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\title[Introduction to lymphatic research]{Introduction to lymphatic research\\
 Part 4}
\subtitle{Model organisms in lymphatic research}

\author[\copyright Michael Jeltsch]{Michael Jeltsch}

\institute{Molecular and Cancer Biology Research Program\\
 University of Helsinki}

\date{\\
October 21, 2011}

\logo{\includegraphics[width=1.2cm]{images/hylogo_transparent.jpg}}

\begin{document}

\section{Intro}

\subsection{Title}

\begin{frame}
  \titlepage
%  \hbox to2cm{\hss\insertlogo\hss}
  \note{Alternative title: Comparative anatomy and physiology of the lymphatic system}
\end{frame}

\subsection{Content}

\begin{frame}{Content of course lectures}
     \begin{enumerate}
        \item<2-> The cardiovascular system vs. the lymphatic system: Anatomy and Physiology
	\item<3-> Molecular make-up of the lymphatic system
	\item<4-> The lymphatic system in disease
        \item<5-> \textcolor{red}{Model organisms in lymphatic research}
        \item<6-> Fundamental techniques in lymphatic research
	\item<7-> Current questions in lymphatic research
     \end{enumerate}
%  \hbox to2cm{\hss\insertlogo\hss}
\end{frame}

\subsection{Target audience}

\begin{frame}{Who is the target audience of this presentation?}
     \begin{itemize}
	\item Students (2nd year and higher) of Biology or Molecular Biology that have a basic undestanding of physiology (course 522043)
        \item All students that have started to work in our laboratory
        \item Anybody interested who has a basic understanding of the lymphatic system
     \end{itemize}
\end{frame}

\subsection{URL}

\begin{frame}{URL}
\centering
This presentation is cc-licensed and can be downloaded both as PDF and as editable .tex file from:\\
\vspace{0.2in}
\href{http://jeltsch.org/presentations_teaching}{http://jeltsch.org/presentations\_teaching} 
  \begin{figure}
  \centering
  \includegraphics[height=6cm]{images/Screenshot2.png}%
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\end{frame}

\subsection{Teaching goals}

\begin{frame}{Teaching goals}
     \begin{itemize}
        \item<2-> Which animals do have a lymphatic system?
        \item<3-> How does the lymphatic system differ between animals?
        \item<4-> What animals are used in lymphatic research?
        \item<5-> What are the advantages and disadvantages of a specific animal model in respect to lymphatic research?
     \end{itemize}
	\note{At the end of this talk you should know, why we do lymphatic research, why we need experimental animals to do so, you should be able to tell which animals have and which don't have lymphatics and you should be able to tell the major differences between the lymphatics of different animal classes.}
\end{frame}

\subsection{Lecture outline}

\begin{frame}{Lecture outline}
  \scriptsize
  \tableofcontents
  \normalsize
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\subsection{Review of lectures 1-3}

\begin{frame}{Anatomy}
  \begin{figure}
  \centering
  \includegraphics[height=6cm]{images/human_cardiovascular_system.png}%
  \includegraphics[height=6cm]{images/human_lymphatic_system.png}%
  \end{figure}
\end{frame}

\begin{frame}{Physiology}
\centering
  \includegraphics[height=6cm]{images/Karpanen_Figure1mod_incl_attr.pdf}%
  \footnotesize
      \begin{itemize}
	\item Drainage system to return leaked fluid into the cardiovascular circulation
	\item Immune surveilance by the lymph nodes
	\item Dietary absorbtion of long-chain fatty acids and other lipophilic substances
      \end{itemize}
      \normalsize
\note{The main function of the lymphatic system in humans is considered to be the return of extravasated fluid from capillaries into the cardiovascular system. Whether this is its primary function in evolutionary terms is unclear. Two of the selective pressures that support its continuing existence nowadays in humans are the high pressure cardiovascular system and a high hydrostatic pressure due to organism height. Now: these selective pressures were most likely not in place when the lymphatic system evolved maybe around 550 Mio. years ago.}
\end{frame}

\setbeamertemplate{background}{\includegraphics[width=\paperwidth]{images/VEGFs_and_Receptors_low.pdf}}
\begin{frame}[plain]{Molecular make-up of the lymphatic system}
\end{frame}
\setbeamertemplate{background}{}

\begin{frame}{Lymphedema}
      \begin{figure}
        \includegraphics[height=6.5cm]{images/Karpanen_Figure7mod.pdf}%
        \\
        Adapted from K\"{a}rp\"{a}nen (2007)
      \end{figure}
\note{R-3 mutations are responsible for some forms of hereditary lymphedema. Some other forms are caused by mutations in the transcription factor FOXC2, but for the large majority of lymphedema syndromes the molecular cause is unknown. Hereditary lymphedemas are relatively rare, secondary lymphedema is more common, mainly in the form of post-surgery edema or edema associated with adbanced stage filariasis. In filariasis worms are literally living in the lymphatics (Wuchereria bancrofti, Brugia malayi, Brugia timori or Loa loa filaria). However the etiology of post-surgical lymphedema are somewhat mysterious as the acute lymphedema e.g. after breast cancer surgery resolves usually rather quickly and the cronic version can appear five to twenty years after the surgical insult.}
\end{frame}

\setbeamertemplate{background}{}

\begin{frame}
  \begin{figure}
  \centering
  \includegraphics[height=4cm]{images/filariasis.png}%
  \includegraphics[height=4cm]{images/bcrl.png}%
  \end{figure}
\end{frame}

\setbeamertemplate{background}{}

\begin{frame}{Lymphatic dissemination of tumour cells}
      \begin{figure}
        \includegraphics[height=6.5cm]{images/Karpanen_Figure6mod.pdf}%
        \\
        Adapted from K\"{a}rp\"{a}nen (2007)
      \end{figure}
\note{At the danger of boring some people, I will spend some time to review some basic questions about lymphatic research. Those basic thoughtsare obvious to the biologist, but because rarely discussed, might escape people without a background in comparative animal physiology, for example medically trained scientists. But because of the medical ramifications of lymphatic research, more and more medically trained scientists became involved in lymphatic research. I am not going to talk about the involvement of the lymphatic system in cancer.
- intratumoral lymphatics versus peritumoral lymphatics
- co-option versus true tumour-lymphangiogenesis
}
\end{frame}

\section{Model organisms in lymphatic research}

\begin{frame}{Model organisms and animal models}
  \begin{block}{Commonly used model organisms and their suitability for lymphatic research}
     \begin{itemize}
        \item Mouse/Rat
        \item Chicken/Quail
        \item Xenopus
        \item Zebrafish
        \item Non-vertebrates: Drosophila, C. elegans, ...
     \end{itemize}
   \end{block}
\note{In the beginning of last century the community of lymphatic researcher were fighting flame wars over the question. Much of the conflicting data might have resulted from the fact that they looked at a great variety of different species. Van der Putte gave in 1975 the probably most detailed account of the lymphatic development in man and mice and he himself admits that his human data is inconclusive. He was almost sure about the centrifugal mechanism for mice. Knowing that we should be careful to choose our own experimental models, especially if we aim to transfer the gained knowledge into medical applications. Therefore let me briefly talk about the commonly used model organisms and their suitability for lymphatic research. And I use the word "suitability" in a very anthropocentric meaning: can the findings be transferred to the species "Homo sapiens".}
 \end{frame}

\subsection{Non-vertebrates}

\begin{frame}{Fruit fly, jellyfish, nematodes}
          \begin{figure}
	  \includegraphics[height=1.8cm]{images/fruitfly.png}%
	\end{figure}
	\center
	  \emph{Drosophila melanogaster}
      \begin{columns}
        \column{.5\textwidth}
          \begin{figure}
            \includegraphics[height=1.8cm]{images/podocoryne_carnea.png}%
          \end{figure}
	\center
	\emph{Podocoryne carnea}
        \column{.5\textwidth}
          \begin{figure}
            \includegraphics[height=1.8cm]{images/nematode.jpg}%
          \end{figure}
		\center
          \emph{Caenorhabditis elegans}
      \end{columns}
\note{}
\end{frame}

\begin{frame}{VEGF-C and VEGF-D are ``ancient VEGFs''}
  \begin{figure}
  \centering
  \includegraphics[height=5.5cm]{images/VEGFdomains.png}%
  \end{figure}
\note{It is a fact that all VEGFs as well as all PDGFs are all multidomain proteins. Including VEGF-A. Some of the auxiliary domains clearly keep the central receptor binding domain in an inactive or partially active state: e.g. PDGFs and VEGF-C/D. Other auxiliary domains are responsible for the heparin binding properties and therefore extracellular distribution and bioavailability. When comparing VEGFs and PDGFs it becomes immediately clear that when it comes to their auxiliary domains VEGF-C and VEGF-D share more similarities with the PDGFs than with the VEGFs.}
\end{frame}

\begin{frame}{Fruit fly, jellyfish, nematodes}
      \begin{columns}
        \column{.3\textwidth}
          \begin{figure}
            \includegraphics[height=2cm]{images/fruitfly.png}%
          \end{figure}
        \column{.7\textwidth}
          \emph{Drosophila melanogaster}
          \\Embryonic hemocyte migration
      \end{columns}
      \pause
      \begin{columns}
        \column{.3\textwidth}
          \begin{figure}
            \includegraphics[height=2cm]{images/podocoryne_carnea.png}%
          \end{figure}
        \column{.7\textwidth}
          \emph{Podocoryne carnea}
          \\Tentacle and gastrovascular tube formation
      \end{columns}
      \pause
      \begin{columns}
        \column{.3\textwidth}
          \begin{figure}
            \includegraphics[height=2cm]{images/nematode.jpg}%
          \end{figure}
        \column{.7\textwidth}
          \emph{Caenorhabditis elegans}
          \\?
      \end{columns}
\note{many other functions have been described since the first description, most of them related to developmental functions which are on the surface unrelated to the vascular or immune system (border cell migration, thorax closure).
The E.elegans PVF-1 RNAi knock-out had no phenotype. Probably a bogus result.}
\end{frame}

\subsection{Do fish have lymphatics?}
\begin{frame}{Fish}
  \begin{figure}
  \centering
  \includegraphics[height=3cm]{images/fish.pdf}%
  \end{figure}
  \footnotesize
  \begin{block}{}
     \begin{itemize}
        \item{No lymphatics in fish (Steffensen \& Lomholt 1992: The Secondary Vascular System. Fish Physiology. Academic Press.)}
        \item{Kuchler et al. Curr Biol 16, 1244 and Yaniv et al. Nat Med 12, 711 describe the discovery of the fish lymphatics in 2006}
	\item{Hoyer 1938: Das Lymphgef\"{a}\ss system. In: Bronns Klassen und Ordnungen des Tierreichs 6/1: Echte Fische, Teil 2. Akademische.}
        \item{But not all fish are alike...}
     \end{itemize}
   \end{block}
 \end{frame}

\note{If you open a textbook of fish physiology the question is clear: They don't. This is a great example of the scientific discourse.}

\begin{frame}{Not all fish are alike}
  \begin{figure}
  \centering
  \includegraphics[height=7cm]{images/vertebrate_classification.pdf}%
  \end{figure}
\end{frame}

\note{While you can be pretty sure that all mammals have lungs and one heart. don't be so sure when it comes to fish! Some have lungs, some don't, some have one heart and others have two hearts. The morphological spectrum within the fish class is much wider than in the mammal class. And this is why many cladistic trees don't consider fish being a vertebrate class. Swimming in water and having a similar body shape is not sufficient to place vertebrates into the same class. Consequently, when Brent Weinstein and Stefan Schulte-Merker describe the lymphatic system of zebrafish, they do not generally claim that fish have lymphatics. They claim at the most that teleost fish have lymphatics and if you read their reports carefully they don't claim novelty. Because...}

\begin{frame}{The lymphatic system of teleost fish}
\begin{figure}
  \centering
  \includegraphics[height=4.5cm]{images/rainbow_trout_small.png}%
  \\ \tiny Hoyer \& Michalski (1922)
\end{figure}
\begin{figure}
  \centering
  \includegraphics[height=1.5cm]{images/zebrafish_weinstein.png}%
  \\ \tiny Weinstein (2006)
\end{figure}
\end{frame}


\begin{frame}{The lymphatic system of teleost fish}
  \begin{figure}
  \centering
  \includegraphics[height=6.5cm]{images/lymphsystem_in_fish_with_lymphatics.pdf}%
  \end{figure}
\note{This is the schematic view of the teleost lymphatic system as described by Hoyer in 1922. Then in 1981 Vogel and Claviez had a close look at the lymphatic system of salmon and came to the conclusion that there is no such thing as a lymphatic system in fish. How is that possible?}
\end{frame}

\begin{frame}{The secondary circulatory system of teleost fish}
  \begin{figure}
  \centering
  \includegraphics[height=6.5cm]{images/lymphsystem_in_fish_without_lymphatics.pdf}%
  \end{figure}
\note{Now Vogel and Claviez did use a bit more advanced techniques that were not available when Hoyer did his research in 1922: electron microscopy. Here is just the schematic bottom line of what they describe. What is the evidence for this model: The evidence is already 100 years old: Paul Meyer described that the flow direction in fish lymphatics is not the same in different parts of the larger lymphatic vessels he also described the existence of the arterioarterial anastomoses which he termed "fine vessels", but he didn't make the leap of giving up the idea of a lymphatic system. But with electron microscopy images like this...}
\end{frame}

\begin{frame}{Arterioarterial anastomoses}
  \begin{figure}
  \centering
  \includegraphics[height=6.5cm]{images/em_secondary_vasculature.jpg}%
  \\ Vogel (1985)
  \end{figure}
\note{...it is difficult to deny them. The openings of these anastomoses towards the primary arterial lumen brushed with cilia and together with the plasma skimming effect prevent red blood cells from entering the secondary arteries.}
\end{frame}

\begin{frame}{Many open questions}
  \begin{itemize}
    \item The piscine secondary circulatory system: a re-labeling of the lymphatic system?
    \item Arterioarterial anastomoses: A modification of the lymphatic system in some fish to increase flowrate or a casting artifact?
    \item Molecular correlates of anatomy?
  \end{itemize}
\note{Lymphatic flow in fish is probably very slow: no high-pressure vascular system, not much capillary filtration. Systolic blood pressure is usually between 5-50 mm mercury with few exceptions and there is no hydrostatic pressure at all; therefore the arterioarterial anastomoses could be a specialization of the lymphatic system to increase flow rate. What advantages would the higher flow rate have? Its main function in fish might not be the return of tissue fluid but its immunological function. At this stage nobody dares to say whether the secondary circulation is identical with the lymphatic system or whether some fish might have three vascular systems. My guess is no, what would be the molecular setup of such third system? Interestingly VEGFR-3 seems not to be restircted to lymphatics is zebrafish or the publications mixed up vessels...}
\end{frame}

\begin{frame}
      \begin{columns}
        \column{.5\textwidth}
	\begin{block}{Homo sapiens}
	    \centering
            \includegraphics<1-2>[height=4.5cm]{images/Figure7A.pdf}%
	\end{block}
        \column{.5\textwidth}
	\begin{block}{Danio rerio}
	    \centering
            \includegraphics<2>[height=4.5cm]{images/Figure7Aduplication.pdf}%
	\end{block}
      \end{columns}
\end{frame}

\subsection{Amphibians}
\begin{frame}{Amphibians}
  \begin{figure}
    \centering
    \includegraphics[height=5cm]{images/toad.pdf}
  \begin{itemize}
    \item Lymphatics function in fluid homeostasis
    \item Dermal lymph sacs in frogs and toads
    \item 50x more lymph formed compared to humans
    \item Many lymphatico-venous communications with lymph hearts
  \end{itemize}
  \end{figure}
\note{skin is very permeable for water, majority of lymph does not originate by capillary filtration, but enters through the skin, necessity of many and strong lymph hearts}
\end{frame}

\subsection{Reptiles}
\begin{frame}{Reptiles}
  \begin{figure}
  \centering
  \includegraphics[height=5cm]{images/snake.pdf}
  \begin{itemize}
    \item Reduction of lymph hearts \& lymphatico-venous anastomoses
    \item VEGF-F in snakes
  \end{itemize}
  \end{figure}
\note{This is more hypothetical as there are no widespread reptile animal models.}
\end{frame}

\subsection{Birds}
\begin{frame}{Birds}
 \begin{figure}
  \centering
  \includegraphics[height=4cm]{images/chick_cropped.jpg}\\
  \tiny
  \href{http://commons.wikimedia.org/wiki/Commons:GNU_Free_Documentation_License_1.2}{Photo by Fir0002/Flagstaffotos}
  \normalsize
  \begin{itemize}
    \item Lymph hearts disappear
    \item Lymph nodes appear in aquatic birds
    \item The chorio-allantoic membrane is rich in lymphatics
  \end{itemize}
  \end{figure}
\note{Lymph hearts still present as transient embryonic organs. But the presence of lymph nodes is of course a big advantage over the xenopus and zebrafish models. Unfortunately this advantage is not present in the mostly used species, the chicken. Ducks would be better models than chicken when it comes to the lymphatic system. Another big plus of the avian system is the accessibility of the embryo to mechanical manipulation including the CAM assay and the existence of the quail chimera system.}
\end{frame}

\hspace{-1.2cm}
\vspace{-1cm}
\begin{frame}[plain]{}
  \begin{figure}
  \includegraphics[width=\paperwidth]{images/amniote_egg.png}
  \end{figure}
\end{frame}

\begin{frame}{The CAM vasculature}
      \begin{columns}
        \column{.5\textwidth}
          \begin{figure}
            \includegraphics[height=6cm]{images/CAM.png}
          \end{figure}
        \column{.5\textwidth}
          \begin{figure}
            \includegraphics[height=6cm]{images/CAM_closeup.png}
          \end{figure}
      \end{columns}
\end{frame}

\subsection{Mammals}
\begin{frame}{Why mice are not men}
  \begin{figure}
  \centering
  \includegraphics[height=3.5cm]{images/mice.pdf}
  \begin{itemize}
    \item Difference in hydrostatic pressure has implications for lymphedema models
    \item Significant differences in the molecular make-up of the VEGFR-3 signalling system
  \end{itemize}
  \end{figure}
\end{frame}

\note{Human lymphedema conditions manifest mainly in the extremities where the hydrostatic pressure is highest. In some mice models of lymphedema this swelling of the extremities was reportedly replicated. Considering that the blood pressure of mice is roughly similar to humans and hydrostatic pressure is virtually non-existant, the mouse should have had swelling all over the place.}

\setbeamertemplate{background}{\includegraphics[width=\paperwidth]{images/VEGFD_and_VEGFR-3.pdf}}
\begin{frame}[plain]{}
\end{frame}
\setbeamertemplate{background}{}

\note{Apart from body size there a more serious reasons why it might not be possible to create good mouse models for human lymphedema conditions. A) The inability of mouse VEGF-D to interact with mouse VEGFR-2 B) The lack of the short splice variant of VEGFR-3, which is exclusive for primates. A retroviral insertion has created this splice variant. Out of the five tyrosine residues that have been shown to be phosphorylated upon stimulation with VEGF-C, three reside in the region that is absent in the short splice variant. This has functional consequences as shown in mouse fibroblasts which can be transformed by transfection with cDNA encoding the long splice variant, but not by the short variant.}

\section{Outro}

\subsection{Summary}

\begin{frame}{Summary}
  \begin{itemize}
  \item Non-vertebrate organisms have no lymphatic system, but they have homologous molecules
  \item Central questions about fish lymphatic anatomy and physiology are unanswered
  \item The most frequently used animal model for lymphatic disease is the mouse despite limitations due to size and molecular differences to humans
  \end{itemize}
\note{How fast?, abandonning human clinical trials}
\end{frame}

\subsection{People}

\setbeamertemplate{background canvas}{\includegraphics[width=\paperwidth]{images/helsinki_grad.jpg}}

\begin{frame}[plain]
  \begin{centering}
    \begin{block}{UNIVERSITY OF HELSINKI}
            \begin{itemize}
            \item Kari Alitalo
            \item and the rest of Molecular/Cancer Biology Laboratory
            \end{itemize}
    \end{block}
    \begin{block}{UNIVERSITY OF G\"{O}TTINGEN}
            \begin{itemize}
            \item J\"{o}rg Wilting
            \end{itemize}
    \end{block}
  \end{centering}
\end{frame}

\setbeamertemplate{background canvas}{}

\subsection{Software}

\begin{frame}
  \begin{centering}
This presentation was entirely created using open source software running under Linux
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        \column{.33\textwidth}
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          \end{centering}
        \column{.34\textwidth}
          \begin{centering}
          \begin{figure}
            \includegraphics[height=1.4cm]{images/LaTeX_logo.pdf}
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          \begin{figure}
            \includegraphics[height=1.1cm]{images/Ubuntu_Logo.pdf}
          \end{figure}
  \end{centering}
        \column{.33\textwidth}
          \begin{centering}
          \begin{figure}
            \includegraphics[height=3cm]{images/inkscape_logo.pdf}
          \end{figure}
  \end{centering}
      \end{columns}
  \end{centering}
\end{frame}

\end{document}