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First published online 25 August 2004
doi: 10.1242/dev.01312

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Expression and release patterns of neuropeptides during embryonic development and hatching of the green shore crab, Carcinus maenas

J. Sook Chung^* and Simon G. Webster

School of Biological Sciences, University of Wales Bangor, Bangor LL57 2UW, UK

View larger version (34K): [in a new window]   Fig. 1. Expression of neuropeptide mRNAs during embryonic development. (A) CHH, (B) MIH, (C) PDH, (D) RPCH; n=3-11 independent measurements. Error bars are ±1 s.e.m.; y-axes are logarithmic. ND, no specific product detected, – not sampled. (E) and (F) amplification and melt curves from standard copy-quantified PDH (107) copies/capillary in comparison with cDNA and mRNA extracted from unfertilised eggs (two egg equivalents/capillary). For definition of developmental stages see Table 1.

View larger version (142K): [in a new window]   Fig. 2. Developmental profiles of CHH- and MIH-expressing neurones during embryonic development. All confocal images are single sections, with the exception of D, which is a stacked and flattened projection image. (A) MIH-IR neurones at mid-eye development. Arrowhead, perikarya; arrow, sinus gland. Inset, higher magnification showing groups of four perikarya in each side of the developing eye. (B) CHH-IR neurones at mid-eye development. Note perikarya (arrowhead) and collaterals (arrow). Inset shows higher magnification of four perikarya. (C) Stacked (40• 1.5 µm), dual immunolabelled projection image of MIH (green, FITC) and CHH (red, Cy3) X-organ perikarya, axon tracts and sinus gland. Note that both peptides are not colocalised, but that the merged view through the sinus gland shows some areas of false colocalisation (yellow) due to processing of the image. (D) Expression of CHH in the pericardial organs at mid-eye development. Immunoreactive structures in the anterior and posterior bars of the pericardial organs are arrowed; sg, sinus gland, xo, X-organ. (E) Detail of the CHH-IR structures in the PO and adjacent areas during late eye development just prior to hatching. Note anastomoses between anterior and posterior bars (arrows; a,p), and five pairs of peripheral IR perikarya (left, small arrows). (F) View of posterior thorax and abdomen of a newly emerged zoea. Anterior and posterior bars of the pericardial organs are shown (a,p). Arrows show four small ventral peripheral thoracic perikarya (arrows) and segmentally iterated lateral abdominal perikarya (arrowheads) in lateral positions close to the anterior insertions of the abdominal flexor muscles. (G) Overview of CHH-IR structures in a newly emerged zoea. Arrows show the X-organ (xo), pericardial organs (po), and abdominal cells (ac). (H-K) Development of CHH-IR cells in the abdomen. (H) 4 days before hatching, (I) 2 days before hatching, (J) at hatching. Arrowheads in J show dorsal cells, which develop at this time, and appear to be close to the posterior insertions of the abdominal extensor muscles. Scale bars: 200 µm (A), 50 µm (B-F), 100 µm (G), 200 µm (H), 50 µm (I-K). Inserts (B,C): 10 µm.

View larger version (126K): [in a new window]   Fig. 3. Developmental profiles of neurones immunoreactive to PDH during embryonic development. All images are stacked flattened projection images (20-40 1 µm slices) (A) PDH-IR neurones 1-2 days after eye anlage formation, five pairs of perikarya can be seen. (B) PDH-IR neurones 2-3 days after eye anlage formation. Note two pairs of anterior-median brain neurones and groups of 2, 4 and 5 perikarya in the eye neuropil. (C) PDH-IR neurones 7-8 days after eye anlage formation. Note that only one pair of anterior-median protocerebral neurones is now evident (arrows) and that single immunoreactive neurones appear in the posterior region of the tritocerebrum (small arrows). (D) PDH-IR neurones in the eye neuropil of a fully developed embryo. Note axons entering and leaving the sinus gland (sg, arrows), and development of a prominent plexus in the lamina ganglionaris (lg). Perikarya numbers (11) remain unchanged. (E) PDH-IR structures 7-8 days after eye anlage formation. Perikarya marked with small arrows correspond to those in C. Note the immunoreactive neurones possibly projecting to the stomatogastric ganglion (arrow), which originate from a small IR structure in the circumoesophageal connectives (arrowheads) Small arrows point to arborising (possibly segmentally iterated) dendrites in the thorax. Abbreviations: lg, lamina ganglionaris; oe, oesophagus; sg, sinus gland. Scale bars: 50 µm.

View larger version (55K): [in a new window]   Fig. 4. Developmental profiles of neurones immunoreactive to RPCH and CCAP during embryonic development. (A) RPCH-IR neurones (X-organ) and immunopositive endings within the sinus gland (sg) in the eye, 5-6 days after eye anlage formation. (B) RPCH-IR structures in the brain and eye 6-7 days after eye anlage formation. Note that immunoreactivity in the X-organ (arrowheads) is weak, but that three pairs of prominent cells in the protocerebrum (arrows) now give rise to descending axons. (C) RPCH-IR structures in an embryo 2-3 days before hatching. Note descending axons, some (if not all) of which project contralaterally, anterior to the oesophagus (oe, arrow), and the prominence of, and connections to the putative post-commissural organs (pco), which dorsally project three digitate extensions (small arrows). (D) CCAP-IR structures associated with the thoracic ganglion and pericardial organs during mid-eye development. Large arrow, the posterior bar of the pericardial organ; small arrows, contralateral projections of axons reminiscent of those seen in the adult. Perikarya were never observed in these preparations. Abbreviations: oe, oesophagus; pco, post-commissural organs; s, segmental nerve; sa, sternal artery; sg, sinus gland. Scale bars: 50 µm.

View larger version (21K): [in a new window]   Fig. 5. Quantification of CHH and CCAP levels during embryonic development, measured by RIA. (A) Developmental profiles for both neurohormones, bars CCAP; line, CHH; n=5-13 for CCAP, 5-23 for CHH. (B) An HPLC-RIA analysis of CHH and CCAP immunoreactive fractions of a Sep-Pak purified sample of 5000 embryos. Filled bar, CCAP; grey bar, PO-CHH; open bar, XO-CHH (see Materials and methods for details of analysis).