fork head: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

Gene name - fork head

Synonyms -

Cytological map position - 98D1-E1

Function - transcription factor

Keyword(s) - selector gene, salivary glands, ventral midline

Symbol - fkh

FlyBase ID:FBgn0000659

Genetic map position -

Classification - forkhead domain

Cellular location - nuclear



NCBI links: | Entrez Gene
Recent literature
Bolukbasi, E., Khericha, M., Regan, J. C., Ivanov, D. K., Adcott, J., Dyson, M. C., Nespital, T., Thornton, J. M., Alic, N. and Partridge, L. (2017). Intestinal Fork head regulates nutrient absorption and promotes longevity. Cell Rep 21(3): 641-653. PubMed ID: 29045833
Summary:
Reduced activity of nutrient-sensing signaling networks can extend organismal lifespan, yet the underlying biology remains unclear. This study shows that the anti-aging effects of rapamycin and reduced intestinal insulin/insulin growth factor (IGF) signaling (IIS) require the Drosophila FoxA transcription factor homolog Fork Head (FKH). Intestinal FKH induction extends lifespan, highlighting a role for the gut. FKH binds to and is phosphorylated by AKT and Target of Rapamycin. Gut-specific FKH upregulation improves gut barrier function in aged flies. Additionally, it increases the expression of nutrient transporters, as does lowered IIS. Evolutionary conservation of this effect of lowered IIS is suggested by the upregulation of related nutrient transporters in insulin receptor substrate 1 knockout mouse intestine. These studies highlights a critical role played by FKH in the gut in mediating anti-aging effects of reduced IIS. Malnutrition caused by poor intestinal absorption is a major problem in the elderly, and a better understanding of the mechanisms involved will have important therapeutic implications for human aging.
Lan, Q., Cao, M., Kollipara, R. K., Rosa, J. B., Kittler, R. and Jiang, H. (2017). FoxA transcription factor Fork head maintains the intestinal stem/progenitor cell identities in Drosophila. Dev Biol 433(2):324-343. PubMed ID: 29108672
Summary:
Understanding how somatic stem cells respond to tissue needs is important, since aberrant somatic stem cell behaviors may lead to tissue degeneration or tumorigenesis. From an in vivo RNAi screen targeting transcription factors that regulate intestinal regeneration, this study uncovered a requirement for the Drosophila FoxA transcription factor Fork head (Fkh) in the maintenance of intestinal stem/progenitor cell identities. FoxA/Fkh maintains the expressions of stem/progenitor cell markers and is required for stem cell proliferation during intestinal homeostasis and regeneration. Furthermore, FoxA/Fkh prevents the intestinal stem/progenitor cells from precocious differentiation into the Enterocyte lineage, likely in cooperation with the transcription factor bHLH/Daughterless (Da). In addition, loss of FoxA/Fkh suppresses the intestinal tumorigenesis caused by Notch pathway inactivation. To reveal the gene program underlying stem/progenitor cell identities, the genome-wide chromatin binding sites of transcription factors Fkh and Da were profiled, and interestingly, around half of Fkh binding regions are shared by Da, further suggesting their collaborative roles. Finally, the genes were identified associated with their shared binding regions. This comprehensive gene list may contain stem/progenitor maintenance factors functioning downstream of Fkh and Da, and would be helpful for future gene discoveries in the Drosophila intestinal stem cell lineage.
BIOLOGICAL OVERVIEW

The process of gastrulation in the fly is a little confusing for those familiar with gastrulation in vertebrates, such as the frog. In the frog, the egg is divided into animal and vegetal poles. The animal pole gives rise to ectoderm, the so-called active developmental tissue, progenitor of the future head, backbone and somatic structures. The vegetal pole gives rise to endoderm, tissue that forms the internal organs such as the digestive system. Seeing the ventral invagination in the fly, one might assume the process is homologous to what occurs in the frog, and that this developmentally driven ventral inturning of cells is also the origin of endoderm in the fly, as it is in the frog. This is not the case however.

The fly's development in this instance seems to be more complex than that of the frog and definitely more alien, when viewed from a vertebrate perspective. The fly has not one but three loci for invagination [Images]: the ventral locus mentioned above, plus one at either end (anterior and posterior) of the fertilized egg. Ventral invagination in the fly is driven by the genes twist and snail, and forms the mesoderm. fork head drives the anterior and posterior loci of invagination. From these are formed the anterior midgut and posterior midgut primordia, which give rise to the endoderm. Therefore, cells that will eventually become the fly's digestive system come from the "head" and "tail" ends of the egg, not the ventral or "stomach" side, as is intuitively suggested by vertebrate development. The vegetal pole in vertebrate eggs may be considered analogous to the amnioserosa in the fly, a tissue that contracts and disappears during germ band extention.

fork head expression marks both the anterior and posterior gut primordia. Gut primordia are the anlagen of the endodermal anterior midgut (anterior pole) and the endodermal posterior midgut (posterior pole). A major part of the invaginating anterior anlage is ectoderm. Anterior derivatives include the foregut (pharynx and esophagus, and the hindgut (proctodeum). The posterior domain abuts the seventh Fushi tarazu stripe and therefore is behind the 15th parasegment (the last FTZ stripe is two parasegments wide).

Posterior fork head expression follows the amnioproctodeal invagination during gastrulation and anterior expression spreads into the invaginating primordia of the anterior midgut. This expression profile suggests that fork head defines both anterior and posterior gut primordia and regulates the invagination process.

An analysis of mutant phenotypes helps clarify fork head's function. In both anterior and posterior domains, fork head mutations cause homeotic transformation of portions of the gut: foregut and hindgut are replaced by ectopic head structures in fkh mutants (Weigel, 1989).

Anterior and posterior fork head expression and function are independently regulated. In one class of regulatory mutants, there is specific failure to form a stomodeal invagination (Weigel, 1990a).

From the vertebrate biologist's viewpoint, the mystery of Drosophila Fork head is its lack of mesodermal expression. Fork head's closest vertebrate homologs, HNF-3alpha, HNF-3beta, HNF-3gamma, XFKH1/XFD1, and XFD1/pintallavis are each expressed in mesoderm, and are involved in fate determination of endoderm, neural ectoderm, notochord and other mesodermal tissues.

Fork head prevents apoptosis and promotes cell shape change during formation of the Drosophila salivary glands

The secretory tubes of the Drosophila salivary glands are formed by the regulated, sequential internalization of the primordia. Secretory cell invagination occurs by a change in cell shape, which includes basal nuclear migration and apical membrane constriction. In embryos mutant for fork head, the secretory primordia are not internalized and secretory tubes do not form. Secretory cells of fkh mutant embryos undergo extensive apoptotic cell death following the elevated expression of the apoptotic activator genes reaper and head involution defective. The secretory cell death can be rescued in the fkh mutants and the rescued cells still do not invaginate. The rescued fkh secretory cells undergo basal nuclear migration in the same spatial and temporal pattern as in wild-type secretory cells, but do not constrict their apical surface membranes. These findings suggest at least two roles for fkh in the formation of the embryonic salivary glands: an early role in promoting survival of the secretory cells, and a later role in secretory cell invagination, specifically in the constriction of the apical surface membrane (Myat, 2000).

FKH mRNA was first detected during embryonic stage 9 in the ventral-posterior cells of the secretory placode. FKH mRNA is detected at approximately equivalent levels in all secretory cells from early embryonic stage 10 and throughout embryogenesis. Fkh is expressed in the salivary glands of late third instar larvae where it activates expression of the Salivary gland secretion protein 3 (Sgs3) and Salivary gland secretion protein 4 (Sgs4) genes. This late expression suggests that fkh is expressed in the salivary gland throughout the larval stages until the onset of metamorphosis (Myat, 2000).

The apoptotic cell death observed in the early secretory primordia of fkh mutants indicates that fkh is required for secretory cell survival. Thus, secretory cells may fail to invaginate in fkh mutants simply because the cells are dead or dying. Indeed, the ectopic expression of rpr and hid, but not grim, is effective in inducing early secretory cell death, which if extensive enough, prevents internalization. Alternatively, fkh may have two separate roles in the salivary gland, one to promote cell survival and another to control invagination of the primordia. To distinguish between these possibilities, the apoptotic secretory cell death was rescued in fkh mutants by generating embryos that were mutant for fkh and also carried Df(3L)H99, a small deficiency that deletes rpr, hid and grim (fkh H99). Normal salivary glands are formed in embryos homozygous for Df(3L)H99 (H99). In the fkh H99 embryos, dCREB-A staining is detected in the entire secretory placode at early stages This staining completely disappears by embryonic stage 13, suggesting that either fkh is required to maintain dCREB-A expression or that the fkh H99 secretory cells are still dying. To address this issue, the expression of another secretory marker protein, PS, whose expression is thought to be fkh independent, was analyzed. In wild-type embryos, PS is expressed at high levels in the salivary glands throughout embryogenesis. In fkh mutant embryos, PS is initially expressed in the entire secretory placode, and at reduced levels in the surviving ring of secretory cells. Importantly, PS is expressed to very high levels in all secretory cells throughout embryogenesis in the fkh H99 embryos. Nonetheless, the PS-expressing cells in the fkh H99 embryos are not internalized and remain at their site of origin on the ventral surface. Therefore, in addition to its early role in promoting secretory cell survival, Fkh is also required for the invagination of the secretory cells (Myat, 2000).

Histological sections and transmission electron microscope analyses of fkh H99 embryos confirm the failure of the secretory cells to be internalized and reveal additional changes in the salivary glands. Cells of the fkh H99 placode are columnar like the wild-type cells, except for cells in the ventral-posterior portion of the placode, which are round and stacked on one another. Interestingly, although coordinate nuclear migration occurs in the secretory cells of all fkh H99 embryos in approximately the same temporal and spatial pattern as in wild type, the apical surface membranes fail to constrict even at very late stages. Consequently, the invaginating pits of fkh H99 salivary glands are wide and shallow compared to those of wild-type embryos. The absence of apical membrane constriction was confirmed by analyzing transmission electron micrographs of invaginating wild-type and fkh secretory cells. Prior to invagination, wild-type secretory cells have broad and flat apices. Wild-type secretory cells at the center of the invaginating pit have constricted apices, and show numerous membrane protrusions at the apical surface. In contrast, secretory cells at the lateral edges of the invaginating pit, have flat and broad apices with few membrane protrusions. In late fkh H99 embryos, the apices of the secretory cells are unconstricted and appear flat and broad, similar to the apices of secretory cells in early wild-type embryos. Fewer apical membrane protrusions are found at the surface of fkh H99 secretory cells, as compared to wild type. Altogether, these findings suggested that fkh is required for secretory cells both to survive and to invaginate. Moreover, the absence of apical membrane constriction may be the underlying defect in the failure of fkh mutant salivary glands to internalize (Myat, 2000).

CrebA, a target of Forkhead, regulates secretory activity in the Drosophila salivary gland and epidermis

Understanding how organs acquire the capacity to perform their respective functions is important for both cell and developmental biology. This study examines the role of early-expressed transcription factors in activating genes crucial for secretory function in the Drosophila salivary gland. Expression of genes encoding proteins required for ER targeting and translocation, and proteins that mediate transport between the ER and Golgi is very high in the early salivary gland. This high level expression requires two early salivary gland transcription factors; CrebA is required throughout embryogenesis and Fkh is required only during late embryonic stages. Because Fkh is required to maintain late CrebA expression in the salivary gland, Fkh probably works through CrebA to affect secretory pathway gene expression. In support of these regulatory interactions, CrebA is shown to be important for elevated secretion in the salivary gland. Additionally, CrebA is required for the expression of the secretory pathway genes in the embryonic epidermis, where CrebA has been shown to be essential for cuticle development. Zygotic mutations in several individual secretory pathway genes result in larval cuticle phenotypes nearly identical to those of CrebA mutants. Thus, CrebA activity is linked to secretory function in multiple tissues (Abrams, 2005).

To test whether regulation of CrebA by Fkh is direct, a 2.8 kb fragment upstream of the CrebA transcription unit was identified that could drive salivary gland expression of a lacZ reporter gene (K. D. Henderson, PhD Thesis, Johns Hopkins University School of Medicine, 2000). Two smaller fragments from this enhancer resulted in salivary gland expression of the lacZ reporter gene either only after invagination had begun and later (CrebA-1100) or prior to invagination and later (CrebA-770). Since the later expression pattern fit the timeframe for Fkh-dependent salivary gland expression of CrebA, the CrebA-1100 construct, which contains six consensus Fkh-binding sites was further characterized. ß-Gal expression in the salivary glands with the CrebA-1100 construct was significantly reduced in fkh homozygotes although expression in the amnioserosa was unaffected, indicating that a Fkh-dependent salivary gland enhancer of CrebA had been identified. Flies were transformed with a CrebA-1100 reporter construct in which all six consensus Fkh-binding sites were mutated (CrebA-1100 fkh1-6 lacZ). Both lines carrying the mutated construct had significantly diminished salivary gland expression of ß-Gal, although ßGal expression in other tissues, including the amnioserosa and hemocytes was unaffected. It is conclude that Fkh functions directly to maintain late high-level expression of CrebA in the salivary gland (Abrams, 2005).

It was predicted that secretory pathway component encoding gene (SPCG) expression is controlled directly by CrebA. As a first step toward testing this possibility, lacZ reporter constructs for six of the 34 SPCGs were analyzed in this study. Each SPCG enhancer fragment spanned the 5' end of the most 5' cDNA for each gene and included ~1-2 kb of DNA further upstream. Transformant lines generated from five of the six constructs resulted in embryonic salivary gland expression. The srp68 lacZ enhancer construct did not express in the embryonic salivary gland. Salivary gland lacZ expression from the spase25 and sec61ß enhancer constructs wasdetected from early stage 12 and throughout embryogenesis. Salivary gland lacZ expression from the p24-1, zCop and SrpRa enhancer constructs was first detected during stage 13 and later. Expression of three of the constructs was examined in CrebA mutants: expression of ß-Gal from both the zCop-lacZ and sec61ß-lacZ constructs was completely absent in the salivary glands, whereas salivary gland ß-Gal expression from the spase25-lacZ construct was significantly reduced. Thus, CrebA-dependent salivary gland enhancers have been identified for at least three of the SPCGs (Abrams, 2005).

A search of the regions immediately upstream of the translation start sites of the SPCGs using MEME revealed a motif that is an excellent match for a mammalian Creb-binding site and that is present within 2 kb upstream of 32 of the 34 SPCGs. (The translation start site is used as a reference point since transcription start sites have not been mapped for any of the SPCGs.) Interestingly, of the two SPCGs that do not contain this consensus, one (sec62) is among the least affected by mutations in CrebA and the other, srp19, is one of only two genes examined that had ubiquitously high levels of expression in all tissues, including the salivary gland. Even more compelling is the finding that 13/32 have the site within 100 bp, another 7/32 have the site within 200 bp and another 5/32 have the site within 500 bp of the translation start site. All of the SPCG reporter gene constructs built contain this consensus site. Thus, not only is it predicted that the site is important for salivary gland expression of the SPCGs, but this could be the site through which CrebA acts to elevate transcription. The proximal location of these putative binding sites with respect to the start site of translation is consistent with the finding that mammalian Creb proteins bind close to the start of transcription. Also of relevance to these studies was the failure to discover consensus Fkh-binding sites conserved among the SPCGs through MEME analysis, further supporting an indirect role for Fkh in SPCG regulation (Abrams, 2005).

This study demonstrates that the Drosophila salivary gland prepares soon after specification to generate the machinery for its high-level secretory activity. The machinery includes components of the early secretory pathway crucial for targeting and translocating proteins into the ER and for vesicle transport between the ER and Golgi. Thus, one way the gland distinguishes itself from surrounding tissues is to greatly increase the relative transcriptional levels of the secretory pathway component genes (SPCGs). The leucine zipper transcription factor CrebA has a crucial and probably direct role in activating increased levels of SPCG expression not only in the salivary gland, but also in the epidermal cells, which secrete the larval cuticle. Fkh, the Drosophila FoxA/PHA-4 homolog, is required to maintain SPCG expression in the salivary gland, but acts indirectly, by maintaining CrebA expression. Hkb, the other early transcription factor examined in this study, is not required for elevated SPCG expression (Abrams, 2005).

Fkh has several roles in salivary gland development and function, including mediating the cell shape changes of invagination, maintaining secretory cell viability and transcriptional activation of the sgs genes in late larval life. In addition to these positive roles, FKH also represses the expression of salivary duct-specific genes in the secretory cells. In this paper, yet another role for fkh in the salivary gland was discovered: the maintenance of SPCG expression (Abrams, 2005).

fkh is a direct transcriptional target of Scr and Exd and the temporal expression of CrebA and the presence of consensus Scr/Exd-binding sites upstream of the gene suggest that CrebA may also be directly controlled by Scr and its co-factors. Late expression of CrebA, however, requires fkh, as does late expression of fkh itself. This study shows that Fkh functions directly to maintain CrebA expression in the salivary gland. Based on the requirement for CrebA for expression of the SPCGs at all embryonic stages and the requirement for fkh only at late stages, these data support a model in which CrebA controls the expression of the SPCGs and Fkh is required only because of its role in maintaining CrebA expression. A direct test of this model would be to express CrebA in the salivary glands of embryos missing fkh function; this experiment, unfortunately, could not be carried out because Fkh-independent drivers capable of providing high-level salivary gland-specific expression of CrebA are not yet available (Abrams, 2005).

A subset of the SPCGs that encode proteins required for retrograde vesicle transport from the Golgi to the ER are still expressed at low levels in CrebA mutants. However, in late but not early fkh mutants, expression of these genes is not above levels in surrounding tissues. It is proposed that the residual expression of the genes observed in the CrebA mutants would be controlled through other early transcription factor genes that, like CrebA, would require Scr and its co-factors for their initial expression and would require Fkh for maintaining late expression. Taken together, these studies suggest that regulation of salivary gland genes does not fit the simple paradigm suggested by studies of the C. elegans pharynx. The genes that specify the salivary gland (Scr/Exd/Hth) are distinct from the genes that activate and maintain gene expression in the organ. Moreover, no single gene takes over for the organ-specifying genes as even Fkh, the homolog of C. elegans PHA-4, is not required for expression of every salivary gland gene. In cases where Fkh is required, it is often indirect, such as with the SPCGs. Fkh does appear to have direct roles, however, much later in development, as demonstrated by regulation studies involving the sgs glue genes. Thus, the involvement of Fkh in salivary gland development and function is complicated and more consistent with the complexity of gene regulation seen in the liver than that suggested for the C. elegans pharynx. The existence of a single 'organ-specifying gene' may be more the exception than the rule (Abrams, 2005).

Single-cell transcriptomes reveal diverse regulatory strategies for olfactory receptor expression and axon targeting

The regulatory mechanisms by which neurons coordinate their physiology and connectivity are not well understood. The Drosophila olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. Each ORN type expresses a unique olfactory receptor, or a combination thereof, and sends their axons to a stereotyped glomerulus. Using single-cell RNA sequencing, this study identified 33 transcriptomic clusters for ORNs, and 20 were mapped to their glomerular types, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN types. Each ORN type expresses hundreds of transcription factors. Transcriptome-instructed genetic analyses revealed that (1) one broadly expressed transcription factor (Acj6) only regulates olfactory receptor expression in one ORN type and only wiring specificity in another type, (2) one type-restricted transcription factor (Forkhead) only regulates receptor expression, and (3) another type-restricted transcription factor (Unplugged) regulates both events. Thus, ORNs utilize diverse strategies and complex regulatory networks to coordinate their physiology and connectivity (Li, 2020).

Using plate-based scRNA-seq, high-quality transcriptomes were analyzed of 1,016 antennal ORNs at a mid-pupal stage, when ORNs are completing their axon targeting to their cognate glomeruli and a subset of ORNs start to express olfactory receptors. The smaller number of transcriptomic clusters compared to glomerular types (44 for antennal ORNs) may result from the following: (1) for some ORN types, not enough cells were captured to reach the minimal requirement of forming a cluster; and (2) closely related ORN types may form one transcriptomic cluster (e.g., cluster 9 corresponds to two ORN types, VM5d and VM5v). Besides olfactory receptor neurons, there are also other sensory cells in the third segment of the antenna; for example, hygro- and thermo-sensory neurons in the sacculus and arista. It has been shown that all those neurons express Ir25a and Ir93a, and different subsets express Ir21a, Ir40a, Ir68a, and Gr28b in adult flies. scRNA-seq data show that Ir25a is broadly expressed in many ORN types, but all other aforementioned genes are not expressed at 48hAPF. Due to the lack of specific markers, these cells could not be identified. Compared to the large number of ORNs, these other cells likely constitute a minority of cells (Li, 2020).

Understanding of how developing neurons coordinately regulate physiological properties and connectivity is limited to only a few examples. This study found that even in the same group of neurons (Drosophila ORNs), the coordination of these two features uses diverse transcriptional strategies. On one hand, the broadly expressed acj6 regulates receptor expression but not wiring in one ORN type and wiring but not receptor expression in a second type. On the other hand, the type-restricted unpg regulates both receptor expression and wiring specificity in all ORN types that express unpg. However, within the V-ORNs, the type-restricted fkh regulates the expression of both co-receptors, but not wiring, whereas unpg regulates only one of the two co-receptors, arguing against a simple regulatory relationship. The complexity of the regulatory network inferred from this study is, perhaps, a result of the evolution of different ORN types in a piecemeal fashion, as reflected by their utilizing three distinct families of chemoreceptors as olfactory receptors. Untangling this complexity requires future studies to systematically identify transcriptional targets of these TFs and investigate their regulatory relationship (Li, 2020).

In conclusion, scRNA-seq in developing Drosophila ORNs enabled us to map 20 transcriptomic clusters to glomerular types. This reinforces the idea that neuronal transcriptomic identity corresponds well with anatomical and physiological identities defined by connectivity and function in well-defined neuronal types. The genetic analyses further suggest that ORNs utilize diverse regulatory strategies to coordinate their physiology and connectivity. Given that each ORN type expresses hundreds of TFs, it is remarkable that the loss of a single TF, unpg, can result in profound disruption of receptor expression and wiring specificity, two most fundamental properties of sensory neurons (Li, 2020).


GENE STRUCTURE

Bases in 5' UTR - 707

Introns - none

Bases in 3' UTR - 1762


PROTEIN STRUCTURE

Amino Acids - 510

Structural Domains

The protein structural domain, termed the Forkhead domain, of which Fork head protein is the archetype, consists of 114 amino acids, having a globular structure with 37% alpha-helix content. Specific interaction with DNA is mediated by two contact regions, separated by one turn of DNA (Kaufman, 1994).


fork head: Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

date revised: 30 Dec 96 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.