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Supplemental material for the chapter "Epidermal Morphogenesis" from WormBook
Andrew Chisholm1, and Jeff Hardin2* 1 Department of Molecular, Cellular and Developmental Biology, University of California-Santa Cruz, Santa Cruz, CA, 95064 USA 2 Department of Zoology, University of Wisconsin, Madison, WI 53706 USA *Author for correspondence: e-mail: jdhardin@wisc.edu
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Introduction
Morphogenesis is the development of form, of tissues, of organs, and of organisms. This chapter describes the major morphogenetic processes of the mid-to-late C. elegans embryo, focusing on the epidermis and its interactions with underlying neurons and muscle. [‘Epidermis’ refers to the outer cellular layer of the organism. In older literature and in WormAtlas the epidermis is named hypodermis, although the terms are synonymous in C. elegans. To avoid needless worm-specific terminology we will use ‘epidermis’]. Morphogenesis of the epidermis involves both autonomously generated changes in epidermal cell shape and position, and interactions with internal tissues, including the developing nervous system and body wall muscles. Molecules required for epidermal morphogenesis thus include components of the epidermal cytoskeleton, epidermal cellular junctions, cell signaling pathways operating between the epidermal cells and underlying tissues, and of the extracellular matrix. In this review we follow the development of the epidermis during embryogenesis, focusing on processes and tissue interactions required for its morphogenesis. Our discussion of genes or molecules involved in these developmental processes is selective, and focuses on pathways whose role in the morphogenetic process has been analyzed in most detail. We begin with the specification of the major epidermal fates, then describe the morphogenetic processes of epidermal enclosure, dorsal intercalation, and elongation. Both enclosure and elongation exemplify organogenesis processes, in that they involve interaction with underlying tissues: ventral neuroblasts are required for epidermal enclosure, whereas body muscles are required for epidermal elongation. We conclude with the role of the extracellular matrix, including the cuticle, in embryonic epidermal development.
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Figures
Figure 1. Developmental timing of morphogenetic events [PDF]
Developmental landmarks are shown with representative Nomarski Differential Interference Contrast (DIC) images of embryos at corresponding stages, based on Fig. 4 of Sulston et al 1983 (PubMed or WormAtlas). Developmental times are relative to the division of the zygote (P0), in minutes at 20 deg C; rates at 25 deg are 1.2 times faster than at 20 deg C; rates at 15 deg C are 1.5 times slower than at 20 deg C. Fertilization occurs ~45 minutes before P0 divides. The stage termed ‘lima bean’ has not been precisely defined but is roughly equivalent to the period of epidermal enclosure when the ventral side of the embryo first becomes visibly concave. Note that unequal growth along the anteroposterior axis forces the embryo to turn within the eggshell between 350 and 400 minutes so that either the left or right side is uppermost, when mounted for observation; images before turning are either ventral or dorsal views, whereas afterwards they are left or right lateral views. All laterally viewed embryos in this and subsequent figures are oriented with anterior to the left, posterior to the right.
Figure 2. Ventral cleft closure [PDF]
Nomarski DIC images (courtesy H.N. Cinar and D. Patel) and schematics of ventral neuroblast positions during closure of the ventral cleft in the wild type; see also Movie 1. The left column includes schematics of ventral views of embryos with the migrating neuroblasts shown in green. The right column includes DIC images or embryos at the corresponding stage. Although the movements of individual cells during ventral cleft closure have not been fully described, the presence of a ventral cleft is well defined and its duration can be quantitated (M. L. Hudson and A.D.C., unpublished results). Scale bar = 10 microns.
Figure 3. Dorsal intercalation [PDF]
Schematics (left column, A-D), Nomarski DIC images (middle column, E-H; dorsal views; see also Movie 2) and DLG-1::GFP images highlighting epidermal cell boundaries (right column, I-L; dorsal view; see also Movie 3) of dorsal intercalation. Different embryos, at corresponding developmental stages, are shown in middle and right columns. Scale bar = 5 microns. For details on DLG-1, which localizes to apical junctions in epithelial cells in C. elegans, see (Koppen et al., 2001; PubMed). Dorsal cells are shown in teal in the left-hand column, seam epidermal cells are shown in yellow, and deirids are shown in white. Dorsal cells born on the right side of the embryo are depicted in a lighter color than those born on the left. For clarity, ventral epidermal cells and non-epidermal tissues are not shown. The last two pairs of dorsal epidermal cells to intercalate have been termed "pointer cells" (Williams-Masson et al., 1998; PubMed), and serve as fiduciary markers for the progress of intercalation in wild-type embryos (C, asterisks). Nomarski images in this Figure and Figure 4 courtesy of E. Williams-Masson; DLG-1::GFP images courtesy of M. Köppen.
Figure 4. Epidermal enclosure [PDF]
Schematics (left column, A-D), Nomarski images (middle column, E-H, ventral view; see also Movie 5) and DLG-1::GFP images (right column, I-L, ventral view; see also Movie 6) of ventral enclosure. Different embryos, at corresponding developmental stages, are shown in middle and right columns. Scale bar = 5 microns. Ventral cells are shown in pink in the left-hand column; seam epidermal cells in yellow, and dorsal cells, which wrap around the tail, in teal. Neuroblasts and other internal cells are depicted in gray. The first two pairs of ventral cells to reach the ventral midline are termed "leading cells" (Williams-Masson et al., 1997; PubMed), which extend long protrusions towards the midline (B). After the leading cells make contact at the ventral midline, cells posterior to these cells, termed "pocket cells" (Williams-Masson et al., 1997; PubMed), reach the midline (C). Finally, the anterior epidermal cells enclose the head (D).
Figure 5. Protrusive activity during morphogenesis [PDF]
Both dorsal (top row, A-C) and ventral (bottom row, D-F) epidermal cells display protrusive activity as they migrate. (A) A schematic of cell wedging during dorsal intercalation, corresponding to Fig. 3B. (B) An embryo expressing an lbp-1::gfp transcription reporter to mark epidermal cells (Heid et al., 2001; PubMed), imaged using spinning disk confocal microscopy (courtesy of R. King; see also Movie 4). Mosaicism has resulted in expression only in the right-hand cohort of posterior dorsal epidermal cells. Rearranging cells extend long protrusions in the direction of migration (arrow). (C) A schematic showing the relationship between such basolateral protrusions and rearrangement of dorsal cells (here and in F, anterior at the top). Protrusions lie basal to the apical junctional domain (pink). In addition, although it is not understood, wedging must presumably remodel junctional proteins as they rearrange (red; see Hardin and Walston, 2004; PubMed for detailed discussion). (D) A schematic of leading cell migration during ventral enclosure, corresponding to Fig. 4B. (E) An embryo expressing a dlg-1::gfp transcriptional reporter (Firestein and Rongo, 2001; PubMed) visualized using spinning disk confocal microscopy (courtesy of M. Sheffield; see also Movie 7). Migrating leading cells extend long protrusions in the direction of migration (arrows); in time-lapse sequences, pocket cells can also be observed with shorter protrusions. Scale bar = 5 microns. (F) A schematic showing the relationship between leading cell protrusions and the underlying cellular substratum of neuroblasts (anterior at the top). Protrusions make contact via cadherin/catenin complex proteins on their surfaces (green). F-actin is abundant within the cytoplasm of extending protrusions and may connect to cadherin complex proteins ("filopodial priming"; see Raich et al., 1999; PubMed).
Figure 6. Requirements for cadherin-based adhesion during morphogenesis [PDF]
The cadherin/catenin complex and associated proteins are required for ventral enclosure and elongation. Left column (A-C), Nomarski images; right column (D-F), phalloidin staining of F-actin imaged using confocal microscopy. (A) Wild-type embryo at approximately the three-fold stage. (B) A hmp-1(zu278) homozygote from a heterozygous mother, displaying the Hmp (Humpback) phenotype. Prominent dorsal bulges in the epidermis are visible (arrows). (C) An embryo from a hmp-1(zu278) germline mosaic mother, which therefore lacks both maternal and zygotic HMP-1, displaying the more severe Hmr (Hammerhead) phenotype. Enclosure failure in the anterior results in rupture of the embryo and ejection of internal cells through the opening in the epidermis. For a multiphoton movie of such enclosure failure, see Movie 8. (D) Phalloidin staining of a wild-type embryo at approximately the three-fold stage of elongation. Circumferential filament bundles (CFBs) are clearly visible; the bright staining is one of the four muscle quadrants. (E) A hmp-1(zu278) homozygote stained with phalloidin. CFBs have detached from cell-cell junctions (arrows). (F) A vab-9(e2016) embryo. CFBs aggregate into abnormally large bundles (arrow). Images in (A, B, D, E) courtesy of M. Costa; (C) courtesy of W. B. Raich, from (Costa et al., 1998; PubMed); used by permission of Rockefeller Press. (F) is courtesy of J. Simske, from (Simske et al., 2003; PubMed); used by permission of Nature Publishing Group. Scale bar = 5 microns.
Figure 7. Embryonic elongation [PDF]
Nomarski images (A-C, left column; lateral view; see also Movie 9) and corresponding DLG-1::GFP images, highlighting epidermal cell boundaries (middle column; D-F), of embryonic elongation. (G, H) Schematic representations of a section of the trunk epidermis corresponding to the boxed regions in panels D and E, respectively. (G, H) are adapted from (Chin-Sang and Chisholm, 2000; PubMed), with permission of Elsevier Publishing. Ventral cells are shown in pink, seam cells in yellow, and dorsal cells in teal. Contractile forces are produced predominantly by seam epidermal cells (arrows pointing toward one another), and transmitted to dorsal and ventral cells via adherens junctions (black ovals), which anchor circumferential actin filaments bundles (CFBs) in dorsal and ventral cells (black lines and arrows within dorsal and ventral cells). CFBs are thought to transmit and distribute the forces of contraction evenly throughout the epidermis. In seam cells, the activity of LET-502/Rho kinase is high, leading to myosin-dependent contraction; conversely, MEL-11/myosin phosphatase activity is low in seam cells and presumed high in non-seam cells. (I) Phalloidin staining of a wild-type embryo at approximately the two-fold stage of elongation (dorsal view). CFBs are clearly visible. The bright staining is one of the four muscle quadrants (image courtesy of E. William-Masson). Scale bar = 5 microns.
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Movies
Movie 1. Ventral cleft closure in a wild-type embryo visualized using Nomarski microscopy.
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Anterior to the left, ventral view. Ventrolateral neuroblasts move towards the ventral midline and close the cleft from posterior to anterior. Frames were acquired at 60 sec intervals. Original movie footage courtesy of H.N. Cinar and D. Patel (unpublished).
Movie 2. Dorsal intercalation in a wild-type embryo visualized using Nomarski microscopy (Williams-Masson et al., 1998; PubMed).
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Cells become wedge-shaped as they begin intercalation, and eventually adopt a ladder-like arrangement straddling the dorsal midline. Anterior to the left, dorsal view. Frames were acquired at 30 sec intervals. Original movie footage courtesy of E. Williams-Masson.
Movie 3. Dorsal intercalation in a wild-type embryo visualized using a dlg-1::gfp translational fusion (Koeppen et al., 2001; PubMed).
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Frames were acquired at 5 min intervals. Original multiphoton movie footage courtesy of M. Koeppen.
Movie 4. Visualizing protrusive activity in wild-type embryos during dorsal intercalation using an lbp-1::gfp transcriptional reporter.
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Frames were acquired at 5 min intervals. To suppress cell fusion, the strain was made homozygous for the eff-1(oj55) allele (Heid et al., 2001; PubMed). Note the basolateral protrusions extended by the dorsal hypodermal cells as they intercalate. The bright ovals are the nuclei of dorsal hypodermal cells, which undergo a contralateral migration as intercalation proceeds. Anterior is to the left. Original spinning disc confocal movie footage courtesy of R. King.
Movie 5. Ventral enclosure in a wild-type embryo visualized using Nomarski microscopy (Williams-Masson et al., 1997; PubMed).
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Anterior to the left, ventral view. Frames were acquired at 2 min intervals. Original movie footage courtesy of M. Koeppen.
Movie 6. Ventral enclosure in a wild-type embryo visualized using a dlg-1::gfp translational fusion (Koeppen et al., 2001; PubMed).
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Frames were acquired at 5 min intervals. Original multiphoton movie footage courtesy of M. Koeppen.
Movie 7. Ventral enclosure in a wild-type embryo visualized using a dlg-1p::gfp transcriptional reporter.
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Frames were acquired at 2 min intervals. For details on the construct used, see Firestein and Rongo (2001; PubMed). Original spinning disc confocal movie footage courtesy of M. Sheffield.
Movie 8. Loss of cadherin function specifically disrupts leading cell adhesion.
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In this example, a hmr-1(RNAi) embryo expressing ajm-1::gfp ruptures during ventral enclosure (Raich et al., 1999; PubMed). As ventral epidermal cells approach the ventral midline, the pocket cells migrate in advance of the leading cells. Epidermal cells that fail to form junctions at the ventral midline retract towards the dorsal surface of the embryo. Anterior to the left, ventral view. Frames were acquired at 5 min intervals.
Movie 9. Elongation in a wild-type embryo imaged using Nomarski microscopy.
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Frames were collected at 60 sec intervals. Anterior to the left, lateral view. Original movie footage courtesy of E. Cox (Pettitt et al., 2003; PubMed).
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