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First published online 16 March 2005
doi: 10.1242/dev.01774

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The myogenic potency of HLH-1 reveals wide-spread developmental plasticity in early C. elegans embryos

Laboratory of Molecular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, NIH, Bethesda, MD 20892, USA

View larger version (121K): [in a new window]   Fig. 1. Ectopic HLH-1 activity converts most somatic blastomeres to a body wall muscle-like fate. (Left) Diagrams of wild-type embryos at the comma stage of development [adapted from Sulston et al. (Sulston et al., 1983)] with four different cell type patterns highlighted by nuclei or tissue area shading; body wall muscle (BWM), intestine (Int); hypodermis (Hyp) and pharynx (Phar). Adjacent to each diagram is a wild-type embryo stained with an antibody recognizing the corresponding tissue and imaged in a single focal plane. The far right column shows terminally arrested embryos in which HLH-1 activity was induced in early development by heat shock treatment. All embryos showed widespread myogenesis as indicated by MHC A staining in a representative focal plane from throughout the embryo (top) and a complete lack of staining for markers of the intestine, hypodermis and pharynx (below). All images in this and subsequent figures are a single focal plane deconvoluted using Hyguens software.

View larger version (87K): [in a new window]   Fig. 2. The myogenic activity of HLH-1 in embryos lacking normal cell fate specification. All embryos shown are transgenic for the heat shock promoter-driven hlh-1 transgene that is inactive in controls (left) or activated by a heat pulse (right). Top: embryos were depleted of PAL-1 and SKN-1 activity by RNAi, allowed to develop overnight, and stained for MHC A. Control embryos (A) have only a small amount of muscle due to zygotic PAL-1 activity that is not completely eliminated by RNAi (Edgar et al., 2001). In the presence of ectopic HLH-1 activity (B), most cells in pal-1, skn-1 double RNAi-treated embryos strongly express MHC A as shown by this focal plane representative of the entire embryo. Bottom: embryos were treated with mex-1, pop-1 double RNAi to convert a majority of anterior blastomeres to ELT-2-positive intestinal cells (C); a small area of MHC A-positive muscle remains in these embryos (D). Heat shock-induced HLH-1 activity, in combination with mex-1, pop-1 double RNAi treatment, results in a complete loss of ELT-2 staining (E) and strong MHC A staining (F) in most blastomeres.

View larger version (99K): [in a new window]   Fig. 3. Ectopic PAL-1 activates HLH-1 and the body wall muscle-like developmental program. The left panels (A) show a wild-type embryo with the body wall muscle cells at 3 hours post-fertilization highlighted by hlh-1::gfp expression (top); descendants of the C and D lineage are visible at this time. A comma stage embryo stained for MHC A (middle) shows the left side focal plane of body wall muscle quadrants; a corresponding diagram is shown below [adapted from Sulston et al. (Sulston et al., 1983)]. The center column of panels (B) show the response of muscle markers to heat shock-induced PAL-1 activity. Strong activation of hlh-1::gfp occurs within 3 hours of treatment in many cells (top). After overnight incubation, most cells in the embryo are MHC A positive (middle) when compared with the DAPI image of the same embryo (bottom). The right set of panels (C) show that the depletion of POP-1 activity by RNAi enhances the myogenic activity of ectopic PAL-1 (compare to B). Embryo staging and staining in C are the same as those in B, and images in these panels are representative of most focal planes of the embryo.

View larger version (11K): [in a new window]   Fig. 4. A schematic representation of the previously published (see text) interactions of several factors in the early embryo. PAL-1, which is negatively regulated by MEX-3, can specify a hypodermal or body wall muscle fate, depending on the level of POP-1. In the presence of high POP-1 (solid black letters), PAL-1 activity results in a hypodermal-like fate. POP-1 can be down-regulated (open lettering) via Wnt/MAP kinase signaling through the combined action of the factors WRM-1 and LIT-1 (see text). PAL-1 specifies a body wall muscle fate when POP-1 levels are low or absent.

View larger version (75K): [in a new window]   Fig. 5. High POP-1 blocks the myogenic activity, and promotes the hypodermal activity, of PAL-1. All embryos were treated with mex-3 RNAi that results in ectopic PAL-1 activity. After overnight incubation, embryos were assayed by antibody staining for the muscle marker MHC A and the hypodermal marker LIN-26. The upper pair of panels show representative focal planes of the robust myogenesis (MHC A) accompanying ectopic PAL-1 activity after co-depletion of POP-1 activity by RNAi; no LIN-26-positive cells are detected. In contrast, the lower pair of panels shows an embryo that was co-depleted of LIT-1 by RNAi to block the down-regulation of POP-1 by Wnt/MAP kinase signaling (see text). After overnight incubation, only a few cells (presumably from the D lineage) adopted a muscle-like fate whereas many cells adopted a hypodermal-like fate as seen in these images that are representative of focal planes throughout the embryo.

View larger version (65K): [in a new window]   Fig. 6. Wnt/MAP kinase signaling plays a role in MS-derived body wall muscle development. The left column (A) shows the comma stage pattern of body wall muscle (MHC A) and pharyngeal muscle (3NB12) in a wild-type embryo. Embryos depleted of MEX-1 activity (B) have a large excess in body wall and pharyngeal muscle due to cell fate transformations that causes a reiteration of MS-like lineages (Mello et al., 1992). The right column (C) shows the effects of blocking Wnt/MAP kinase signaling using wrm-1 RNAi, in an embryo co-depleted of mex-1. There is a severe reduction in the number of body wall muscle-like cells and concomitant increase in pharyngeal-like muscle cells demonstrating the role of Wnt/MAP kinase signaling in body wall formation within the MS lineage. Images in B and C are representative of focal planes throughout the embryo.

View larger version (60K): [in a new window]   Fig. 7. Ectopic PAL-1 activates the muscle-like program independent of HLH-1. The left panels (A) shows the MHC A pattern on the left side of a comma stage wild-type embryo (top) and a diagram of the body wall muscle nuclei (bottom; adapted from Sulston et al., 1983). In the center (B), treatment of wild-type embryos (top) with mex-3, pop-1 double RNAi results in widespread myogenesis as indicated by MHC A staining. The same treatment in a presumed hlh-1(cc450) mutant null genetic background (bottom: see text for details) results in similar amounts of ectopic muscle, although MHC A expression appears weaker. The right panels (C) show similar results in embryos treated with heat shock-induced PAL-1 and pop-1 RNAi in either a wild-type (top) or an hlh-1 RNAi (bottom) embryo. Images in B and C are representative of focal planes throughout the embryo.