Arrowhead

DEVELOPMENTAL BIOLOGY

Embryonic

Awh transcription is first detected in cells located at the position of the neuroblasts in stage 9 embryos, when neuroblasts begin to segregate. Loss-of-function mutations in the neurogenic gene Delta result in hypertrophy of the nervous system. Embryos carrying a loss-of-function mutation in Delta have about twice as many Awh-expressing cells as wild-type embryos, suggesting that the Awh expressing cells are neuroblasts. Awh is also transcribed at stage 11 in cells in the procephalon that may correspond to cells of the supraesophageal ganglia (brain). During embryogenesis, Arrowhead is expressed in each abdominal segment and in the labial segment, consistent with its role in establishing the proper numbers of abdominal histoblasts and salivary gland imaginal ring cells (Curtiss, 1995). AWH mRNA expression during stage 10 of embryogenesis, the extended germ band stage, is found in bilateral stripes in the three thoracic segments and in all 10 abdominal segments. As the gnathal buds become apparent during stage 11, the Awh transcript is also detected in a stripe and a few additional cells at the center of the labial bud. At stage 14, Awh is not expressed in cells in the region of the thoracic segments in which the developing prothoracic, wing, haltere, and leg discs are located. escargot, a gene involved in imaginal disc cell cycle regulation. is expressed in the developing discs from which Awh expression is excluded. Late in embryonic development, expression is refined to the abdominal histoblasts and salivary gland imaginal ring cells themselves (Curtiss, 1997).

Larval

By the late third instar, most excorporate imaginal cells have completed an intense period of proliferation and are undergoing the final stages of organization before the dramatic events of metamorphosis begin. The abdominal histoblasts, which have remain mitotically quiescent throughout the larval stages, are about to enter a period of especially intense proliferation, migration, and differentiation to generate the adult abdominal epithelium. At this stage, the Awh transcript is expressed in cells on both sides of the border separating the larval salivary gland from the salivary gland imaginal ring. Awh is also transcribed in specific areas of wing, leg and eye-antennal discs. Awh is transcribed on the medial edge of the wing disc, in a stripe on the anterolateral edge of the leg disc, which extends in a spiral toward the center of the disc, and in cells extending down the ventral edge of the eye-antennal disc, from approximately the middle of the antennal portion, to approximately the middle of the eye portion. No defects in the structures derived from these imaginal discs have been detected in Awh mutants (Curtiss, 1997).

Effects of mutation or deletion

Metamorphosis in Drosophila melanogaster requires synchronization of numerous developmental events that occur in isolated imaginal precursor tissues. The imaginal primordia are established during embryonic stages and are quiescent for much of larval life. The Arrowhead gene is necessary for the establishment of proper numbers of cells within a subset of imaginal precursor tissues. Loss-of-function mutations in Arrowhead reduce the number of abdominal histoblasts and salivary gland imaginal ring cells before the proliferative stages of their development. The number of abdominal histoblasts in mutant animals is approximately half that of wild-type, as might result from failure of a single early division of these cells. A neomorphic Arrowhead allele, Awh1, results in the specific loss of the retinal precursors by the early third instar, before they have begun to differentiate. Since Arrowhead mutations affect only subsets of imaginal tissue, there must be distinctions in the developmental regulation of different imaginal precursors. Arrowhead may be part of a regulatory pathway responsible for establishing the proper number of abdominal histoblasts and salivary gland imaginal ring cells. The neomorphic Arrowhead allele, which may cause misexpression of the Arrowhead gene in the eye-antenna imaginal disc, interferes with the establishment or proliferation of retinal precursor cells (Curtiss, 1995).

The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. The transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. Two LIM-homeodomain proteins, Arrowhead and Lim1, are shown to be expressed in the region of the eye-antennal disc affected in Chip mutants, and both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue (Roignant, 2009).

Regionalization of the eye-antennal disc is a progressive process in which selector genes and signaling pathways specify the fates of different head structures. Clones of eye-antennal disc cells induced during the second larval instar can contribute to multiple organs, indicating that these cells retain developmental plasticity at this stage. The anteroposterior boundary of the wing disc is established much earlier; expression of the selector gene engrailed (en) specifically in the posterior cells during embryogenesis generates an affinity border that keeps the two compartments clonally separated. By contrast, the eye selector gene ey is uniformly expressed throughout the early eye-antennal disc, and only retracts to the eye field in the second instar. It was initially proposed that localized Notch signaling controls this retraction, as expression of dominant-negative forms of Notch in the eye disc abolishes ey expression and leads to antennal duplications. However, a later study demonstrated that loss of Notch function does not affect ey expression directly, but reduces cell proliferation in the retinal field, preventing the initiation of eya expression. This study shows that Chip and Lim1 are both necessary to repress ey expression in the anterior of the antennal disc. Additional factors probably help to restrict ey expression to the eye disc, because ey expression does not extend throughout the normal Lim1 expression domain in Lim1 or Chip mutant clones in the antennal disc (Roignant, 2009).

Since Lim1 mutant clones always misexpress Ey, but rarely misexpress Eya and never differentiate ectopic photoreceptors, additional proteins must interact with Chip to repress retinal differentiation. Awh is a good candidate because it is expressed at the ventral margin of the eye-antennal disc, its misexpression in the retina represses photoreceptor differentiation in a Chip-dependent manner, and loss of both Lim1 and Awh leads to ectopic photoreceptor differentiation in the ventral eye-antennal disc. Since ectopic photoreceptors differentiate only in the absence of both Lim1 and Awh, whereas Ey expansion is observed in Lim1 single mutants, Awh must control the expression of target genes other than ey. It may negatively regulate other genes involved in retinal determination, such as eya, or positively regulate genes important for head capsule development, such as Deformed and odd-paired (Roignant, 2009).

Like Chip, Hth is required to prevent retinal differentiation at the ventral eye-antennal disc boundary. Investigation of the relationship between Chip and Hth indicates that Chip is not required for Hth expression or activity. The ability of Hth to repress photoreceptor differentiation in Chip mutant clones rules out the possibility that Chip acts as a co-factor for Hth or an essential downstream mediator of its effects. The normal expression of Hth and its target gene wg in Chip mutant clones also make it unlikely that Chip controls the expression of Hth or its co-factor Exd. However, the possibility that Hth and Chip act in parallel poses the paradox that misexpressed Hth is sufficient to repress photoreceptor development in the eye field in the absence of Chip, but endogenous Hth is insufficient to do so in the head field. It is possible that Hth expression levels in the head field early in development are too low to repress the eye fate in the absence of Chip. Consistent with this hypothesis, it was found that overexpression of Hth in Chip mutant cells prevents ectopic photoreceptor differentiation. Similarly, overexpression of Awh or Lim1 prevents ectopic photoreceptor differentiation in hth mutant cells, suggesting that endogenous levels of these LIM-HD proteins are not sufficient to compensate for the absence of Hth. The two classes of transcription factors may normally act on different sets of target genes, but show some cross-regulatory ability when overexpressed (Roignant, 2009).

The boundary between the eye and the dorsal head appears to be established differently from the boundary in the ventral region. The LIM-HD gene tup is expressed at the dorsal eye-antennal disc boundary, in a pattern resembling the mirror image of the Awh pattern, and is capable of repressing photoreceptor development in a Chip-dependent manner. However, loss of Chip in this region does not lead to ectopic eye formation, although it can cause overgrowth and mispatterning of the head. In the absence of Chip, the GATA transcription factor Pannier (Pnr) and its target gene wg may be sufficient to maintain dorsal head fate. The ventral margin of the eye-antennal disc may be particularly susceptible to ectopic photoreceptor differentiation because of the high level of Dpp signaling there. A 5' enhancer element has been shown to direct dpp expression specifically in the ventral marginal peripodial epithelium of the eye-antennal disc. The ability of Dpp and Ey to synergize to drive retinal differentiation therefore makes it critical to repress Ey in this region, which is fated to form head capsule (Roignant, 2009).

In addition, this domain of Dpp overlaps with Wg present at the anterior lateral margin of the eye disc; the combination of these two growth factors induces proximodistal growth of the leg. One function of Chip and its partner proteins might thus be to repress the outgrowth that would otherwise be triggered by the combination of Dpp and Wg. Unlike growth of the wild-type eye disc, growth of Chip mutant regions appears to be Notch-independent, as they do not contain a fng expression boundary and do not show activation of the Notch target genes E(spl)mβ or eyg. Notch has been thought to trigger growth by inducing the expression of the JAK/STAT ligand Unpaired (Upd); however, a recent report describes an earlier function for Upd upstream of Notch, raising the possibility that upd expression is activated independently of Notch in Chip mutant clones. As hth mutant clones, or clones lacking the Odd skipped family member Bowl, frequently show ectopic ventral photoreceptor differentiation but rarely induce outgrowths like those seen in Chip mutants, the functions of Chip in growth and differentiation are likely to be separable (Roignant, 2009).

LIM-HD proteins also set developmental boundaries in other imaginal discs, acting in concert with other classes of transcription factors. In the wing disc, Tup specifies the notum in collaboration with homeodomain transcription factors of the Iroquois complex, and Ap specifies the dorsal compartment. Ap interacts with the homeodomain protein Bar and Lim1 with Aristaless to establish specific tarsal segments within the leg disc. LIM-HD proteins have also been implicated in vertebrate eye development, although those that have been studied appear to play positive roles. The Ap homologue Lhx2 is expressed within the mouse retinal field at the neural plate stage, and contributes to the expression of Pax6, Six3 and Rx. Lmx1b, the homologue of CG32105, is required for the development of anterior eye structures such as the cornea and iris, and is mutated in human patients with nail-patella syndrome, often characterized by glaucoma. Within the retina, loss of Lim1 results in mispositioning of horizontal cells within the amacrine cell laye. Drosophila Lim3 shows photoreceptor-specific expression, and might therefore have a positive function in eye development (Roignant, 2009).

In the central nervous system, LIM-HD proteins act combinatorially to specify different neuronal cell fates. In both Drosophila and vertebrates, combinations of Islet and Lhx3/4/Lim3 proteins regulate motoneuron specification and pathfinding. The ability of Chip to interact with LIM-HD proteins and other transcription factors as well as to dimerize enables it to form heteromeric transcription factor complexes. In the wing disc, the active complex is a tetramer containing two subunits each of Chip and Ap, whereas in motoneuron development the Chip homologue NLI can form either a tetramer with Lhx3 or a hexamer containing both Isl1 and Lhx3. The finding that Lim1 and Awh act redundantly to prevent eye development in the ventral head primordium, whereas Chip is absolutely required, seems most consistent with regulation of distinct subsets of target genes by independent Chip-Awh and Chip-Lim1 complexes; however, a contribution from a complex containing all three proteins, or even additional transcription factors, cannot be ruled out. The role of the Chip co-factor may be to coordinate multiple transcriptional regulatory complexes to restrict developmental fates within the eye-antennal imaginal disc, allowing it to give rise to the head cuticle as well as distinct external sensory structures (Roignant, 2009).

Curtiss, J. and Heilig, J. S. (1995). Establishment of Drosophila imaginal precursor cells is controlled by the Arrowhead gene. Development 121(11): 3819-3828. PubMed Citation: 8582291

Curtiss, J. and Heilig, J. S. (1997). Arrowhead encodes a LIM homeodomain protein that distinguishes subsets of Drosophila imaginal cells. Dev. Biol. 190(1): 129-141. PubMed Citation: 9331336

Kylsten, P and Saint, R. (1997). Imaginal tissues of Drosophila melanogaster exhibit different modes of cell proliferation control. Dev. Biol. 192: 509-522. PubMed Citation: 9441685

Roignant, J. Y., Legent, K., Janody, F. and Treisman, J. E. (2010). The transcriptional co-factor Chip acts with LIM-homeodomain proteins to set the boundary of the eye field in Drosophila. Development 137(2): 273-81. PubMed Citation: 20040493

Sagasti, A., Hobert, O., Troemel, E. R., Ruvkun, G. and Bargmann, C. I. (1999). Alternative olfactory neuron fates are specified by the LIM homeobox gene lim-4. Genes Dev. 13(14): 1794-806. 10421632

date revised: 10 April 2010  

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