BIOLOGICAL OVERVIEW

The subdivision of the lateral mesoderm into a visceral (splanchnic) and a somatic layer is a crucial event during early mesoderm development in both arthropod and vertebrate embryos. In Drosophila, this subdivision leads to the differential development of gut musculature versus body wall musculature. biniou, the sole Drosophila representative of the FoxF subfamily of forkhead domain genes, has a key role in the development of the visceral mesoderm and the derived gut musculature. (The biniou koz is a high-pitched bagpipe unique to Brittany). biniou expression is activated in the trunk visceral mesoderm primordia downstream of dpp, tinman, and bagpipe and is maintained in all types of developing gut muscles. biniou activity is essential for maintaining the distinction between splanchnic and somatic mesoderm and for differentiation of the splanchnic mesoderm into midgut musculature. biniou is required not only for the activation of differentiation genes that are expressed ubiquitously in the trunk visceral mesoderm but also for the expression of dpp in parasegment 7, which governs proper midgut morphogenesis. Activation of dpp is mediated by specific Biniou binding sites in a dpp enhancer element, which suggests that Biniou serves as a tissue-specific cofactor of homeotic gene products in visceral mesoderm patterning. Based upon these and other data, it has been proposed that the splanchnic mesoderm layers in Drosophila and vertebrate embryos are homologous structures whose development into gut musculature and other visceral organs is critically dependent on FoxF genes (Zaffran, 2001).

Similar to vertebrates, the musculature of the Drosophila midgut is composed of an inner layer of circular muscles and an outer layer of longitudinal muscles. Vertebrate and Drosophila visceral musculatures have additional similarities, including the features that the fibers are spindle-shaped and display slow, supercontracting properties. Although Drosophila visceral muscles are striated, their striation is atypical, and several of their ultrastructural features are reminiscent of vertebrate smooth muscles. In both vertebrates and arthropods, the visceral mesoderm is largely derived from the lateral (splanchnic) mesoderm, which is under the inductive influence of BMP/Dpp signals. Dpp, which is secreted from the dorsal ectoderm, is critically required for the induction of visceral muscle in the underlying lateral (in insects, dorsal) mesoderm. The mesodermal response to Dpp consists of the transcriptional activation of at least two important regulatory genes in the dorsal mesoderm; these are the NK homeobox genes tinman (tin) and bagpipe (bap). tin is genetically required for the formation of all dorsal mesodermal derivatives (heart, dorsal body wall muscles, and midgut visceral muscles), whereas bap is required exclusively for the specification of the visceral (specifically the circular) muscles of the midgut and functions downstream and as a direct target of tin. bap is therefore the first example of a regulator that is uniquely involved in the specification of visceral muscles (Zaffran, 2001 and references therein).

bin requires the combined activities of bap and dpp for its normal activation: this suggests that bin is positioned furthest downstream in the known regulatory hierarchy of transcription factors during visceral mesoderm development. Another important conclusion is that inductive Dpp signals are active not just once in the dorsal mesoderm but rather operate successively during each of the three known steps of gene activation that participate in the pathway leading to trunk visceral mesoderm. These activation steps (tin -> dorsal tin -> bap -> bin) occur in rapid succession, apparently within minutes after the mesoderm has spread beneath the dorsal ectodermal domain of Dpp expression (Zaffran, 2001).

Induction of tin expression by Dpp in the dorsal mesoderm involves the combined binding of Smad proteins (Medea and Mad) and Tin itself to a Dpp-responsive enhancer of the tin gene. Genetic data for bin suggest an analogous mechanism for bin, in which the combined binding of Smads and Bap (an NK homeodomain protein related to Tin) may activate a Dpp-responsive bin enhancer. However, in contrast to the induction of tin and bap by Dpp, which stringently requires tin activity, low levels of bin can be induced by Dpp even in the absence of tin and bap. This observation indicates that there is at least one other mesoderm-intrinsic factor, in addition to Tin and Bap, which helps to provide the mesoderm with the competence to respond to Dpp and allow bin induction (Zaffran, 2001).

The relatively normal spatial pattern of residual bin expression in the absence of bap indicates that bin may also receive direct inputs from striped regulators. This regulation could be analogous to that of bap as well and involve the negative activities of wg and slp. Lastly, there is feedback regulation in which bin regulates prolonged expression of bap. However, this indirect feedback loop is only operative until early stage 12, when bap expression ceases in the trunk visceral mesoderm. The maintenance of bin expression during later stages of visceral mesoderm development could involve direct autoregulation (Zaffran, 2001).

The similarities in the early expression patterns of bin and bap in the trunk visceral mesoderm primordia raise the question of the relative contribution of these two genes to visceral mesoderm development. Are all functions of bap in this developmental process mediated through bin, or does bap also fulfill bin-independent roles? In the absence of either bin or bap activity, a large portion of the cells that are normally destined to form visceral muscles become incorporated into body wall muscles. Likewise, the expression of several trunk visceral mesoderm markers is affected similarly in bin and bap mutants. The similarities of these phenotypes as well as the partial rescue of bap phenotypes upon forced expression of bin suggest that most of the activities of bap in the trunk visceral mesoderm involve the activation and function of its downstream gene bin. Because of the temporal overlap of Bap and Bin expression during stage 10 to early stage 12, it is also possible that both proteins are required in combination to activate some target genes. Based upon lineage tracing data, the transformation from visceral into somatic muscle fates appears to be more complete in bap mutants compared to bin mutants, and defects in the migration behavior and morphogenesis of the trunk visceral mesoderm are evident at an earlier stage in bap mutants. It is therefore likely that bap has some additional targets in the visceral mesoderm that do not require bin (Zaffran, 2001).

The lineage tracing experiments with the bap-lacZ marker also show that the earliest steps of trunk visceral mesoderm, which consist of the segregation of primordial cells from the mesodermal monolayer towards the interior, do not require bap or bin activity. This behavior is unlikely to be due to functional redundancy between the two genes, because bin expression is largely missing in bap mutants. Rather, this observation points to the existence of regulatory gene(s) in addition to bap and bin that are present in the 11 clusters of dorsal mesodermal cells and control their segregation towards the interior to form visceral mesoderm. The emerging picture is that multiple regulatory genes are activated in metameric clusters of dorsal mesodermal cells that define the trunk visceral mesoderm primordia. Based on the available genetic data, it is likely that the induction of all of these regulators requires Dpp and is prevented by slp. In addition to Dpp, bap is strictly and bin largely dependent on tin, whereas induction of yet unknown genes that regulate visceral mesoderm segregation may be largely or fully independent of tin. While each of these regulatory genes appears to have some unique functions in visceral mesoderm development, three of the Dpp-induced genes, tin, bap, and bin, are part of a mesoderm-intrinsic cascade of gene activations, which also involves feedback regulation. It is proposed that a major outcome of this regulatory cascade is the activation of bin in the primordial cells of the trunk visceral mesoderm and its maintenance in the developing circular midgut muscles. It appears that bin plays a key role in activating multiple, if not the majority of, patterning and differentiation genes that define the morphological and functional features of midgut muscles and prevent visceral mesodermal cells from fusing with somatic muscle precursors. Molecular and genetic studies show that at least two of these downstream genes, dpp and ß-3tubulin, are direct targets of bin (Zaffran, 2001).

Spatially restricted expression of dpp in PS7 of the trunk visceral mesoderm plays an important role in normal midgut morphogenesis. Previous studies have identified Ubx and Exd as directly and positively acting upstream regulators of dpp in the visceral mesoderm. Because these regulators are present in other tissues where they do not activate dpp expression, it has been proposed that they require tissue-specific cofactor(s) for activating dpp in the visceral mesoderm. Further evidence of the involvement of cofactor(s) that are predicted to be expressed uniformly in the visceral mesoderm comes from the dissection of the visceral mesodermal dpp enhancer. In particular, truncations or specific mutations within this enhancer cause an expansion of enhancer activity beyond the Ubx domain and in some cases throughout the trunk visceral mesoderm. These data suggest that, in the absence of repressing activities, uniformly expressed visceral mesodermal factor(s) are able to activate the dpp enhancer without a requirement for Ubx and Exd (Zaffran, 2001 and references therein).

The FoxF protein Bin corresponds to such a visceral mesoderm-specific factor during the activation of dpp expression. Bin is expressed in the visceral mesoderm prior to dpp, is genetically required for dpp expression, and the Bin binding sites within the dpp enhancer are essential for enhancer activity in PS7 of the visceral mesoderm. Of note, the data suggest that Bin has a key role in the activation of a previously identified general visceral mesoderm enhancer of dpp, that is active throughout PS7 to PS12 of the visceral mesoderm. In the normal context, Bin binding to these sequences is required for the enhancement of dpp expression in PS7. However, the presence of additional functionally important Bin binding sites that are interdigitated with Ubx and Exd sites demonstrates that Bin is an integral component of PS7-specific enhancer activation as well and that a clean separation between general and PS7-specific enhancer elements does not exist. The available data suggest that PS7-specific expression of dpp in the visceral mesoderm is regulated by an exquisite balance between positive and negative activities. Negative regulators have been shown to include dTCF (or a factor with related binding specificity), which acts in a wg-independent manner in the entire trunk visceral mesoderm, and Abd-A, which is only active between PS8 and PS12. Based on these combined data, a model is proposed in which the activator bin is neutralized by the negative factors to provide a sensitized equilibrium of gene activities, which is set below the threshold level of dpp activation. In this model, the role of Ubx and Exd would be to disrupt this equilibrium and shift it towards the active state in PS7 (Zaffran, 2001).

An early binary decision during mesoderm development in both insect and vertebrate embryos results in the splitting of a single layer into the two separate layers of somatic and splanchnic mesoderm, which become associated with lateral ectoderm and endoderm, respectively. In Drosophila and other insects, the splanchnic mesoderm develops exclusively into gut musculature, whereas in vertebrates it contributes to many additional internal organs. Interestingly, the overt morphological and developmental similarities in the splanchnic mesoderm of insects and vertebrates extend to the molecular level. Similar to Drosophila bin, the two vertebrate orthologs FoxF1 and FoxF2 (previously termed FREAC1/2 [mouse, rat, human Foxf1/2], HFH-8 [mouse Foxf1], lun [mouse Foxf2], and XFD-13 [frog FoxF1]) are predominantly expressed in the splanchnic mesoderm and mesenchyme that line the digestive tract. At later stages, expression continues in various tissues that are derived from splanchnic mesoderm, including smooth muscles of the intestine, lung, and liver capsule. In addition to similarities in their expression patterns, the phenotype of mouse embryos with a targeted disruption of Foxf1 suggests similarities in developmental functions of the Drosophila and vertebrate genes. In Foxf1^-/- embryos, splitting of the lateral plate mesoderm into splanchnic and somatic layers is frequently incomplete or absent, and ectopic expression of the somatic mesoderm marker Irx1 indicates that the splanchnic mesoderm assumes characteristics of somatic mesoderm (Mahlapuu, 2001a). Both of these alterations are strongly reminiscent of the bin phenotype. Other striking similarities with Drosophila bin include the requirement of Foxf1 for the activation of BMP4 expression in the lateral plate and allantois (Mahlapuu, 2001b) as well as its coexpression with the bap ortholog Bapx1 in the splanchnic mesoderm. Moreover, the demonstration that the lateral plate mesoderm is under the influence of BMP signaling could imply that, analogous to bin, which is initially a target of Dpp and at later stages activates dpp expression, FoxF genes are both downstream and upstream of BMPs. Collectively, these data suggest that the splanchnic mesoderm in insect and vertebrate embryos is composed of homologous tissues and that their development is controlled by related genetic circuits in which members of the FoxF class of forkhead domain genes occupy a central position (Zaffran, 2001).

GENE STRUCTURE

cDNA clone length - 2413

Bases in 5' UTR - 209

Exons - 4

Bases in 3' UTR - 194

PROTEIN STRUCTURE

Amino Acids - 667

Structural Domains

Biniou protein includes a forkhead (winged helix) domain in its central portion. Sequence comparisons show that Biniou (Bin) is the only fly representative of the FoxF subfamily of forkhead domain proteins and is equally related to FoxF1 and FoxF2. The sequence similarities between Bin and its vertebrate orthologs are confined to the forkhead domains (Zaffran, 2001).

date revised: 20 December 2001

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