Worm Breeder's Gazette 12(3): 83 (June 15, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We are interested in understanding how neurons and hypodermal cells are generated. Based on the analysis of the expression of a lin-26 /lacZfusion and on the study of animals hemizygous for the mutation lin-26 ( n156 ),we concluded (see 1991 Meeting on C. elegans) that lin-26 is likely to be a hypodermal differentiation factor.
We have sought to confirm the lin-26 expression pattern using other methods, and towards this end have raised polyclonal antibodies directed against a portion of the lin-26 protein (the first two-thirds, which excludes the zinc fingers). We affinity-purified the antibodies over a bacterially expressed recombinant lin-26 protein column. The purified antibodies recognize two polypeptides on Western blots of N2 and n156 extracts with apparent molecular weights of 55 kD and 58 kD (expected size for the lin-26 protein is 53.2 kD). We believe that both polypeptides are encoded by lin-26 ,since an insertion of a small piece of DNA in the middle of the lin-26 coding sequence that should result in a 90 amino acid insertion within the lin-26 protein shifts both bands by 10 kD. The nature of the difference between the 55 kD and 58 kD polypeptides is not yet known. We used the antibodies in conjunction with the monoclonal antibody MH27 (Francis and Waterston), which recognizes belt desmosomes, to determine which cells express lin-26 .
Our anti lin-26 antibodies appear to stain the nuclei of all hypodermal cells and of all socket and sheath cells, plus the nuclei of a few other cells. Staining starts in embryos shortly after the birth of the cells that stain. More precisely, the following nuclei are stained:
- the nuclei of all hyp cells;
- the nuclei of the seam cells (H0, H1 , H2 , V1 -V6,T) and of their hypodermal descendants (see also below); the nuclei of the two Q cells (until they divide);
- the nuclei of the P cells and of their hypodermal descendants;
- the nuclei of the cells that form the anus and rectum, B, F, K (and then K.a), K', Y (until it differentiates into PDA), U; the three rep nuclei are also stained but very faintly and not before the L2 /L3stage;
- the nuclei of the cells that form the excretory system with the exception of the excretory gland cell, that is the excretory cell, the excretory duct cell, G1 and W (until they divide), G2 (and then G2 .p);
- the nuclei of the socket and sheath cells that are associated with ciliated sensillae, that is the amphids, the CEPs, the ILs, the OLLs and the OLQs in the head, the deirids and the postdeirids in the body, the phasmids in the tail;
- certain nuclei of the somatic gonad, that is Z1 and Z4 ,then Z1 .pand Z4 .aand their descendants (staining becomes very faint after the L2 stage).
These results are in agreement with what we had seen previously by studying the expression of lin-26 /lacZfusions. Although expression of the gene fusions in the socket and sheath nuclei as well as in the somatic gonad had initially escaped our attention, we have since looked for and found ß-galactosidase activity in those cells. The antibodies also stain the germline nuclei very brightly in adults. However, we are not certain of the significance of this latter staining, because oocytes are not stained and staining in the germline appears nucleolar, whereas in all of the cells mentioned above the nucleolus is not stained. In addition, we never observed B-galactosidase expression from the lin-26 /lacZfusions in the germline. Similarly, we see some variably weak staining in the nucleolus of many large nuclei, particularly in adult animals and in animals where the background is high, but have not detected any ß-galactosidase activity in those cells so far. We suspect, although we do not have have proof for it, that nucleolar staining might be caused by non-specific reaction of the antibodies to some abundant protein(s) in the nucleolus.
Socket and sheath cells are support cells for certain neurons and in some ways can be considered as the nematode equivalent of glial cells. In other animal species, glial cells seem to be more closely related (by lineage and by growth factors dependence) to neurons than to epidermal cells, and in C. elegans, socket and sheath nuclei (with the exception of the amphid sheath nucleus) have the same stippled appearance by Nomarski microscopy that neuronal nuclei have. We were thus surprised to observe lin-26 to be expressed both in hypodermal cells and in the socket and sheath cells. To determine if this staining is biologically important, we have studied the structure and function of the ciliated sensory organs of lin-26 ( n156 )animals. It appears that the amphid, the phasmid and the cephalic sensory organs are defective in n156 animals. Electron microscopic studies of serial sections through the nose and tails of n156 animals show defects in the socket and sheath cells associated with the amphids, phasmids and cephalics sensory organs: the socket cells do not always form a channel and the sheath cells form large deposits of matrix material. In over 60% of n156 animals, the six amphidial neurons and the two phasmidial neurons on each side of the animal, which are normally exposed to the environment and which are stained by the lipophilic dye DiO in control animals, are not stained. We have tested whether the amphids are functional in n156 animals by examining three behaviors that are mediated by the amphids. It appears that n156 animals are partially dauer defective (Daf) and fail to avoid high concentration of glycerol (Osm), much as do animals with socket and sheath defects (see Albert et al., Dev. Biol. 126, 270-293,1981; J. Thomas and 8. Horvitz, WBG 10 #1, page 89,1987); however n156 animals chemotax normally to the volatile odorant benzaldehyde, a behavior mediated principally by the neuron AWC, which is not exposed to the environment (C. Bargmann et al., WBG 11 # 4, page 107,1990). The cephalic neurons control certain aspects of the foraging behavior of C. elegans; it appears that n156 animals tend to forage with their nose up, much as do animals ablated for the CEP neurons (see abstract by J. Kaplan et al.).
In summary, the gene lin-26 is generally expressed in the cells that are affected by the mutation n156 .Based upon these observations, we believe that lin-26 determines, at least in part, the cell fates of all non-neuronal ectodermally derived cells in C. elegans, that is the hypodermal cells, socket cells and sheath cells.
Albert et al., Dev. Biol. 126, 270-293,1981
J. Thomas and 8. Horvitz, WBG 10 #1, page 89,1987
C. Bargmann et al., WBG 11 # 4, page 107,1990
abstract by J. Kaplan et al.