Deformed

DEVELOPMENTAL BIOLOGY

Embryonic

In Drosophila, the Deformed and Sex combs reduced genes determine the developmental pathways followed by the most anterior metameric units. The spatial distributions of DFD RNA accumulates in parasegments 0 and 1, corresponding to maxillary and mandibular segments [Images], behind cells expressing labial (Mahaffey, 1989). SCR RNA accumulation shows a dynamic pattern spanning parasegments 2 and 3. The expression of Dfd and Scr seems to change from parasegmental to segmental during formation of the gnathal appendages. Both genes are transcribed during imaginal development: Dfd in a portion of the eye-antennal disc. (Martinez-Arias, 1987).

The spatial accumulation of the protein product of the proboscipedia locus overlaps partially with the distribution of the DFD and SCR proteins in the maxillary and labial segments, respectively. SCR and DFD have different dorsal and ventral patterns of accumulation. Dorsally, these proteins are expressed in segmental domains while, within the ventral region, a parasegmental register is observed (Mahaffey, 1989).

An interactive network of zinc-finger proteins contributes to regionalization of the Drosophila embryo and establishes the domain of Deformed protein function

During animal development, the HOM-C/HOX proteins direct axial patterning by regulating region-specific expression of downstream target genes. Though much is known about these pathways, significant questions remain regarding the mechanisms of specific target gene recognition and regulation, and the role of co-factors. From studies of the gnathal and trunk-specification proteins Disconnected (Disco) and Teashirt (Tsh), respectively, evidence is presented for a network of zinc-finger transcription factors that regionalize the Drosophila embryo. Not only do these proteins establish specific regions within the embryo, but their distribution also establishes where specific HOM-C proteins can function. In this manner, these factors function in parallel to the HOM-C proteins during axial specification. In tsh mutants, disco is expressed in the trunk segments, probably explaining the partial trunk to head transformation reported in these mutants, but more importantly demonstrating interactions between members of this regionalization network. It is concluded that a combination of regionalizing factors, in concert with the HOM-C proteins, promotes the specification of individual segment identity (Robertson, 2004).

disco was initially identified in a screen for mutations affecting neural development. It was not until the discovery of disco-related (disco-r) that a patterning role was uncovered. The phenotype of terminal embryos lacking disco and disco-r is similar to those lacking the gnathal HOM-C genes Dfd and Scr; that is, structures from the gnathal segments (mandibular, maxillary and labial) are missing. This phenotype is due to reduced expression of Dfd and Scr target genes. Since HOM-C protein distribution is normal in disco, disco-r null embryos, and vice versa, these factors appear to act in parallel pathways (Robertson, 2004).

These studies have been extended and it is shown that: (1) Dfd can only direct maxillary developmental when Disco and/or Disco-R are present; (2) Tsh represses disco (and disco-r), helping to distinguish between trunk and gnathal segment types, and thereby establishing domains for appropriate HOM-C protein function, and (3) when ectopically expressed in the trunk, Disco represses trunk development and may transform these segments towards a gnathal segment type (Robertson, 2004).

Though HOM-C genes have a clear role in establishing segment identities, ectopic expression often has only a limited effect. The data indicate that, for Dfd, this restriction arises because of the limited distribution of Disco in the trunk segments. There are two important conclusions from these observations: (1) the spatial distribution of Disco establishes where cells can respond to Dfd, and this is probably true for Scr as well. Cells expressing disco develop a maxillary identity when provided with Dfd, even though this may not have been their original HOM-C-specified fate. This highlights (2) -- the combination of Disco and Dfd overrides normal trunk patterning, without altering expression of tsh and trunk HOM-C genes. As with the maxillary segment, identity is lost in the mandibular and labial segments when embryos lack disco and disco-r. This indicates that Disco and Disco-R may have similar roles in all gnathal segments. That co-expression of Disco and Scr in the trunk activates the Scr gnathal target gene pb strengthens this conclusion. Therefore, it is proposed that Disco defines the gnathal region, and establishes where the gnathal HOM-C proteins Dfd and Scr can function (Robertson, 2004).

Alone, ectopic Disco significantly alters development, indicating that Disco has a morphogenetic ability, separate from gnathal HOM-C input. Since Disco is required for normal gnathal development, it is suspected that disco specifies a general gnathal segment type. Definitive identification is difficult because of the lack of morphological or molecular markers that denote a general gnathal segment type. Yet, there is support for the conclusion that disco expression establishes a gnathal segment type. Ectopic Disco can, to some extent, override the trunk specification system and repress trunk development (repressing denticles, oenocytes and trachea). Furthermore, ectopic Disco blocks dorsal closure, which is similar to the role of endogenous Disco in the gnathal segments (Robertson, 2004).

Perhaps the most compelling evidence that Disco specifies a gnathal segment type comes from the observation that disco is activated in the trunk segments when embryos lack Tsh. The identity of the trunk segments in tsh mutant embryos is somewhat uncertain. It has been suggested that some aspects of the tsh phenotype indicate the trunk segments acquire gnathal characteristics; for example, the ventral neural clusters appear to be transformed to a gnathal-like identity. Mutations in the tsh gene can therefore be interpreted in two ways -- either they partially transform the trunk segments into a gnathal-like identity, and in particular the prothoracic segment into a labial one, or they cause a non-specific change in segmental identity perhaps due to cell death. However, the loss of tsh and the trunk HOM-C genes may transform the trunk cuticle toward anterior head cuticle. Again, the difficulty in assigning an identity is due to the lack of a readily discernable gnathal morphological or molecular marker. Evidence is presented that disco and disco-r are reliable molecular markers for gnathal identity, and disco mRNA is shown to be present in the ventral and lateral regions of the trunk segments in tsh mutant embryos. This expression of disco coincides, spatially, with the region of the trunk that is transformed in tsh mutant embryos. UAS-driven disco does mimic some aspects of tsh mutants, denticles are reduced and the ventral chordotonal neurons do not develop, but since Tsh is still present, the transformation caused by ectopic disco may be incomplete. Finally, Dfd cannot induce maxillary structures, even in tsh mutants, when disco and disco-r are absent. This reinforces the role for Disco in establishing gnathal identity, and indicates that the ectopic Disco present in embryos lacking Tsh is functional. Therefore, considering these arguments, it is proposed that Disco and Disco-R establish the gnathal region of the Drosophila embryo, and in this regard, they function similarly to Tsh, which specifies the trunk region (Robertson, 2004).

There are other parallels between Disco/Disco-R and Tsh. They are regionally expressed zinc-finger transcription factors, and they are required in parallel with the HOM-C proteins for proper segment identity. Furthermore, the distribution of these proteins establishes domains in which specific HOM-C proteins can properly direct embryonic development. The data reveal a regulatory relationship between Tsh and disco (and disco-r), indicating they are part of an interacting network that helps regionalize the Drosophila embryo. The HOM-C proteins then establish specific segmental identities in the appropriate region. In the trunk segments, Tsh, along with the trunk HOM-C proteins, specifies the trunk segment characteristics, in part by repressing disco and, thereby, preventing gnathal characteristics from arising in the trunk segments. The presented model requires that tsh expression be limited to the trunk segments, and it is proposed this is accomplished by another C2H2 zinc-finger protein, Salm. tsh expression has been shown to expand into the posterior gnathal and posterior abdominal segments in embryos lacking Salm. Therefore, Salm establishes the boundary between the Tsh and Disco domains. It is stressed that, at this time, it is not known what parts of this regulation are direct. Interestingly, other zinc-finger transcription factors are responsible for positioning salm expression, so that a more extensive hierarchy of zinc-finger transcription factors leads to regionalization, eventually establishing the domains of HOM-C protein function. It is also noted that Tsh has other roles than just repressing disco. Tsh actively establishes the trunk region, just as Disco does the gnathal. It is also noteworthy that ectopic Tsh activates disco in the labial sense organ primordia, leading to a Keilin's Organs fate, as occurs in the thoracic segments. Therefore, for unknown reasons, Tsh changes from a repressor of disco to an activator in these cells. This observation highlights the complex interplay between factors like Tsh and Disco, and it will be interesting to determine what causes these opposing roles (Robertson, 2004).

Many other questions remain. For example, how are the expression domains for these factors established? It is clear that Salm could form a boundary separating gnathal from trunk, but in salm mutants, tsh is only ectopically activated in the posterior labial segment, not in every gnathal segment. This implies that Salm forms a boundary, not by repressing tsh throughout the head, but by, in a sense, drawing a line between the head and trunk regions. What then prevents tsh expression from crossing that line and extending further into the gnathal segments in salm mutants? Is there an activator of tsh that is limiting, another gnathal repressor, or is something else involved? Likewise, what activates tsh and disco? It is unlikely that lack of Tsh is the only requirement for disco expression. More likely, this relies on the prior segmentation pathway. With regard to the HOM-C specification of segment identity, questions remain as to how the zinc-finger proteins establish where specific HOM-C proteins can function. Are the zinc-finger proteins co-factors or simply a parallel pathway? Furthermore, if they are co-factors for the HOM-C proteins, how can different HOM-C proteins establish different segment identities with the same co-factor (for example, Dfd and Scr with Disco), or how can different co-factors alter the role of a HOM-C protein (Scr with Disco or Tsh) (Robertson, 2004)?

Finally, the question remains of whether or not factors such as Disco and Tsh establish head/trunk domains and delimit HOM-C protein function only in the Drosophila embryo, in all stages of Drosophila or in other animals as well. Though this remains to be tested experimentally, there are indications that this may be a general mechanism. homologs of these zinc-finger genes are found in vertebrates and in other invertebrates, and, although only limited data are currently available, expression data indicate that these genes may have similar roles to their Drosophila counterparts during embryonic patterning. In an informative experiment the Tribolium Dfd homolog, Tc-Dfd, has been expressed in Drosophila embryos lacking the endogenous Dfd gene; persistent expression of Tc-Dfd rescues maxillary development. Though at present, it is not known whether or not a direct interaction is required between Disco and Dfd, this result would indicate that the Tribolium Dfd protein can fulfill the same roles as the Drosophila protein, and, therefore, it must be able to function with the Drosophila regionalization system. In any case, it will be important to investigate and interpret the role of the regionalizing genes as they relate to development and evolution of body pattern in other animals, and to ask whether a similar network is involved in patterning all animals (Robertson, 2004).

Larval and Pupal Stages

The cephalopharyngeal skeleton or pupal "mouth," is composed of four major cuticular structures: mouth hooks, median tooth, H-piece and cephalopharyngeal plates. These chitinous structures are secreted by cells of the atrum and pharynx of the developing embryo. In Deformed mutants these structures, specified by mandibular and maxillary epidermis, are absent, disrupted or duplicated. Antennal and maxillary sense organs are also disrupted (Regulski, 1987).

Dfd+ is required in three embryonic cephalic segments to form a normal head. Mutant embryos of Dfd display defects in derivatives of the maxillary segment, of the mandibular segment, and of some more anterior segments. In the adult fly, defects are seen in the posterior aspect of the head when the gene is mutant. A transformation from head to thoracic-like tissue is seen dorsally and a deletion of structures is seen ventrally. The gene product is necessary during at least two periods of development: during embryonic segmentation and head involution, and during the late larval and pupal stages (Merrill, 1987).

A screen was performed for second site mutations that reduce the viability of animals carrying hypomorphic mutant alleles of Deformed. Genes mapping to six complementation groups on the third chromosome were isolated as modifiers of Deformed function. Products of two of these genes, sallimus and moira , have been previously proposed as homeotic activators since they suppress the dominant adult phenotype of Polycomb mutants. Mutations in hedgehog, which encodes secreted signaling proteins, were also isolated as Deformed loss-of-function enhancers. Hedgehog mutant alleles also suppress the Polycomb phenotype. Mutations were also isolated in a few genes that interact with Deformed but not with Polycomb, indicating that the screen identified genes that are not general homeotic activators. Two of these genes, cap'n' collar and defaced, have defects in embryonic head development that are similar to defects seen in loss-of-function Deformed mutants (Harding, 1995).

Ectopic expression of homeotic genes, Dfd, Scr and Antp, results in the disruption of the developing PNS in the abdomen. Thus homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression (Heuer, 1992).

Deformed mutants dying during metamorphosis share defects of CNS reorganization, ventral adult head development, and adult salivary gland morphogenesis. Specifically, the shared phenotypes show failure to separate the subesophageal ganglion (SEG) from the thoracic ganglion (TG); structural and functional abnormalities of the proboscis and maxillary palps, innervated by the SEG; and failure of the adult salivary glands to extend into the thorax. Broad Complex/Deformed double mutants show synergistic enhancement of the ventral head defects. This genetic interaction suggests that the segment identity and steroid hormone-sensitive regulatory hierarchies intersect during postembryonic development (Restifo, 1994).

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