A nematode embryo is surrounded by chitinous eggshell. It has been reported that, in C.elegans, about 75% of eggs without eggshell at the stage later than the 2-cell stage can develop to hatch1. Therefore, it has been believed that eggshell does not play any particular role in the development at later stages except the mechanical protection of the embryo. However, our earlier studies showed that the body length of the first-stage larva (L1) hatched from the eggs without eggshell and with the permeability barrier inside the eggshell was about 10% shorter than that from intact eggs2. This result suggests that eggshell plays some role in epidermal morphogenesis. To investigate mechanical functions of eggshell, we have developed a microdevice as artificial eggshell. We have shown that incubation of eggs without eggshell in our microdevice recovers the defects in body length3. In the present study, we further investigated the effects of eggshell on L1 morphologies. Arrangements of epidermal cells were observed by using the strain in which cell boundaries were visualized by the AJM-1::GFP. As a result, the number and the line-pattern arrangements of seam cells were unchanged but contacts between some seam cells (H1-H2 cells and V1-P1/2 cells) were altered. The variation was observed mainly at an anterior half of the L1 larva hatched from the eggs without eggshell. In contrast, cell-cell contacts of seam cells in the embryo from the comma to the 3-fold stages showed no noticeable difference between intact eggs and eggs without eggshell. The eggshell would have functions at the very last stage of embryonic development such as the stage just before hatching. To manipulate mechanical environments around embryos during development, we fabricated microwells with various sizes (20 -200 m in width) and shapes (round, oval and rectangle), which adapt to our device. When eggs without eggshell were incubated in oval-shaped microwells (well size 23 x 36
m2), which was smaller in size than normal eggs, no recovery was found in the body length, unlike the case when oval-shaped microwells (well size 43x59
m2), as small as normal embryos, were used. We speculate that mechanical environments around an embryo affect efficiencies in mechanotransduction during morphogenesis. 1. Schierenberg E and Junkersdorf B, 1992, Roux's Arch Dev Biol, 202, 10-16. 2. Hatakeyama A and Onami S, 2017, 21th International C. elegans Conference Abstracts, LA. 3. Hatakeyama A et al, 2018, EMBO Workshop: C. elegans Development, Cell Biology, and Gene Expression Abstracts, Barcelona.