BIOLOGICAL OVERVIEW

How do muscle cells made in the mesoderm become attached to overlying ectodermal cells in a precise geometric array? Before dealing with the role of stripe in this process, a look at what goes on prior to stripe's involvement is in order.

During germ band shortening, somatic mesodermal cells migrate to their final position in each segment and begin to fuse to founder cells, forming myotubules. Nascent myotubules elongate and migrate toward ectodermal segmental groove cells, the sites of their future attachment. How do these myotubules know where to attach?

The patterning of the epidermis influences the patterning of the mesoderm. It is said that the epidermis provides positional information which controls the oriented migration of myotubes. There is a segmental variation in the expression of twist in the mesoderm that mirrors the segmentation of the ectoderm. High levels of mesodermally located twist are expressed adjacent to ectodermal segmental borders, sites where engrailed is expressed in posterior compartments and wingless (wg) is expressed in adjacent anterior compartments. The level of mesodermal twist sharply declines beneath the epidermal cells expressing engrailed, but gradually expression increases to reach a peak under the cells expressing wg and dpp in the next segment. It is these cells, underneath the cells expressing wg and dpp at the segmental border that are fated to attach to the segmental border. Along with other signals, the chemical signals (DPP and WG) emitted by the segmental border cells and their neighbors communicate information about the site of attachment to the underlying mesodermal cells. (Dunin-Borkowski, 1995).

The stripe gene is involved in the process of epidermal cell recognition of myotubules. stripe is expressed in the epidermis, and mutants exhibit a disruption in myotubule patterning. Stripe is a member of the early growth response family of zinc finger proteins that also includes Krox20, a protein involved in mammalian hindbrain and neural crest development (Lee, 1995).

Another protein expressed in segmental border cells is Groovin. This appears to be the contact protein for myotubules. Groovin immunoreactivity is abolished in both wingless and naked mutant embryos. patched mutants show a duplication in groovin expressing cells and a reduced disruption of muscle pattern. groovin expression is independent of stripe, but apparently their coordinated activity is involved in epidermal interation with mesoderm (Volk, 1994).

Further analysis of Stripe and Groovin reveals the existence of reciprocal signaling between Drosophila epidermal muscle attachment cells and their corresponding muscles. stripe is both necessary and sufficient to initiate the developmental program of epidermal muscle attachment (EMA or segmental border) cells. In stripe mutant embryos, these cells do not differentiate correctly. Ectopic expression of Stripe in various epidermal cells transforms these cells into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific markers. These markers include goovin, delilah, and beta1 tubulin. The EMA-specific genes induced by Stripe can be divided into two groups: genes that follow Stripe ectopic expression in all embryonic stages or genes that can not be detected in early (stage 10-11) or late (older than stage 14) developmental stages, but only in intervening stages. groovin and alien represent the first group (all stages) and delilah and beta1 tubulin represent the second group (intervening stages) and are expressed only in stages 12-14 (Becker, 1997).

The ectopic epidermal muscle attachment cells are capable of attracting somatic myotubes from a limited distance, providing that the myotube has not yet been attached to or been influenced by a closer wild-type attachment cell. Analysis of the relationships between muscle binding and differentiation of the epidermal muscle attachment cell has been performed in mutant embryos in which either loss-of-muscles or ectopic muscles were induced. This analysis indicates that although the initial expression of epidermal muscle-attachment cell-specific genes including stripe and groovin is muscle independent, continuous gene expression is maintained only in epidermal muscle attachment cells that are connected to muscles. Normally, the expression of beta1 tubulin is restricted to the final stage of gene expression in tendon-like cells, supporting the idea of a distinct mechanism regulating gene expression within the tendon cells as a result of muscle interactions. These results suggest that the binding of a somatic muscle to an epidermal muscle attachment cell triggers a signal affecting gene expression in the attachment cell. Thus there exists a reciprocal signaling mechanism between the approaching muscles and the epidermal muscle attachment cells. First the epidermal muscle attachment cells signal the myotubes and induce myotube attraction and adhesion to their target cells. Following this binding, the muscle cells send a reciprocal signal to the epidermal muscle attachment cells inducing their terminal differentiation into tendon-like cells (Becker, 1997).

The muscle pattern of the Drosophila abdomen depends on a subdivision of the anterior compartment of each segment

In the past, segments were defined by landmarks such as muscle attachments, notably by Snodgrass, the king of insect anatomists. This study shows how an objective definition of a segment, based on developmental compartments, can help explain the dorsal abdomen of adult Drosophila. The anterior (A) compartment of each segment is subdivided into two domains of cells, each responding differently to Hedgehog. The anterior of these domains is non-neurogenic and clones lacking Notch develop normally; this domain can express stripe and form muscle attachments. The posterior domain is neurogenic and clones lacking Notch do not form cuticle; this domain is unable to express stripe or form muscle attachments. The posterior (P) compartment does not form muscle attachments. In vivo films indicate that early in the pupa the anterior domain of the A compartment expresses stripe in a narrowing zone that attracts the extending myotubes and resolves into the attachment sites for the dorsal abdominal muscles. The tendon cells were mapped precisely, and it was show that all are confined to the anterior domain of A. It follows that the dorsal abdominal muscles are intersegmental, spanning from one anterior domain to the next. This view is tested and supported by clones that change cell identity or express stripe ectopically. It seems that growing myotubes originate in posterior A and extend forwards and backwards until they encounter and attach to anterior A cells. The dorsal adult muscles are polarised in the anteroposterior axis: the hypothesis is disproved that muscle orientation depends on genes that define planar cell polarity in the epidermis (Krzemien, 2012).

All the dorsal muscles of a typical segment of the abdomen, including the larval persistent muscles and the MOL, attach only to a small subregion: part of the anterior (A) compartment of each adult segment. This discussion first considers the mechanisms responsible for the muscle pattern and then the general significance of what was found (Krzemien, 2012).

It appears that the pupal A compartment contains two distinct types of cells even before Hh arrives to pattern it. In an anterior domain of the A compartment, the cells develop normally without N and do not form bristles and, above a certain level, Hh specifies a1 cuticle. By contrast, when presumptive a3 cells of the posterior domain of the A compartment receive Hh, they now make a4-a6 cuticle as well as the appropriate types of bristles. Here, it was confirmed that epidermal cells of this posterior domain absolutely depend on N activity as N^- clones do not form cuticle in that territory and instead make clusters of neurons. More evidence is added for the two domains: the anterior domain appears to be competent to express sr and to form muscle attachments whereas the posterior domain does neither. Also, in the thorax of Drosophila, there is earlier evidence that sensory bristles and tendon cells cannot be both formed by the same progenitors. If sr is overexpressed in the thorax, bristle formation is inhibited. Reciprocally, expression of achaete-scute in the sr domain leads to impairment of the flight muscles. This antagonism between achaete-scute and sr is conserved in many dipteran groups. These findings provide a genetic correlate with the two domains of the A compartment (Krzemien, 2012).

In general, the pattern of attachment of muscles is related to the patterned expression of sr in the ectoderm. In the embryo, as soon as the myotubes arrive nearby, they become attracted to the sr-expressing cells and contact them. This is crucial; only the epidermal cells that establish contact with a myotube can maintain a high level of sr expression and can mature properly. In the pupal abdomen, as only the anterior domain of the A compartment can express sr, one would expect muscle attachments to be limited to those cells, as was found. The P compartment is a special case; N^- clones form normally showing the cells to be non-neurogenic, consistent with an absence of bristles and sensilla in P compartments. However, muscle attachments do not form there either, perhaps because the action of en on cell identity abrogates the expression of sr (Krzemien, 2012).

Consistently, it was find that ectopic expression of either srA or srB bypasses the original positional identity, even P compartment cells will attach muscles if they express ectopic sr. In these circumstances, muscles may lose their normal anteroposterior orientation and bend to reach the clones expressing sr. This is more evidence that the orientation of the dorsal abdominal muscles is a response to an Sr-dependent signal and not a response to polarity information in the epidermis (Krzemien, 2012).

The pattern of muscle attachments therefore depends on information specified in the ectoderm, raising the question of how far the mesodermal founder cells are also programmed. In pupal myogenesis, the myoblasts and histoblasts develop in close association and the movies illustrate this in vivo suggesting that they interact. The most concrete evidence for some interaction between the ectoderm and the mesoderm comes from the 'muscle of Lawrence' (MOL). The MOL is longer than other adult muscles, extending further both at the front and at the back, even though both ends attach within the tendon-competent zones. The peculiar attachment sites and size of the MOL depend entirely on the intervention of a neuron that is active only in the fifth abdominal segment of males; without this intervention, these myotubes form the same attachment sites as the normal dorsal abdominal muscles (Krzemien, 2012).

This study presents some additional evidence that signals are sent from the epidermis to the developing myotubes. Clones in the epidermis with changed cell identities were induced in third instar larvae, before the formation of myotubes. These clones affected the growing myotubes: if the tendon-competent cells of the anterior domain of the A compartment were replaced by tendon-incompetent cells of the posterior domain, the muscles passed over the transformed cells to attach to anterior domain cells further away, suggesting that a signal emanating from these tendon-competent (sr-expressing) cells had attracted the extending myotubes (Krzemien, 2012).

Films show that precise anteroposterior orientation of the developing myotubes is maintained from the beginning. How is this achieved? One possibility is that polarity information originating in the epidermis might orient the muscle migration but this is shown to be not correct, at least with regard to the PCP genes. The films also show that, just as the myotubes are forming, a band of cells in the anterior region of each A compartment expresses sr. Probably, as in the embryo, these sr-expressing cells of the pupa attract and orient the growing myotubes. Later still, sr expression is increased at the actual sites of attachment, presumably as a result of short-range interaction between the specified myoblasts and the epidermal cells; again, as in the embryo (Krzemien, 2012).

For many decades, the definition of a segment was contentious -- scientists argued using anatomical criteria about the homology of parts of one species with similar parts of another. Most had been content with Snodgrass' perspective that the integumentary plates of harder cuticle (tergites in the dorsal abdomen of Drosophila) form the essential segments and these are separated by more flexible cuticle he called the intersegmental membranes (Snodgrass, 1935). Muscle attachments were traditionally used as landmarks for the borders of segments, particularly in juvenile and primitive soft-bodied insects. Using these criteria, it was concluded that most of the longitudinal muscles were 'intrasegmental'. But, in adults or in more advanced arthropods, Snodgrass noted that most muscles spanned from one integumentary plate to the next, and he therefore described those muscles as 'intersegmental'. He concluded that the muscle attachments could shift during development and evolution. Following Brenner, one could call this rather vague picture an 'external description' of a segment. However, a different way of defining a metamere came from studies of cell lineage during development and from mapping domains of gene expression, and these methods redefined a segment as the sum of two precisely defined group of cells: the A and the P compartments. This definition amounted to an 'internal description' (Brenner, 1999; Krzemien, 2012 and references therein)

This study shows that the dorsal abdominal muscles of the abdomen can attach to a limited anterior domain of the A compartment at the front of one segment and, as they extend, they cross over the posterior domain of the A compartment as well as the P compartment to reach the anterior domain of the next segment. It follows that all the three types of muscles of the dorsal abdomen that were studied are intersegmental in extent. It is likely that these same fundamental subdivisions of the segments are conserved in other insects and, if so, would confine most muscle attachment sites to particular locations, thereby constraining the range of muscle patterns that can be built by evolution (Krzemien, 2012).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - at least 512

Structural Domains

There are three zinc finger domains in SR. The variable domain in SR, lacking the egr domain of EGR proteins, is a glutamine rich region implicated in transcriptional activation (Lee, 1995).

The Klumpfuss sequence is characterized by four zinc-finger domains of the C2H2 class; part of the N-terminus is negatively charged; the C-terminus, including the zinc-fingers, is positively charged. The N-terminal region contains glutamine-, histidine- and proline-rich stretches, features found in transcriptional activation and repression domains. There are three poly-alanine stretches, two in the N- and one in the C-terminal region. Such stretches are implicated in transcriptional repression. Three putative nuclear localization sites are found in the protein. Klu has a high degree of similarity to the zinc-finger domains of the members of the EGR family. As in WT-1, Klu contains an additional zinc finger (the first) that is only distantly related to those of the EGR proteins. Besides WT-1, Klu is the only other member of the family with four zinc fingers. There is complete conservation of the amino acids that contact the DNA-binding consensus sequence. So far only two other proteins are described as EGR-like in invertebrates, the Drosophila genes stripe and huckebein. Whereas the zinc fingers of Stripe have the characteristic amino acids for the binding of the DNA consensus sequence, in the zinc fingers of Huckebein, four out of the six amino acids are not conserved. However, the overall homology of the Huckebein finger region shows no greater homology to the EGR sequences than does the finger region of Klumpfuss. In comparison, the Stripe sequence shows a much more marked homology to the vertebrate EGR proteins than do the sequences of either Huckebein or Klu (Klein, 1997).

A subset of zinc finger transcription factors, including SR, contain amino acid sequences that resemble those of Krüppel. They are characterized by multiple zinc fingers containing the conserved sequence CX2CX3FX5LX2HX3H (X is any amino acid, and the cysteine and histidine residues are involved in the coordination of zinc) that are separated from each other by a highly conserved 7-amino acid inter-finger spacer, TGEKP(Y/F)X, often referred to as the H/C link.

Each 30-residue zinc finger motif folds to form an independent domain with a single zinc ion tetrahedrally coordinated beween an irregular, antiparallel, two stranded ß-sheet and a short alpha-helix. Each zinc finger of mouse Zif268 (which has three fingers) binds to DNA with the amino terminus of its helix angled down into the major groove. An important contact between the first of the two histidine zinc ligands and the phosphate backbone of the DNA contributes to fixing the orientation of the recognition helix. Although the two fingers of Drosophila Tramtrack interact with DNA in a way very similar to those of Zif268, there are important differences. Tramtrack has an additional amino-terminal ß-strand in the first of the three zinc fingers. The charge-relay zinc-histidine-phosphate contact of Zif268 is substituted by a tyrosine-phosphate contact. In addition, for TTK, the DNA is somewhat distorted with two 20 degree bends. This distortion is correlated with changes from the rather simple periodic pattern of amino base contacts seen in Zif268 and finger 2 of TTK (Klug, 1995 and references).

Zif268-like zinc fingers are generally regarded as independent DNA-binding modules that each specify three base pairs in adjacent, but discrete, subsites. However, crystallographic evidence suggests that a contact also can occur from the second helical position of one finger to the subsite of the preceding finger. For the three-finger DNA-binding domain of the protein Zif268, and a panel of variants, it has been shown that deleting the putative contact from finger 3 can affect the binding specificity for the 5' base in the adjoining triplet, which forms part of the binding site of finger 2. This finding demonstrates that Zif268-like zinc fingers can specify overlapping 4-bp subsites, and that sequence specificity at the boundary between subsites arises from synergy between adjacent fingers. This has important implications for the design and selection of zinc fingers with novel DNA binding specificities (Isalan, 1997).

date revised: 2 January 2023

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