Histology and Embryology Lecturers: Aleš Hampl, D.V.M., Ph.D., Assoc. Prof., Head of the Dept. Petr Vaňhara, RNDr., Ph.D., Assist. Prof. Brno, 2019 Lecture 1 Introduction • The object and significance of histology. • Relevance of histology to other biomedical disciplines. • History, current state, and future of histology. • Methodologies to study a structure of cells and tissues. Cytology • The cell - definition, characteristics, compartmentalization. • Cell nucleus - ultrastructure and function, chromosomes, nucleolus. • Endoplasmic reticulum • Golgi apparatus • Centrosome Histology Microscopic and submicroscopic structure of the body (cells, extracellular matrix, fluid substances) Microscopic anatomy Composition and structure of organ systems & individual organs Which tissue types and how organized? Which special cell types? Which special structures? (e.g. tubules) How does it all work? Cytology General aspects of the structures composing the cells and their functioning General histology What are the main types of tissues? What are their functions? What cell types these tissues are made of? All this mirrors hierarchical organisation of living organisms Histology is no longer a static discipline dealing with just the structure !!! Histology Cell biology Physiology Pathology Biochemistry Molecular biology Gross anatomy Embryology Have the histology in action & in motion Learn thinking „histologically“ Studying histology was first made mandatory for medical students in 1893 by John’s Hopkins Medical School ! Most histologists are Germans primarily because they made great microscopes. Eponymously theirs….. Marcello Malpighi 1628 - 1694 Italian physician Founder of microscopic anatomy and the first histologist • Discovered taste buds • Discovered capillaries • Maybe first to see red blood cells undermicroscope Malpighian layer of the skin Term for basale and spinosum layers of epithelium Malpighian corpuscles in the kidney & spleen • Invented the solar microscope • Also invented a reflector to view opaque specimens easily Main histological contribution was discovering the glands of the small intestine and colon-the crypts of Lieberkuhn Johan Nathanael Lieberkuhn 1711 - 1756 German anatomist and physician Jan Evangelista Purkyně 1787 - 1869 Bohemian physiologist • Pioneer in histological techniques First to use something like a microtome • Introduced the term plasma • Found Purkinje fibers of the heart • Found Purkinje cells of the cerebellar cortex Schwann + Schleiden – 1839 – cell theory “Once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. In the adult centers, the nerve paths are something fixed, ended, and immutable. Everything may die, nothing may be regenerated. It is for the science of the future to change, if possible, this harsh decree.” Santiago Ramón Y Cajal 1852 - 1934 Spanish physician and anatomist He established the neuron as the primary structural and functional unit of the nervous system. Nobel Prize in 1906 Making unexpected discoveries The existence of multipotent self-renewing progenitors residing in the postantal and adult nervous system • Subventricular zone of the lateral ventricle • Subgranular zone of the dentate gyrus of the hippocampus DEFINITELY IN: • Cortex ? • Amygdala ? POSSIBLY IN: (since early 1990s) Our view on the organization of the central nervous system has been dramatically changed !!! Many questions on NSC remain to be answered Combination of developmental biology, histology, cell biology, and molecular biology approaches is required. • exact position in the tissue ? • proliferative activity and migration ? • developmental potential ? • involvement in disease developement ? • others Gleason et al., Neuroscience, 2008. Any practical use of such discovery ? (1) Helping brain regenerate after the stroke Promote endogenous neurogenesis and improve histological structure and function Jin et al., Brain Research, 2011. • experiment on rats • MCAO – middle cerebral artery occlusion to induce infarction • human neural precursors tranplanted into the site of infarction • histologically evaluated Options: • drugs • growth factors • cell implantation Jin et al., Brain Research, 2011. Any practical use of such discovery ? (2) 3 months 24 months Transplanted human cells in the site of infarction HuN – human nuclei DCX – doublecortin (marker of early neuronal lineage) HuNHuN Neurogenesis in SVZ of rat brain becomes stimulated DCX DCX Non-transplated Cells transplanted Neocytogenesis occurs before day 60 after transplantation Pulse-labelling with BrdU at day 60 after transplantation Tissue & Cell transplantation No permanent cure – Transplantation ? - Immunosuppression DiabetesDysregulated glucose metabolism Damage to b cells of pancreatic islets of Langerhans Surviving islets Rejected islets IHC Insulin - brown Glucagon - blue Haematoxilin & Eosin Badell et al., J. Clin. Invest., 2010 Lymphocyte function–associated antigen 1 (LFA-1) Short-term treatment with the LFA-1–specific Ab Tissue and organ engineering is not novel in its principle but we develop new approaches based on our understanding of tissue composition Egyptian mummy Tissue engineering 1 The first report of the construction of 3D vascularized human cardiac tissue that may have unique applications for studies of cardiac development, function, and tissue replacement therapy Caspi et al., Tissue Engineering of Vascularized Cardiac Muscle From Human Embryonic Stem Cells, Circulation Research, 2007 (group of Shulamit Levenberg, Israel) (stay with the infarction) Histological and functional analysis Endothelial cells Human stem (naive) cells differentiated in vitro into cardiac muscle lineage 3-dimensional polymeric scaffold Fibroblasts Combined together Tissue engineering 2 Cardiomyocytes Cardiomyocytes + Endothelia Cardiomyocytes + Endothelia + Fibroblasts Troponin I Sarcomeric actinin CD 31 Markers of cardiac muscle Markers of cardiac endothelia Caspi et al., Circulation Research, 2007 Tissue engineering 3 Caspi et al., Circulation Research, 2007 Myofibrils Z bands T tubules Sarcoplasmic reticulum Gap junctions Conexin – Gap junctions Troponin - cardimyocytes Ultrastructural characteristics of the engineered cardiac tissue Baseline 1-Heptanol Engineered cardiac tissue propagates synchronous surges of Ca2+ Laser scanning cofocal microscopy (Gap junctions uncoupler) Methodologies to study cells and tissues 1 Making it observable Stabilization of the structure Fixation Making the objects smaller – transmissible for the light Embedding + Sectioning Making the structures well visible „Staining“ Enlargement Utility of Microscopes Light (optical) microscopes (interaction of photons with a matter) Resolution 0.1 mm • Equipped for visible light only • Equipped for fluorescence • Confocal laser scanning Electron microscopes (interaction of electrons with a matter) Resolution 0.1 nm (in practice 1 nm) • Transmission • Scanning Methodologies to study cells and tissues 2 Fixation (denaturation) • Organic solvents (EtOH, MetOH, Aceton,…) • Aldehydes (form-, paraform-, glutar-aldehyde, …) • Organic acids (acetic, picric, …) • Heavy metals (salts of mercury, chrome, osmium, …) Embedding + Sectioning • Paraffine wax • Celloidine (=cellulose nitrate) • Durcupan (synthetic polymer) • LR White (synthetic polymer) • others „Staining“ Chemical stains (H+E, Azan, van Gieson, …) Histochemical stains (for proteins/enzymes, sugars, lipids, …) Immunochemical visualization (labeled antibodies) Heavy metals (for TEM – salts of uranium, lead, wolfram, …) Understanding the complex systems can only be built on understanding its components Tissues Cells Cells Cells Fluids Extacellular matrix The cells make it all ! Fluids • Intersticial fluid • Plasma (in blood) • Lymph (in lymph vessels) • Cerebrospinal fluid • Intracellular fluid (cytosol) Living organisms are composed of cells Long way to this discovery: Robert Hooke 1665 He for the first time observed the structure of cork - cell Antonie van Leeuwenhoek 1678 He for the first time observed microscopical organisms (bacteria, protozoa) Rudolph Virchow 1855 Cell can develop only from preexisting cells „Omnis cellula e cellula“ Matthias Schleiden Theodor Schwann All organisms are composed of one or more cells 1839 Current cell theory – 6 principles on which it is built • Cell is the smallest structural and functional unit capable of life functions • Function of each cell is given by its specific structure • Cells are bulding units of all multicellular organisms – cells are responsible for all processes taking place in the organisms • Structure and function of all organisms is based on structural and functional properties of cells from which they are composed • All new cells originate from preexisting cells • Thanks to the continuity of life on the Earth, all cells are in principle the same (universal genetic code and its expression) Muscle fibre Neuron Neuromuscular junction Anaphase Telophase Cell is unifying theme/element of life (cells are very similar among each other: small + specialized functions) Despite of their common scheme, structural and functional diversity is a typical feature of all eukaryotic cell types The cells of human tissues and organs are also structurally and functionally very diverse Egg Sperm Neuron in brain Fat cell Cell of gut epithelium Blood cells Muscle cell Bone cells Such diversity is critical for an ability of cells to serve various functions in human body No cell is exactly like all others, but cells do have many common structural and functional features. Keep in mind that not all cells contain all the structures we will discuss ! All cells have 3 major parts: 1. PLASMA MEMBRANE 2. CYTOPLASM 3. NUCLEUS (eukaryotic) 1 2 3 Cellular organization is based on COMPARTMENTALIZATION Specialized functions can be carried out in different locations Membranes make up boundaries between the compartments Unique protein and lipid components and unique function Unique control of the movement of molecules Unique composition of the surrounded interior Compartments & Membranes Many small compartments are better More space for: • regulation • nutrients exchange • waste removal More membrane surface per volume surrounded Surface area is proportional to the square (r2) of its diameter. Volume is proportional to the cube (r3) of its diameter. Amplification X Reduction of selected compartments Cell differentiation Specialization of cells for different functions Rough ER in secretory cells Mitochondria in cardiac musle cells Biological membrane structure 1 ~2.5 nm ~2.5 nm 7-10 nm ~2.5 nm Electron dense Electron opaque Electron dense Unit membrane common to all membranes Cell membranes seen in electron microscope (pseudocolored) Biological membrane structure 2 Fluid mosaic - A bilayer of lipids with mobile globular proteins Polar phospholipid head Cholesterol Non-polar phospholipid tail Hydrophilic portion Hydrophobic portion Membrane structure 3 Membrane lipids Make up 90-99% of molecules in membrane (in numbers). • Phospholipids - 75% of lipids • Cholesterol - 20% • Glycolipids - 5% - only on cytoplasmic membrane - GLYCOCALYX Membrane proteins Constitute 1-10% of total molecules but 50% of the weight because of their larger size. Sensing signalsTransport Cell identity Cell adhesion Enzymatic activity PeripheralIntegral + Organelles Membranous • Endoplasmic reticulum • Golgi apparatus • Lysosomes • Endosomes • Peroxisomes • Mitochondria Specialized internal structures with specialized functions Non-membranous • Ribosomes • Centrosomes • Centrioles • Basal bodies Related to specific structure and function of the cell e.g., much energy needed  many mitochondria Nucleus 1 membrane-bounded structure Liver cell nucleus Mostly: • Spheherical (5-10 mm) (lobular, twisted, disk-shaped,…) • Located centrally • One per cell (osteoclast more, erythrocyte none) Nucleus Perinuclear cisterna Unit membrane Lamina Unit membrane 20-50 nm 80-100 nm Outer nuc. membr. Inner nuc. membrane Nucleus 2 Continuation on nuclear membrane Perinuclear cisterna Unit membrane Lamina Unit membrane 20-50 nm 80-100 nm Outer nuc. membr. Inner nuc. membrane Lamins: • Intermadiate filament proteins (A, B, C) • Form meshwork inside of INM, some extend into nucleoplasm • Nuclear strength and architecture • Anchorage sites for chromatin • DNA replication and mRNA transcription • Involved in apoptosis Laminopathies • Human diseases (at least 13 known) • Mutations in lamin genes (almost 200 mutations known) • Deregulated gene expression • Premature aging Hutchinson-Gilford progeria Rare - 1-4 per 8 milion of newborns Missense mutation in A-type lamin Nucleus 3 Nuclear pore complex Diameter ~ 100 – 125 nm Three rings (8 subunits each) Inner filamentous basket Distal ring Nuclear ring Cytoplasmic ring Transport via nuclear pores (Nucleocytoplasmic shuttling) • Proteins, RNAs, ribosome subunits • Bidirectional • Needs nuclear localization/export signals • Helped by importins/exportins • Regulated by Ran GTPases Nucleus 4 Chromatin Heterochromatin Feulgen positive – dark in light microscope Dark/dense granular in TEM Transcriptionally inactive Euchromatin Invisible in light microscope Relaxed uncoiled chromosomes Transcriptionally active Interphase nucleus 2 nm DNA double helix 30 nm Chromatin fiber of packed nucleosomes Nucleus 5 Nucleolus nucleolusnuclear membrane nucleoplasm cytoplasm non-membrane-bounded structure Main functions Synthesis of rRNA Assembly of ribosomes Pars fibrosa Primary transcripts of rRNA Pars granulosa Assembly of ribosomes Fibrilar centers Non-transcribed DNA Nucleolar-organizing regions of DNA on five chromosomes in human cells (chrs. 13, 14, 15, 21, 22) Endoplasmic reticulum 1 „within cell“ „net“ Majority of the membrane within cells. Interconnected tubuli and vesicles Cisterns Endoplasmic reticulum 2 Rough ERSmooth ER 200 nm NO attached ribosomes  No protein-synthesis functions! Manufactures phospholipids and cholesterol • Liver – lipid and cholesterol metabolism, breakdown of glycogen and, along with the kidneys, detoxification of drugs • Testes – synthesis of steroid-based hormones (testosterone) • Intestinal cells – absorption, synthesis, and transport of lipids • Skeletal and cardiac muscle – storage and release of calcium (sarcoplasmic reticulum) External surface has ribosomes attached • Manufactures all secreted proteins • Synthesizes integral membrane proteins • Modifies proteins Ribosomes ribosomes mRNA 100 nm POLYRIBOSOME (cluster of ribosomes translating certain segment of mRNA) mRNA 5` AUG 3` START kodon 3`UAC 5` Met-tRNA Beginning of translation mRNA 5` UAG 3` End of translation mRNA 5` UAA 3` mRNA 5` UGA 3` STOP kodony bind „release factor“E P A 5` 3` reading of mRNA = movement of ribosomes on mRNA codons Aminoacyl tRNA free tRNA Growing polypeptide chain Ribosomes - Translation Golgi apparatus – Transgolgi pathway Cis Trans Centrosome Diameter – 0.2 mm Length - 0.5 mm 1 2 3 4 56 7 8 9 Thank you for your attention ! ahampl@med.muni.cz Building A1 – 1st floor Histology lectures Key elements of the microscopic structure of tissues and organs and their relevance to the function Very latest discoveries in the field of tissue structure and maintenance and their relevance to the disease development and therapy