Morphogenesis of the somatic musculature in Drosophila melanogaster

Abstract

In Drosophila melanogaster, the somatic muscle system is first formed during embryogenesis, giving rise to the larval musculature. Later during metamorphosis, this system is destroyed and replaced by an entirely new set of muscles in the adult fly. Proper formation of the larval and adult muscles is critical for basic survival functions such as hatching and crawling (in the larva), walking and flying (in the adult), and feeding (at both larval and adult stages). Myogenesis, from mononucleated muscle precursor cells to multinucleated functional muscles, is driven by a number of cellular processes that have begun to be mechanistically defined. Once the mesodermal cells destined for the myogenic lineage have been specified, individual myoblasts fuse together iteratively to form syncytial myofibers. Combining cytoplasmic contents demands a level of intracellular reorganization that, most notably, leads to redistribution of the myonuclei to maximize internuclear distance. Signaling from extending myofibers induces terminal tendon cell differentiation in the ectoderm, which results in secure muscle-tendon attachments that are critical for muscle contraction. Simultaneously, muscles become innervated and undergo sarcomerogenesis to establish the contractile apparatus that will facilitate movement. The cellular mechanisms governing these morphogenetic events share numerous parallels to mammalian development, and the basic unit of all muscle, the myofiber, is conserved from flies to mammals. Thus, studies of Drosophila myogenesis and comparisons to muscle development in other systems highlight conserved regulatory programs of biomedical relevance to general muscle biology and studies of muscle disease.

FIGURE 1

Drosophila larval body wall muscles. (a) Stage L3 Drosophila larva expressing tropomyosin (TM1)-GFP (grayscale). Anterior, left; Dorsal, up. (Left) Whole larva. Yellow box highlights one hemisegment shown at higher magnification to the right. Scale bar, 100 μm. (Right) One hemisegment. Scale bar, 25 μm. (b) Schematic diagram of the 30 distinct muscles within each hemisegment. External view, as seen in A. (c) Internal view, internal-most and external-most muscles in B reversed. Muscle identities are as follows: Muscle position (D, dorsal; V, ventral; L, lateral) followed by orientation (A, acute; L, longitudinal; O, oblique; T, transverse); SBM, segment border muscle. Former muscle numbering system shown in parentheses.

FIGURE 2

Overview of myoblast fusion. (a) A founder cell (FC, purple) and a fusion-competent myoblast (FCM, gray) recognize and adhere to each other via cell type-specific Immunoglobin (Ig) domain-containing transmembrane proteins (yellow/orange, blue/green). Recognition between FCs and FCMs is thought to promote clustering of Ig domain-containing proteins at the cell surface to facilitate adhesion. (b) Cytoskeletal rearrangements (actin, red) in both cells lead to pore formation in the cell membranes. (c) Cytoplasmic mixing occurs upon fusion. (d) The previously naïve (white) nucleus of the FCM adopts the transcriptional profile of the FC nucleus (pink), aiding in the development of a particular muscle fate. (e) The FC/growing myofiber recycles Ig domain-containing transmembrane proteins to the cell surface to prepare for additional rounds of myoblast fusion.

FIGURE 3

Intracellular signaling cascades necessary for cytoskeletal remodeling and myoblast fusion. The extracellular domains of cell type-specific Immunoglobin (Ig) domain-containing proteins (blue) on both the founder cell (FC, left) and the fusion-competent myoblast (FCM, right) interact to adhere to each other. PI(4,5)P2 (PIP₂, yellow) becomes enriched in the membranes at the contact site, and via adaptor proteins (orange), the intracellular domains of the Ig domain-containing proteins induce downstream signaling events (green) that lead to activation of the Arp2/3 complex, which mediates branched actin polymerization. These activities facilitate the formation of a dense actin focus (red) in the FCM and a thin sheath of actin (red stripe) in the FC that apposes the focus. Continued actin remodeling, mediated by proteins required to dissolve the actin focus (white), induces actin filaments to protrude into the FC, leading to pore formation in the cell membranes and fusion of the two cells. Duf, Dumbfounded; Rst, Roughest; Sns, Sticks-n-stones; Hbs, Hibris; Crk, Crack; Dock, Dreadlock; Rols, Rolling pebbles; Blow, Blown fuse; Sltr/dWIP, Solitary/Drosophila WASp-interacting protein; WASp, Wiskott-Aldrich Syndrome protein; Mbc, Myoblast city. Not shown is Pak1 and Pak3 activity downstream of Rac, which regulates further actin rearrangements for invasive podosome formation.

FIGURE 4

Overview of myonuclear positioning. (a) In multinucleated myofibers (green), microtubules (black dotted lines) originate at the nuclear periphery (minus (−) ends) and extend outwards (plus (+) ends). Following myoblast fusion, Kinesin (pink) and Ensconsin (Ens, gray triangle) work together to separate adjacent myonuclei (white) by crosslinking microtubules and sliding them past one another. Kinesin forces also extend the leading edge of moving nuclei (white teardrop) while Dynein activity (dark blue) at the rear of the nucleus allows forward motion to complete a translocation step. Activation of the JNK signaling pathway (red, curved black arrows) phosphorylates (yellow) Sunday Driver (Syd, light blue rod), which facilitates Kinesin-dependent relocation of Dynein to the cell periphery. Rapsynoid (Raps, purple) anchors Dynein to the cell cortex, and cortical Dynein activity pulls microtubules and the attached nuclei closer to the muscle end. During these movements, CLIP-190 (orange) is necessary to support the integrity of the microtubule network by ensuring proper contact with the cell cortex. Straight thin black arrows denote the direction of motor protein activity. Large silver arrows highlight the direction of net force exerted on nuclei. (b) Within the perinuclear space (PS) of the nuclear envelope, SUN proteins (light green) in the inner nuclear membrane (INM) interact with KASH proteins (black, brown) in the outer nuclear membrane (ONM) to link the nucleoskeleton (light blue, Lamin) and the cytoskeleton (red, Actin) to anchor nuclei in place. It has also been proposed that SUN and KASH proteins may directly link the nucleus to the motor proteins, Kinesin (pink) and Dynein (blue).

FIGURE 5

Overview of muscle attachment to tendon cells. (a) Fields of tendon precursor cells are specified by Wingless (Wg) and Hedgehog (Hh) signaling gradients in the overlying ectoderm. All equally competent cells (blue) express StripeB (SrB, red), which auto-regulates its own expression as well as the expression of How(L), a SrB inhibitor. A balance of SrB and How(L) maintains a low level of SrB expression in all tendon precursor cells, preventing further differentiation. Tendon precursor cells secret Slit (orange), a chemoattractant for extending myofibers. The transmembrane receptor, Roundabout (Robo), on the surface of the myofiber responds to Slit secretion, and the myofiber extends filopodia toward the field of tendon precursor cells. (b) Upon Robo activation, the myofiber secretes Vein (purple), an Egfr ligand that binds to DERs on one tendon precursor cell. Interactions between Robo and Leucine-rich tendon-specific protein (Lrt), a transmembrane receptor expressed by the tendon precursor cell, solidify the attachment to the extending muscle end. (c) DER-mediated Ras signaling occurs in only the selected tendon precursor cell bound to the muscle. This leads to dedifferentiation of neighboring cells (faded blue) and upregulation of StripeA (SrA) and How(S) in the selected cell. How(S) stabilizes SrA transcripts, and SrA mediates terminal differentiation into a mature tendon cell (dark blue). A preliminary attachment between the tendon cell and the myofiber mediates the secretion of extracellular matrix proteins from both cell types (Thrombospondin (Tsp), Laminin (Lam), and Tiggrin, colored respectively), which contribute to the formation of the myotendinous junction (MTJ, gray). (d) Both cells express βPS (purple) Integrin, while the tendon cell specifically expresses αPS1 (pink) and the myofiber expresses αPS2 (orange) Integrin adhesion molecules. These transmembrane proteins heterodimerize on the surfaces of their respective cell types and bind to ECM proteins (black). α-β Integrin dimers also bind to the intracellular cytoskeletons of each cell type, forming stable connections between the tendon cell, the MTJ, and the myofiber, which can withstand the contractive forces of the mature muscle. Kon, Kon-tiki; Drl, Derailed (additional transmembrane proteins for targeting muscles to tendon cells). Dei, Delilah; β1-tub, β1-tubulin (markers of terminal tendon cell differentiation). Slow, Slowdown (secreted by tendon cell to ensure proper temporal regulation of MTJ formation). (Modified with permission from Ref . Copyright 2010 Company of Biologists)

FIGURE 6

Diagram of sarcomere structure and formation. (a) Sarcomeres are composed of intercalated actin thin filaments (red) and myosin thick filaments (yellow) that slide past one another to compress the muscle in the longitudinal direction. Myosin filaments are held together by Myomesin (light blue) and Obscurrin (gray) at a structure known as the M-line. Actin filaments are held together by α-actinin (white) and Zasp (magenta) at a structure called the Z-line (or Z-disk). The distance between two Z-lines constitutes one sarcomere. Zasp in the Z-line also connects sarcomeres to the cell membrane by interacting with Integrin adhesion complexes (purple/orange). D-Titin (dark blue, black) is a component of both of both the Z-line and the M-line, connecting the two structures to modulate sarcomere length during contraction. (b) One proposed model of sarcomerogenesis in which individual components are sequentially added. (c) Alternative model of sarcomerogenesis in which lattices of actin or myosin filaments are formed separately and subsequently intercalated together.^,

FIGURE 7

Myoblast fusion in the adult Drosophila musculature. (a) Three muscles (blue) in the second thoracic hemisegments (T2) persist into metamorphosis and serve as scaffolds for the development of the dorsal longitudinal muscles (DLMs). Myoblasts are associated with the wing imaginal disk. (0 h after puparium formation, APF) (b) A wave of histolysis destroys the other larval muscles early in pupal development. The persistent muscles are surrounded by myoblasts (6–8 h APF). (c) The myoblasts begin to fuse and the larval muscles split to give rise to the six DLM fibers (14–18 h APF). (d) The six DLMs grow to fill the thoracic space and are one-third their adult size at 36 h APF. (Reprinted with permission from Ref . Copyright 1996 Company of Biologists)