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First published online March 28, 2008
Journal of Experimental Biology 211, 1344-1351 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012013

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CD14 and TLR4 are expressed early in tammar (Macropus eugenii) neonate development

Kerry A. Daly^1,2, Christophe Lefévre^2,3, Kevin Nicholas^2,3, Elizabeth Deane⁴ and Peter Williamson^1,2,*

¹ Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia
² Cooperative Research Centre for Innovative Dairy Products, Australia
³ Department of Zoology, University of Melbourne, Parkville, Victoria 3010, Australia
⁴ School of Environmental and Life Sciences, Macquarie University, Ryde, NSW 2109, Australia

View larger version (55K): [in this window] [in a new window]   Fig. 1. Multiple alignment of the amino terminal end of CD14. Boxes 1–4 represent hydrophobic motifs that are part of the lipopolysaccharide (LPS) binding site. Boxes 5 and 6 represent motifs involved in Toll-like receptor 4 (TLR4) interactions. Asterisks indicate the shared potential N-linked glycosylation sites; marsupial-specific sites are underlined. Plus signs indicate the residues involved in the LPS binding pocket. Sequences used in the alignment are detailed in Table 2.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Multiple alignment of the toll/interleukin receptor (TIR) domain of TLR4. The number suffix indicates the corresponding TLR of that species. The alpha helices and beta sheets are indicated beneath the alignment. TM, transmembrane region; HR, hydrophobic region; asterisk, site of LPSd mutation; 1, residues important in the BB protein interaction site; and 2, residues important in the DD loop protein interaction site. Sequences used in the alignment are detailed in Table 2.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Kyte–Doolittle hydrophobicity plots of the TIR domain of TLR4. Similar scores were seen over the entire domain, reflecting the high levels of conservation, including in the transmembrane (TM) and hydrophobic regions (bar).

View larger version (29K): [in this window] [in a new window]   Fig. 4. Neighbour joining phylogenetic trees for TLR4 (A) and CD14 (B) calculated from protein sequences. Bootstrap values (1000 replicates) are indicated on each tree. In both trees, species are indicated by common names. In A, the number suffix indicates the corresponding TLR of that species.

View larger version (12K): [in this window] [in a new window]   Fig. 5. Expression of CD14 (A), TLR4 (B) and TNF- (C) in adult tammar leukocytes stimulated with either LPS (solid line) or LTA (broken line). FC expression, fold change in expression relative to the control non-stimulated tammar leukocytes. Relative expression was calculated using the 2–Ct method (Livak and Schmittgen, 2001). Y error bars indicate ±1s.d.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Average expression of CD14 (A,B) and TLR4 (C,D) in organs of the tammar pouch young throughout the first 30 days of life. (A,C) Expression in cervical thymus, spleen, liver and bone marrow. (B,C) Expression in skin, lung and jejunum. Relative expression was calculated using the 2–Ct method (Livak and Schmittgen, 2001). Y error bars indicate ±1s.d.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Expression of CD14 (A,B) and TLR4 (C,D) in organs of the tammar pouch young up to 120 days of life. (A,C) Expression in cervical thymus, spleen, liver and bone marrow. (B,C) Expression in skin, lung and jejunum. Relative expression was calculated using the 2–Ct method (Livak and Schmittgen, 2001). Y error bars indicate ±1s.d.