Thermal experience during embryogenesis contributes to the induction of dwarfism in whitefish <i>Coregonus lavaretus</i>

<div><p>Ecotype pairs provide well-suited model systems for study of intraspecific phenotypical diversification of animals. However, little is still known about the processes that account for the development of different forms and sizes within a species, particularly in teleosts. Here, embryos of a normal-growing ‘large’ form and a dwarf form of whitefish <i>Coregonus lavaretus</i> were incubated at two temperatures that are usually experienced at their own spawning sites (2°C for the normal and 6°C for the dwarf form). All fish were subjected to similar thermal treatment after hatching. The present data demonstrate for the first time that different thermal experience in embryonic life has lasting effects on body and muscle growth of this ecotype pair and contributes to the development of the dwarf form. Thus, juvenile fish of the regular form are much smaller and have less muscle mass when pre-hatching thermal conditions were similar to those typical for the spawning sites of the dwarf form (6°C) than when subjected to conditions of their own spawning sites (2°C). Surprisingly, fish of the dwarf form exhibit a similar pattern of response to thermal history (2°-fish much larger than 6°-fish), indicating that in their case, normal spawning site temperature (6°C) is indeed likely to act as a growth limiting factor. Results also demonstrate that the hypertrophic and hyperplastic muscle growth modes are similarly affected by thermal history. Immunolabelling experiments for Pax7, H3P and Mef2 provide evidence that the cellular mechanisms behind the increased growth rates after cold incubation in both ecotypes are increased proliferation and reduced differentiation rates of muscle precursor cells. This is of major significance to aspects of ecological and developmental biology and from the evolutionary perspective.</p></div

Quantification of muscle precursor cells (MPCs).

<p>Numbers of labelled cells (means + s.e.) in double-immunostained 10 μm myotomal cross-sections of fish of the normal-sized form (NF, open bars) and dwarf fish (DF, dashed bars) imprinted at 2° (blue) and 6°C (red) at hatching and at 80 dph. Values at bottom of bars provide total numbers of evaluated quadrants (fish at hatching) and somite/myotome areas delimited by 2 successive myosepta (fish at 80 dph), respectively. Data at hatching derived from 16 2°-fish and 6°- fish each in the normal form, and from 8 2°-fish and 6°-fish each in the dwarf form; data at 80 dph derived from 7 individuals in all four groups. (A) Total numbers of Pax7+ cells per 100 μm distance within the DM and the lateral fast muscle. (B,C) Percentages of Pax7+ cells that have entered proliferation (Pax7+/H3P+) (B) or differentiation (Pax7+/Mgn+) (C). (*) Intergroup differences significant at p≤0.05.</p

Development of body lengths and muscle mass.

<p>(A) Total body lengths of normal-sized fish (NF, open bars) and dwarf fish (DF, dashed bars) imprinted at 2 (blue) and 6°C (red) at the end of the imprinting period (0 dph) and in the juvenile stage (80 dph). Values at bottom of bars provide number of individuals included in length measurement. Total fast (B) and slow (C) muscle csa in one half of the trunk (8 individuals per thermal group of each ecotype); whiskers indicate s.e., significant differences are assigned at p≤0.05 (*). (D) Correlation of slow muscle relative proportion (fast-to-slow muscle ratio) with fish size as given by total muscle csa; regression line equations: NF-2: y = 7.3x + 3.9 (r² = 0.87), NF-6: y = 12.2x + 7.6 (r² = 0.95), DF-2: y = 11.7x + 7.2 (r² = 0.94), DF-6: y = 15.9x + 10.3 (r² = 0.97).</p

Development of slow and fast muscle fibre numbers.

<p>Relationship between fibre number in one epaxial trunk quadrant of normal-sized fish (NF, open bars) and dwarf fish (DF, dashed bars) imprinted at 2° (blue) and 6°C (red) and developmental time (8 individuals per thermal group of each ecotype). (A) Fast fibres, (B) slow fibres, (C) fast fibres > 200 μm², (D) fast fibres ≤ 50 μm²; whiskers indicate s.e., differences between thermal groups significant at p≤0.05 (*).</p

Relationship of nuclei to fibre volume of isolated fast fibres.

<p>Myonuclear densities at hatching (A) and 80 dph (B). Regression line equations: (A) NF-2: y = 3.9e^-6x + 5.5 (r² = 0.25) (356 fibres from 6 individuals), NF-6: y = 3.0e^-6x + 6.5 (r² = 0.38) (126 fibres from 6 individuals), DF-2: y = 8.0e^-6x + 5.1 (r² = 0.48) (303 fibres from 6 individuals), DF-6: y = 2.9e^-6x + 5.9 (r² = 0.26) (197 fibres from 6 individuals); (B): NF-2: y = 1.3e^-6x + 15.4 (r² = 0.28) (376 fibres from 8 individuals), NF-6: y = 3.5e^-6x + 8.2 (r² = 0.27) (236 fibres from 7 individuals), DF-2: y = 9.4e^-6x + 11.0 (r² = 0.59) (552 fibres from 8 individuals), DF-6 y = 2.6e^-6x + 10.0 (r² = 0.29) (332 fibres from 8 individuals).</p

New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and <i>In Vitro</i> Generation

<div><p>Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. <i>In vitro</i> induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.</p></div

Ultrastructure of <i>in vitro</i> generated NETs.

<p>TEM images of <i>in vitro</i> generated NETs immunogold stained for citH3. A: Thick NET trajectory with attached electron-dense substance and small branches giving rise to loosely structured fibrous meshwork. Frames depict details shown in B–D. B: Patch of dense substance adhering to a thick NET strand with parallel fibrous subarchitecture (black arrowhead). C,D: Examples of NET strand disintegration into a network of randomly oriented small fibres with diameters down to about 2 nm (arrowheads). Some accretions at fibre intersections and along the strands are labelled for citH3 (arrows).</p

Detailed TEM analysis of NETs from COPD sputum (A–C,F) and <i>in vitro</i> generated NETs (D,E,G,H).

<p>A,D: Immunogold staining for NE. Amorphous attachments and ‘grid-point’ aggregates of both COPD NETs (A) and <i>in vitro</i> NETs (D) are all strongly labelled. B,E: Immunogold staining for citH3. Only some of the aggregates adhered to COPD NETs (B) and <i>in vitro</i> NETs (E) are stained (white arrow). Arrowheads indicate fibres consisting of single DNA double-helices. C,F: Sectioned COPD sputum. C: NET attachment to epithelial cell surface. F: Individual NET fibres (arrowheads) and bundles of such fibres in parallel alignment (asterisk) interspersed with granules and pieces of cell debris. The bundles are abundantly clotted with amorphous substance. G,H: Structure of fully spread NETs. Bundles of parallel fibres (black arrowheads) provide a scaffold for an irregular network of much finer fibres with diameters down to about 2 nm (white arrowheads) some of which are equidistantly decorated with protrusions of about 10 nm, likely corresponding to nucleosome cores (black arrows in H). Immunolabelling for citH3 recognises sites along the fibrous bundles (white arrows) and on detached substance (open arrowheads in G), but in no case the presumed nucleosomes.</p

Semi-quantitative evaluation of NET abundance in induced sputa from hospitalized COPD patients with acute exacerbations and from controls.

<p>Images depict characteristic microscopic appearance conforming to the categories ‘large amounts’ (A) and ‘minor traces’ (B).</p

Electron microscopic characterisation of NET components.

<p>SEM (A,C) and TEM (B,D–H) micrographs from COPD sputum (A–E,H) and <i>in vitro</i> induced NETs (F,G). A: Fibrous strands diverge from a PMN cell body with a sculptured surface and attached granulae and merge into loosely structured texture. B: Motif corresponding to that of A. Thinly spread areas (inset) exhibit a fibrous texture with attached globules (arrows). C: Bacterium (open arrow) entrapped by NET fibres with globular protrusions. D,E: Sections of COPD sputum. D: Bacterium (open arrow) surrounded by a fibrous network that embeds spherical granules with amorphous content (arrow). E: Undisturbed PMN with normal nuclear chromatin and amorphous vesicular inclusions (arrows). F–H: Sections of <i>in vitro</i> induced NETs. F: NETs attached to the remnants of their cell of origin. NET fibres exhibit less accretions than those from COPD sputum NETs (cf. E); arrows indicate neutrophilic granula. G: Fibres and neutrophilic granula of the NETs shown in F. H: Membrane coated in COPD sputum vesicle containing NET-like structures.</p