradient of macronutrients, trace metals and siderophores at 21 stations near the West Antarctic Peninsula (WAP). Offshore Drake Passage stations had low dissolved iron (0.33 ± 0.15 nM), while the stations closer to the continental margin had higher dissolved iron (5.05 ± 1.83 nM). A similar geographic pattern was observed for macronutrients and most other trace metals measured, but there was not a clear inshore-offshore gradient in siderophore concentrations. The diatom and bacteria assemblages, determined using 18S and 16S rDNA sequencing respectively, were similar by location sampled, and variance in both assemblages was driven in part by concentrations of soluble reactive phosphorous, dissolved manganese, and dissolved copper, which were all higher near the continent. Some of the most common diatom sequence types observed were Thalassiosira and Fragilariopsis, and bacteria in the plankton size fraction were most commonly Bacteroidetes and Gammaproteobacteria. Network analysis showed positive associations between diatoms and bacteria, indicating possible in situ mutualisms through strategies such as siderophore and vitamin biosynthesis and exchange. This work furthers the understanding of how naturally occurring gradients of metals and nutrients influence diatom-bacteria interactions. Our data suggest that distinct groups of diatoms and associated bacteria are interacting under different trace metal regimes in the WAP, and that diatoms with different bacterial partners may have different modes of biologically supplied trace metals

Interactions of Bioactive Trace Metals in Shipboard Southern Ocean Incubation Experiments

In the Southern Ocean, it is well‐known that iron (Fe) limits phytoplankton growth. Yet, other trace metals can also affect phytoplankton physiology. This study investigated feedbacks between phytoplankton growth and dissolved Fe, manganese (Mn), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), and cadmium (Cd) concentrations in Southern Ocean shipboard incubations. Three experiments were conducted in September–October 2016 near the West Antarctic Peninsula: Incubations 1 and 3 offshore in the Antarctic Circumpolar Current, and Incubation 2 inshore in Bransfield Strait. Additions of Fe and/or vitamin B12 to inshore and offshore waters were employed and allowed assessment of metal (M) uptake relative to soluble reactive phosphorus (P) across a wide range of initial conditions. Offshore, treatments of \u3e1 nmol L−1 added Fe were Fe‐replete, whereas inshore waters were already Fe‐replete. Results suggest Mn was a secondary limiting nutrient inshore and offshore. No Fe‐vitamin B12 colimitation was observed. Overall, M:P uptake in the incubations was closely related to initial dissolved M:P for Fe, Mn, Co, Ni, and Cd, and for Cu inshore. Final concentrations of Fe and Zn were similar across light treatments of the experiments despite very different phytoplankton responses, and we observed evidence for Co/Cd/Zn substitution and for recycling of biogenic metals as inventories plateaued. In dark bottles, the absence of Mn oxidation may have allowed more efficient recycling of Fe and other trace metals. Our results provide insight into factors governing trace metal uptake, with implications for phytoplankton community composition locally and preformed micronutrient bioavailability in Southern Ocean water masses

Accumulation of the Vitamin D Precursor Cholecalciferol Antagonizes Hedgehog Signaling to Impair Hemogenic Endothelium Formation

Summary Hematopoietic stem and progenitor cells (HSPCs) are born from hemogenic endothelium in the dorsal aorta. Specification of this hematopoietic niche is regulated by a signaling axis using Hedgehog (Hh) and Notch, which culminates in expression of Runx1 in the ventral wall of the artery. Here, we demonstrate that the vitamin D precursor cholecalciferol (D3) modulates HSPC production by impairing hemogenic vascular niche formation. Accumulation of D3 through exogenous treatment or inhibition of Cyp2r1, the enzyme required for D3 25-hydroxylation, results in Hh pathway antagonism marked by loss of Gli-reporter activation, defects in vascular niche identity, and reduced HSPCs. Mechanistic studies indicated the effect was specific to D3, and not active 1,25-dihydroxy vitamin D3, acting on the extracellular sterol-binding domain of Smoothened. These findings highlight a direct impact of inefficient vitamin D synthesis on cell fate commitment and maturation in Hh-regulated tissues, which may have implications beyond hemogenic endothelium specification

Filipino physical therapists’ practice and perspectives on non-treatment physical activity for older adults

Introduction: Studies have already investigated the practice of physical therapists (PTs) in promoting non-treatment physical activity (NTPA). However, these were done in the context of mostly western settings or were not specific to older adult practice. It is still unclear if a similar level of practice and perspectives on NTPA promotion exists in a setting where physical therapy is more associated with rehabilitation than health promotion, such as in the Philippines. Therefore, this study aimed to describe Filipino PTs\u27 knowledge of WHO physical activity (PA) guidelines and their use of behavioral change techniques (BCTs) and theories when promoting PA in older adults. It also explored factors that potentially influenced their engagement in PA promotion. Methods: Filipino PTs who were handling or interested in handling older adult clients from March to April 2020 answered an online/printed survey. Results: More than half of 72 respondents were unaware (59.72%) and most were unable to recall the WHO guidelines (98.61%) correctly. Respondents used a limited range of BCTs when promoting PA. While some (66.66%) were aware of at least one behavior change theory, a number were unfamiliar with all (33.33%) and only a small proportion reported regular use in practice. A number of factors and respondent characteristics were found to have a statistically significant positive relationship with PA promotion. Discussion: Study results can serve as preliminary basis for programs that improve promotion of NTPA on older-adult clients by Filipino PTs, specifically regarding knowledge on WHO PA guidelines and their use of a wide range of BCTs and theories. This can potentially place them at the forefront of addressing this health concern in the aging population

Mutation mapping and identification by whole-genome sequencing

Genetic mapping of mutations in model systems has facilitated the identification of genes contributing to fundamental biological processes including human diseases. However, this approach has historically required the prior characterization of informative markers. Here we report a fast and cost-effective method for genetic mapping using next-generation sequencing that combines single nucleotide polymorphism discovery, mutation localization, and potential identification of causal sequence variants. In contrast to prior approaches, we have developed a hidden Markov model to narrowly define the mutation area by inferring recombination breakpoints of chromosomes in the mutant pool. In addition, we created an interactive online software resource to facilitate automated analysis of sequencing data and demonstrate its utility in the zebrafish and mouse models. Our novel methodology and online tools will make next-generation sequencing an easily applicable resource for mutation mapping in all model systems.Harvard Stem Cell Institute (Junior Faculty Grant)National Institutes of Health (U.S.) (Grant 1R01DK090311)National Institutes of Health (U.S.) (Grant 5R01MH084676

The influence of climatic change, fire and species invasion on a Tasmanian temperate rainforest system over the past 18,000 years

We aim to understand how did cool temperate rainforest respond to changes in climate and fire activity over the past 18 kcal yrs, interrogating the role that flammable plant species (such as Eucalyptus) have in the long-term dynamics of rainforest vegetation. We used high-resolution pollen and charcoal analysis, radiometric dating (lead and carbon), modern pollen-vegetation relationships, detrended correspondence analysis, rarefaction (palynological richness), rate of change and granger causality to understand the patterns and drivers of change in cool temperate rainforest from the sediments of Lake Vera, southwest Tasmania through time. We record clear changes in key rainforest taxa in response to climatic change throughout the record. The spread of rainforest through the lake catchment in the early and mid- Holocene effectively negated disturbance from fire despite a region-wide peak in fire activity. An anomalously dry period in the late-Holocene resulted in a local fire that facilitated the establishment of Eucalyptus within the local catchment. Granger causality tests reveal a significant lead of Eucalyptus over fire activity in the Holocene, indicating that fires were enhanced by this pyrogenic taxon following establishment

Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse betaglobin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein Cerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models

Next-generation plasmids for transgenesis in zebrafish and beyond

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models