Researchers have identified several methods for treating acute myocardial infarction (AMI) patients affected by ischemia and reperfusion injury. Some of these therapies include thrombolysis, balloon angioplasty, and coronary arterial bypass graft (CAGB). This lab has previously demonstrated that transplantation of mitochondria into the ischemic zone of a rabbit heart during reperfusion significantly improved recovery as compared to current techniques. In order for this therapy to be translated into the clinic a rapid isolation method for producing highly pure and functional mitochondria will be required. Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 minutes to complete. Herein, a method for rapid isolation of mitochondria from mammalian tissue biopsies is described. In this protocol, manual homogenization is replaced with the tissue dissociator's standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters which eliminates repetitive centrifugation steps. Mitochondrial isolation time is less than 30 minutes compared to 60-100 minutes using alternative methods. This isolation protocol yields approximately 2 x 10^10 viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.

Panarthropoda is a monophyletic group comprised of arthropods and lobopods, molting animals with a segmented body, paired appendages, dorsal brain, and ventral nerve cords. Evolutionary Developmental Biology (EvoDevo) is an interdisciplinary field that seeks to understand how changes in development form the basis for variations in morphology and phenotypic evolution, including the genetic network underlying these processes. To study the evolution of panarthropods from such an EvoDevo perspective, one typically uses standard molecular techniques. A first step here is to investigate the expression of a gene of interest in order to find out where and when it is transcribed during development. A hallmark of EvoDevo studies is its comparative character, often with respect to model organisms such as the fruit fly Drosophila melanogaster. Recently developed single-cell RNA sequencing technologies allow the profiling of a plethora of gene expression on the level of individual cells, and thus provide a much more detailed insight into gene expression. In Paper I, I applied standard molecular techniques used in EvoDevo research such as PCR, gene cloning, probe synthesis and whole mount in situ hybridization, to investigate the embryonic expression patterns of the tiptop/teashirt (tio/tsh) and spalt (sal) genes in a range of arthropods representing all main groups of this phylum, and an onychophoran. In the arthropod model Drosophila, these genes act as trunk-specifiers, and the objective of my work was to find out if this is conserved in Arthropoda or even Panarthropoda as a whole. I provide comprehensive data on arthropod tio/tsh and sal expression, including the first data from an onychophoran. The results support the idea that tio/tsh genes are involved in the development of ‘trunk’ segments by regulating limb development. In addition, my data suggest that the function of Sal is unlikely to be conserved in trunk vs head development. Early expression of sal, however, is in line with a potential homeotic function of this gene, at least in Arthropoda. In Paper II, I provide an embryonic tissue dissociation protocol for embryos of the common house spider Parasteatoda tepidariorum that I developed and that I successfully applied for single-cell RNA sequencing. In addition, I report on the progress of this experiment, and provide and discuss preliminary results.

RNA ist ein zentrales Molekül in der Zelle und essentiell für ihre Lebensfunktionen. Die durchschnittliche Halbwertszeit von RNA-Molekülen limitiert jedoch die zeitliche Auflösung herkömmlicher RNA-Sequenzierung, da geringe Änderungen im Transkriptom kaum zu erkennen sind, bis eine gewisse Anzahl an Molekülen akkumuliert. Durch metabolische Markierung von RNA (SLAMseq) kann die Auflösung deutlich erhöht werden. Hierfür werden der Probe markierte Nucleotide (4sU/4sUTP) zugesetzt, die dann zufällig in neu transkribierte RNA inkorporiert werden und eine Unterscheidung zwischen ‚neuer‘ und ‚alter‘ RNA erlauben. In dieser Arbeit werden eine der ersten Einzelzell-SLAMseq-Methoden, die dazugehörige Datenanalyse-Software sowie drei Anwendungen der entwickelten Methoden vorgestellt. Die erste Anwendung verwendet Einzelzell-SLAMseq, um zwischen maternaler (alter) und zygotischer (neuer) RNA in sich entwickelnden Zebrafischembryos bis zur Gastrulation zu unterscheiden. Im Rahmen des Projekts entstand der erste Einzelzell-SLAMseq-Datensatz in einem vollständigen Wirbeltier, der es außerdem erlaubt, im Vorfeld identifizierten lokalisierten maternalen Transkripten zeitlich zu folgen. Diese – vorher uncharakterisierten –Transkripte wurden während der Gastrulation in den Keimzellen angereichert gefunden, was Rückschlüsse auf ihre mögliche Funktion erlaubt. Die zweite Anwendung konzentriert sich auf die neu transkribierte RNA und verwendet (Einzelzell-)SLAMseq, um Transkripte, die in Reaktion auf Stress während der Probenaufbereitung hergestellt wurden, zu identifizieren und rechnerisch zu entfernen. Die Vorteile der Methode werden in mehreren Systemen und Geweben (Mausherz, Zebrafischlarve, Maus-Microglia) demonstriert. In der dritten Anwendung wird eine Machbarkeitsstudie für in vivo SLAMseq zur Identifikation der initialen Immunantwort nach Makrophagenstimulation präsentiert, die auf einen deutlichen Gewinn an zeitlicher Auflösung durch SLAMseq hindeutet. / RNA is a central molecule in the cell and essential to its life functions. With the average RNA half life being multiple hours, regular RNA sequencing has an intrinsic limit on temporal resolution, where small changes in the transcriptome are not picked up until a certain amount of transcripts has build up. This resolution can be greatly improved using RNA metabolic labelling (SLAMseq), where labelled nucleotides (4sU/4sUTP) are added to the samples. These nucleotides are randomly incorporated into nascent transcripts and allow distinction between RNA produced before and after introduction of the labelling agent. This thesis presents one of the first high throughput single cell SLAMseq protocols, an accompanying computational pipeline for data analysis as well as three applications for the developed methods. The first application uses single cell SLAMseq to distinguish between maternal (unlabelled) and zygotic (labelled) transcripts in early zebrafish development (up to mid-gastrulation). This project generated the first single cell SLAMseq dataset in a whole vertebrate. Additionally the data allows to follow a previously discovered set of vegetally localised maternal transcripts in time and determine that these specific transcripts are mainly enriched in the primordial germ cells at gastrulation, therefore ascribing a potential function to a set of so far uncharacterised genes. The second application focuses on newly transcribed RNA and uses (single cell) SLAMseq as a technique to identify and remove transcripts generated in response to sample preparation stress. The method’s benefits are demonstrated in multiple systems and tissues, among them mouse cardiomyocytes, zebrafish larvae and mouse microglia. Finally as the third application an in vivo proof of concept study of SLAMseq to identify first response genes in macrophage stimulation is presented, where the introduction of 4sU shows clear advantages in temporal resolution compared to unlabelled data.