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First published online 14 November 2007
doi: 10.1242/dev.004697

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Lymph heart in chick - somitic origin, development and embryonic oedema

¹ School of Biological Sciences, University of Reading, Reading RG6 6AJ, UK.
² Royal Veterinary College, London NW1 0TU, UK.
³ Institute of Anatomy I, University Erlangen-Nuremberg, Krankenhausstrasse 9, 91054, Germany.
⁴ Pediatrics I, Children's Hospital, Robert-Koch-Strasse 40, 37075 Goettingen, Germany.
⁵ Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK.

View larger version (124K): [in this window] [in a new window]   Fig. 1. Early lymph heart has only striated musculature. (A) Schematic representation of musculature in the caudal region of E10 chick embryo. Lateral view showing the lymph heart (arrow) and selected labelled muscles: Lev, musculus levator caudae; Lat, m. lateralis caudae; for other muscles see Valasek et al. (Valasek et al., 2005). (B-D) MyoD whole-mount in situ hybridisation; lateral views below the hind limb bud. (B) E6 showing single myogenic cells detaching from myotomes into the more superficial layers. Myotomes 35 and 38 are labelled. (C) At E8, the cells coalesce into the anlage of the lymph heart (asterisk). (D) By E10, the lymph heart musculature (arrow) is well organised. (E) E10: confocal immunofluorescence with pan-myosin heavy chain (MyHC) shows the branching structure of the muscle fibres. Scale bar: 500 µm. (F-I) Longitudinal sections through the lymph heart with adjacent epaxial muscle. At E10 there is robust MyHC expression (F; brown) and absence of SMA expression (G; counterstaining with eosin, pink). At E18 there is robust MyHC expression (H; brown) and distinct SMA expression adjacent to the endothelium (I; arrow. Counterstained with eosin, pink). Scale bar: 500 µm. (J-L) Electron micrographs of lymph heart wall. (J) At E10, myoblasts containing mostly immature contractile myofilaments (red arrowhead) with few Z-disc striations are directly underneath the endothelial lining (arrow). They have many adherent contacts with each other. Scale bar: 10 µm. (K) Motor endplate at E10 with distinct postsynaptic density (arrow), a shallow postsynaptic fold, basal lamina in the synaptic cleft, discrete presynaptic dense projections (spike-like electron dense areas) and an omega-shaped profile (red arrowhead) suggesting exocytotic release of transmitter. Scale bar: 200 µm. (L) At E19, the subendothelial layer is occupied by cells with extensions containing secretory vesicles (black arrowhead) and darker cytoplasm with smooth muscle proteins (red arrowhead). Lymph heart striated muscle fibres situated in a more peripheral region have more mature myofilaments with Z-discs (arrow) compared to those at E10. Loose connective tissue separates smooth and striated muscle layers. Scale bar: 10 µm.

View larger version (108K): [in this window] [in a new window]   Fig. 2. Chick-quail somitic chimeras show hypaxial origin of lymph heart. (A) Immunohistochemistry with QCPN shows the localization of grafted quail cells 6 hours after transplantation of the dorsal part of a newly formed somite I (outlined). (B,C) Similar grafts of the dorsal parts of somites 34-41 give rise to lymph heart tissue after 7 days (see Table 2) including the skeletal muscle cells with darker cytoplasm (B, arrow) and the endothelial QH1-positive cells lining the lumen (C). Ectoderm, associated with some somitic tissue grafts, never contributes to the lymph heart (see Fig. S1 in the supplementary material). (D) QCPN immunohistochemistry detects quail nuclei in grafted tissue 5 hours after transplantation of the medial portion of the dermomyotome (outlined) of somite VII. (E) Grafting of the medial third of the dermomyotome gives rise only to the local epaxial m. levator caudae and dermis (above black line), but not to the hypaxially located tissues (below black line, including the lymph heart; arrow). (F) QCPN immunohistochemistry detects quail nuclei in grafted tissue 4 hours after transplantation of the lateral third of the dermomyotome (outlined) of somite V. (G) Transplantation of lateral dermomyotome gives rise to quail cells in the lymph heart (arrow). Scale bars: 100 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Lymph heart innervation. (A) Whole-mount neurofilament immunofluorescence showing a lateral view of the tail at E10 with hypaxial branches overlying the lymph heart (white outline) that also supply local dermis and musculature. Segmental epaxial branches (white arrows) lie dorsal to the lymph heart. (B-E) Immunofluorescence for neurofilament (green) and alpha-bungarotoxin binding (red) in the lymph heart (LH) wall (thickness indicated by double-headed arrow). (B,C) Scattered bungarotoxin-positive acetylcholine receptors (red arrow) and neurofilaments (green arrow) at E10 in lymph heart and striated muscle, respectively. (D) At E18 the lymph heart is practically devoid of any neurofilaments while retaining the ACh receptors (red arrow). (E) In body muscle, neuromuscular junctions are now regularly organised in bands (red arrow) and form intimate contacts with the neurofilaments (green arrow). (F) At E15, acetylcholinesterase reaction product (brown) is diffusely seen in the striated muscle of the lymph heart wall. (G) At E15, body muscle fibres have similar diffuse activity; however, their ACh receptors are organised in bands (brown arrows), and display significantly higher activity, which is not blocked by iso-OMPA (not shown). (H) At E15, parasympathetic efferent innervation, assessed by NADPH-diaphorase histochemistry (blue reaction product), failed to give a signal in the wall of the lymph heart, with red blood cells in its lumen. (I) At E15, the wall of the hindgut has robust parasympathetic NADPH-diaphorase activity in the nerve fibres and in the ganglia of the myenteric plexus (arrow). (J,K) Sympathetic efferent innervation determined by immunoreactivity to tyrosine hydroxylase (TH; in red) and sensory axons through immunoreactivity for CGRP (green). (J) At E15, lymph heart tissue expressed neither marker. (K) At E15, robust expression of both markers was detected in the nerve tissue in the wall of the hindgut (red and green arrows). Scale bars: 100 µm in A-J; 50 µm in K.

View larger version (108K): [in this window] [in a new window]   Fig. 4. Lymph heart dysfunction results in embryonic oedema. (A) Normal E10 chick embryo (dorsal view). Arrow denotes flank skin adherent to the trunk. (B) E10 embryo following tail ablation at HH20 showing oedema (arrow). (C) MyoD whole mount in situ hybridisation of embryo in B showing absence of lymph heart (white arrow) and all local tail muscles. Absent tail makes the cloacal tubercle appear more prominent (arrowhead in B and C). (D) Spontaneous mutant Araucana rumpless chick at E10 is tailless and oedematous. (E) Mechanical blockage of the lymph hearts injected at HH36 with Mercox resin (white arrows) results in oedema (yellow arrow). (F) E10 embryos treated with decamethonium develop oedema (arrow).

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