heartless/FGF receptor 1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - heartless

Synonyms - DFR1, Fibroblast growth factor receptor 1, DFGF-R2

Cytological map position - 90C-D

Function - Receptor tyrosine kinase

Keywords - FGF pathway, central nervous system, heart, muscle, hindgut, foregut, wing, leg, eye and brain

Symbol - htl

FlyBase ID:FBgn0010389

Genetic map position - 3-[62]

Classification - Immunoglobulin - C2-type-domain - FGF receptor homolog

Cellular location - surface



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Rothenbusch-Fender, S., Fritzen, K., Bischoff, M. C., Buttgereit, D., Oenel, S. F. and Renkawitz-Pohl, R. (2017). Myotube migration to cover and shape the testis of Drosophila depends on Heartless, Cadherin/Catenin, and myosin II. Biol Open 6(12):1876-1888. PubMed ID: 29122742
Summary:
During Drosophila metamorphosis, nascent testis myotubes migrate from the prospective seminal vesicle of the genital disc onto pupal testes and then further to cover the testes with multinucleated smooth-like muscles. This study shows that DWnt2 is likely required for determination of testis-relevant myoblasts on the genital disc. Knock down of FGFR Heartless by RNAi and a dominant-negative version revealed multiple functions of Heartless, namely regulation of the amount of myoblasts on the genital disc, connection of seminal vesicles and testes, and migration of muscles along the testes. Live imaging indicated that the downstream effector Stumps is required for migration of testis myotubes on the testis towards the apical tip. After myoblast fusion, myosin II is needed for migration of nascent testis myotubes, in which Thisbe-dependent FGF signaling is activated. Cadherin-N is essential for connecting these single myofibers and for creating a firm testis muscle sheath that shapes and stabilizes the testis tubule. Based on these results, a model is proposed for the migration of testis myotubes in which nascent testis myotubes migrate as a collective onto and along the testis, dependent on FGF-regulated expression of myosin II.
Irizarry, J. and Stathopoulos, A. (2015). FGF signaling supports Drosophila fertility by regulating development of ovarian muscle tissues. Dev Biol [Epub ahead of print]. PubMed ID: 25958090
Summary:
The thisbe (ths) gene encodes a Drosophila FGF, and mutant females are viable but sterile suggesting a link between FGF signaling and fertility. Ovaries exhibit abnormal morphology including lack of epithelial sheaths, muscle tissues that surround ovarioles. This study investigated how FGF influences Drosophila ovary morphogenesis. Heartless (Htl) FGF receptor was found expressed within somatic cells at the larval and pupal stages, and phenotypes were uncovered using RNAi. Differentiation of terminal filament cells was affected, but this effect did not alter ovariole number. In addition, proliferation of epithelial sheath progenitors, the apical cells, was decreased in both htl and ths mutants, while ectopic expression of the Ths ligand led to these cells' over-proliferation suggesting that FGF signaling supports ovarian muscle sheath formation by controlling apical cell number in the developing gonad. Additionally, live imaging of adult ovaries was used to show that htl RNAi mutants, hypomorphic mutants in which epithelial sheaths were present, exhibited abnormal muscle contractions. Collectively, these results demonstrate that proper formation of ovarian muscle tissues is regulated by FGF signaling in the larval and pupal stages through control of apical cell proliferation and is required to support fertility.
BIOLOGICAL OVERVIEW

Fibroblast growth factor receptor 1, called Heartless, to reflect a complementary function to Breathless, the FGF-R homolog expressed in trachea, is expressed in putative muscle precursor cells, cells in the central nervous system (CNS), and in cells surrounding the hindgut and foregut (Shishido, 1993). Messenger RNA is also detected in the morphogenetic furrow and in the posterior region of the eye disc and around the proliferation center of the brain. These results suggest that FR1 is involved in the development of mesodermal and neuronal cells (Emori, 1993).

Breathless, the second FGF-R homolog in Drosophila is involved in differentiation of trachea cells and central nervous system glial cells. In both cases, interruption of breathless function results in altered cell mobility (Reichman-Fried, 1995). The involvement of BTL in cell mobility is a common theme for FGF-R receptors in other species. A C. elegans FGF-R homolog (EGL-15) regulates cell migrations of the sex myoblasts. The C. elegans receptor is not required for motility of the sex myoblasts but rather for the normal guidance of myoblast migration. The signal mediated by EGL-15 may be an instructive one: for example, receiving an attractive gonadal signal that guides the sex myoblasts to their final positions. Another possibility: the EGL-15 mediated signal may permit other signals to determine the directionality of the migration process (DeVore, 1995). Several FGF-Rs are thought to allow signaling through other receptors, such as in activin-mediated mesoderm induction in Xenopus embryos (Cornell, 1995), and Sonic hedgehog-mediated limb bud formation (Laufer, 1994). Drosophila Breathless plays a permissive rather than an instructive role in the migration of tracheal branches (Reichman-Fried, 1995).

Because absence of the heart represents the most pronounced and defined phenotype of Fr1, Beiman (1996) and Gisselbrecht (1996) have called the gene Heartless. In late stage 11 embryos, the dorsal-most two rows of mesodermal cells split from the visceral mesoderm and give rise to the heart precursors. From stage 12 until after dorsal closure, these cells move dorsally together with the overlying ectoderm until they meet and fuse with the cells migrating from the opposite side. The cells of the dorsal row, which form the dorsal vessel, are contractile. They express Myosin, and are termed cardioblasts. The cells in the second row are rounded, and give rise to the pericardial cells that flank and support the dorsal vessel. In Fr1 mutant embryos, no Even-skipped positive heart precursors or dorsal somatic muscles can be detected, no expression of Mef2 in cardioblasts can be detected, and cardial cells, detectable through the use of anti-Myosin staining are absent (Beiman, 1996).

Fr1, or Heartless, is involved in the spreading of mesoderm over ectoderm. In mutants, cell fates are not induced in several lineages, including the visceral mesoderm, heart and the dorsal somatic muscles. The defects in the induction of cell fates are likely to result from failure of the mesoderm to spread over the ectoderm and receive patterning signals. The defective spreading can be circumvented in Fr1 mutant embryos by providing an ectopic Decapentaplegic patterning signal, leading to the formation of heart and dorsal muscle cells. Fr1 also appears to be required subsequently during the migration and morphogenesis of the different lineages. Expression of a dominant-negative Fr1 construct after the initial induction of cell fates gives rise to aberrant migration and organization of visceral mesoderm, heart and somatic muscles. Thus, a common role for Fr1 in cell migration and tissue organization may account for the pleiotropic defects of the Fr1 mutation (Beiman, 1996)

Ras1 is an important downstream effector of FR1 because an activated form of Ras1 partially rescues the Fr1 mutant phenotype. Ectopic expression of activated Ras1 in a wild-type genetic background leads to the marked overproduction of segmentally repeated eve-expressing cells. This effect is a manifestation of the involvement of Ras1 in signaling by another RTK, the Drosophila EGF receptor, which is also essential for mesodermal eve expression. Most significantly, the rescued eve-expressing cells are confined entirely to the dorsal mesoderm, implying that activated Ras1 is capable of at least partially inducing proper dorsolateral migration of the mesoderm in the absence of normal Fr1 receptor function (Gisselbrecht, 1996).

Although the intracellular signaling pathways mediated by FGF-R tyrosine kinases are not well established, phospholipase C-gamma has been shown to bind directly to human FGFR1, and members of the Ras pathway have been implicated in mesoderm induction in Xenopus (references in DeVore, 1995). Do Breathless and FR1 use the same or different ligands?

Heartless is required for the differentiation of a variety of mesodermal tissues in the Drosophila embryo, yet the identity of its ligand(s) has remained a mystery over the years. Two FGF genes, thisbe (ths; FGF8-like1) and pyramus (pyr; FGF8-like2), have been identified which probably encode the elusive ligands for this receptor. The two genes were named for the 'heartbroken' lovers described in Ovid's Metamorphoses because the genes are linked and the mutant phenotype exhibits a lack of heart. The genes exhibit dynamic patterns of expression in epithelial tissues adjacent to Htl-expressing mesoderm derivatives, including the neurogenic ectoderm, stomadeum, and hindgut. Embryos that lack ths+ and pyr+ exhibit defects related to those seen in htl mutants, including delayed mesodermal migration during gastrulation and a loss of cardiac tissues and hindgut musculature. The misexpression of Ths in wild-type and mutant embryos suggests that FGF signaling is required for both cell migration and the transcriptional induction of cardiac gene expression. The characterization of htl and ths regulatory DNAs indicates that high levels of the maternal Dorsal gradient directly activates htl expression, whereas low levels activate ths. It is therefore possible to describe FGF signaling and other aspects of gastrulation as a direct manifestation of discrete threshold readouts of the Dorsal gradient (Stathopoulos, 2004; Gryzik, 2004).

In Drosophila two known FGF receptors, Breathless (Btl) and Htl, are required for cell migratory events, tracheal migration, and mesodermal cell migration, respectively. Despite the annotation of the Drosophila genome sequence, only Bnl, the ligand of Btl, has thus far been identified. Bnl is not expressed in the right temporal-spatial pattern to serve as a ligand for Htl, and bnl mutant embryos do not display defects in early mesoderm morphogenesis. In addition, expression of dominant-negative forms of Btl does not produce mesoderm migration defects, and upon ectopic expression of Bnl within the ectoderm, only Btl-expressing cells show ectopic activation of MAP kinase. Together, these data suggest either that the ligand of Htl was missed in the genome annotation or that Htl might not be activated by FGF-like ligands. The identification is reported of two genes encoding for novel fly FGF homologs, which exhibit features consistent with being ligands for Htl (Gryzik, 2004).

The early expression of FGF8-like1 and FGF8-like2 is restricted to the neuroectoderm and thus corresponds to the predicted source of ligands required for Htl activation. The knockdown of the function of these genes by RNAi leads to a mesoderm cell phenotype that is very similar to that produced by mutations in the Htl receptor. Thus, FGF8-like1 and FGF8-like2 act non-cell-autonomously in the early embryo and are required for mesodermal cell shape changes during gastrulation. In addition, deletion of the two genes blocks activation of MAP kinase in early mesoderm cells. Genetic mapping using isogenic deletions shows that an interval containing 14 genes is responsible for the observed phenotypes. Because RNAi experiments are able to reproduce the migration phenotype of the deletion embryos, it is proposed that FGF8-like1 and FGF8-like2 together are responsible for the mesoderm defects observed in the deletion. In conclusion, these results strongly suggest that FGF8-like1 and FGF8-like2 represent ligands for Htl (Gryzik, 2004).

Although only one FGF ligand has been identified, Drosophila contains two FGF receptors, Breathless and Heartless (Htl). The Htl receptor is essential for the development of various mesoderm lineages, including cardiac tissues, hindgut visceral musculature, and the body wall muscles. Htl is initially expressed throughout the mesoderm of early embryos, and its activation is thought to trigger the spreading of the mesoderm across the internal surface of the neurogenic ectoderm. The mesoderm cells that come into contact with the dorsal ectoderm receive an inductive signal, Dpp, which triggers the expression of genes such as tinman (tin) and even-skipped (eve) that are required for the differentiation of cardiac and pericardial tissues, respectively. However, the mechanism of Htl activation is uncertain. It has been suggested that localized FGFs emanating from the neurogenic ectoderm might be responsible for Htl activation and provide an instructive cue that guides the migration of the mesoderm. An alternative view is that Htl plays a permissive role in migration by rendering the mesoderm competent to respond to an unknown localized signal (Stathopoulos, 2004).

Htl may be required both for the spreading of the mesoderm and the subsequent specification of cardiac tissues. The misexpression of Dpp throughout the ectoderm, in both dorsal and ventral regions, causes widespread activation of tin expression within the mesoderm. However, eve expression is not expanded, and it has been suggested that its activation depends on both Dpp signaling (normally achieved through spreading) and a second dorsally localized signal, possibly FGF. The analysis of the hindgut visceral musculature provides evidence for this dual role of FGF signaling in movement and specification. The activation of Htl is required for the initial spreading of the visceral mesoderm around the hindgut, as well as the subsequent differentiation of the hindgut musculature (Stathopoulos, 2004).

To investigate the function of FGF signaling in the early embryo, Htl ligands, which have eluded intensive genetic screens, have been identified. This study identified two closely linked genes, thisbe (ths) and pyramus (pyr), which encode FGF signaling molecules that appear to function in a partially redundant fashion to activate Htl. Ths and Pyr are most closely related to the FGF8/17/18 subfamily, which controls gastrulation as well as heart and limb development in vertebrates. Both ths and pyr are expressed in the neurogenic ectoderm during the spreading of the internal mesoderm in gastrulating embryos. These two genes also exhibit dynamic expression in the stomadeum, hindgut, and muscle attachment sites of older embryos. These sites of expression closely match the genetic function of htl described in previous studies. Moreover, a small deletion that removes both ths and pyr causes a variety of patterning defects, including delayed spreading of the mesoderm during gastrulation, the loss of cardiac tissues and hindgut visceral musculature, and abnormal patterning of the body wall muscles. These defects are similar to those seen for htl mutants. The ectopic expression of Ths in the early mesoderm of gastrulating embryos causes an expansion in the domain of Htl activation and a corresponding expansion in the eve expression pattern. These observations suggest that Htl controls both the spreading of the mesoderm and (along with Dpp and Wingless) the specification of pericardial cells. Computational methods were used to identify a mesoderm-specific enhancer for htl that is directly activated by peak levels of the maternal Dorsal gradient. Because ths is directly activated by low levels of the gradient, it is possible to describe gastrulation as a direct manifestation of discrete threshold readouts of the Dorsal gradient (Stathopoulos, 2004).

Several lines of evidence suggest that Ths and Pyr correspond to ligands for the Htl FGF receptor. (1) The two genes exhibit dynamic patterns of expression in tissues that influence the development of different mesoderm lineages, including the neurogenic ectoderm (early mesoderm spreading), muscle precursors (dorsal muscles, visceral muscles, and heart), hindgut (visceral musculature), and neuroblasts. (2) Mutant embryos that lack both ths+ and pyr+ gene activity exhibit defects that are quite similar to those seen in htl mutants, including a delay in mesoderm spreading during gastrulation, a reduction in dorsal mesoderm lineages, the loss of pericardial and cardial cells, the absence of hindgut musculature, and disruptions in the ventral oblique muscles. Misexpression of Ths throughout the early mesoderm causes an expansion in the Eve expression pattern, consistent with expanded induction of pericardial and/or dorsal muscle founder cells. (3) Expression of activated Htl or Ths rescues the loss of dorsal mesoderm lineages in mutant embryos. Pyr and Ths might also activate the Htl receptor at later stages of the life cycle. For example, a recent microarray screen identified CG13194 (pyr) and CG12443 (ths) transcripts in the body wall muscle of wing imaginal disks, where htl is also expressed (Stathopoulos, 2004).

Previous genetic screens failed to identify ths and pyr, possibly because of overlap in the activities of the encoded proteins, which are closely related members of the FGF8/FGF17/FGF18 subfamily of FGF signaling molecules. Mutations in either gene alone might be insufficient to produce robust dorsal-ventral patterning defects, as seen for htl mutants. Indeed, two related FGF genes, FGF8 and FGF24, are required for the patterning of the posterior mesoderm in zebrafish embryos. A mutation in the FGF8 gene alone causes a relatively mild phenotype, but a severe loss of the posterior mesoderm is observed when FGF24 activity is also diminished. Similarly, a small chromosome deficiency that removes both ths and pyr produces severe embryonic patterning defects (Stathopoulos, 2004).

It is conceivable that the spreading of the mesoderm across the internal surface of the neurogenic ectoderm is a simple manifestation of cell-cell contact. FGF signaling might cause each mesoderm cell to make maximal contact with the neurogenic ectoderm. According to this view, the Ths and Pyr ligands are permissive, and simply promote cell adhesion. An alternative view is that Ths and Pyr are spatially activated in a manner that promotes a temporal gradient of information that guides the movement of the mesoderm toward the dorsal ectoderm. The expression of dpERK is consistent with an early requirement of FGF signaling acting permissively to activate Htl and allow the mesoderm to start spreading. Staining is first seen throughout the mesoderm that is in contact with the ectoderm during early phases of gastrulation when these individual mesoderm cells come into contact with the neurogenic ectoderm. Later, dpERK staining is restricted to the leading edge of the mesoderm as it spreads into the dorsal ectoderm. These data support a model in which the FGF ligands, Ths and/or Pyr, activate Htl in an instructive manner that guides the mesoderm during the later stage of spreading. The expression of ths and pyr is consistent with this model of early permissive and late instructive roles of the ligands in Htl activation. Early, ths is expressed in a broad staining pattern, which might reflect a role in the promotion of initial contact between the mesoderm and neurogenic ectoderm. The restricted staining of both ths and pyr seen later may reflect a Ths/Pyr activity gradient emanating from increasingly more dorsal regions of the neurogenic ectoderm (Stathopoulos, 2004).

The combined ths and pyr expression profiles might produce a dynamic FGF activity gradient within the neurogenic ectoderm that guides the spreading of the mesoderm into the dorsal ectoderm. pyr expression is particularly dynamic, and rapidly lost in the neurogenic ectoderm, whereas ths expression is progressively lost first in ventral regions and then in more dorsal regions of the neurogenic ectoderm. In principle, this putative FGF gradient could provide a precise guidance cue for the coordinated spreading of the mesoderm into the dorsal ectoderm. However, it is also conceivable that the production of an FGF signaling gradient depends on post-transcriptional regulation, such as the translational regulation of mRNA expression or differential processing of FGF precursor proteins. For instance, the early embryonic enhancer isolated for ths does not support expression during germ-band elongation even though ths mRNA can be detected by in situ hybridization at this same stage. One interpretation of these results is that the ths mRNA is not synthesized during mesoderm migration. Differential degradation might help shape an FGF ligand activity gradient, as observed for FGF8. In addition, negative regulators of signaling downstream of the Htl receptor could contribute to the production and sharpening of an FGF signaling activity gradient (Stathopoulos, 2004).

Ths and Pyr are related to FGF signaling molecules that control both cell movement and differentiation. For example, EGL-17 directs the movement of the sex myo-blasts in the gonad and FGF8 is required for the migration of the mesoderm into the primitive streak of vertebrate embryos and later for heart development (Burdine, 1998; Sun, 1999; Reifers, 2000). Ths and Pyr are required both for the spreading of the mesoderm along the internal surface of the neurogenic ectoderm during gastrulation, as well as the subsequent induction of the dorsal mesoderm to form pericardial tissues (Stathopoulos, 2004).

Evidence that neurogenic expression of Ths and Pyr is important for the orderly spreading of the mesoderm was obtained by misexpressing Ths. Embryos misrepresenting Ths in the mesoderm exhibit a variety of defects including mild twisting of the germ band, abnormal patterning of the body wall muscles, and an expansion of cardiac tissues. The latter phenotype can be explained on the basis of expanded induction of dorsal mesoderm (there is at least a threefold increase in the number of Eve-expressing cells). A more uniform rescue phenotype was obtained when exogenous Ths products were expressed in the ectoderm using the 69B-gal4 transgene (Stathopoulos, 2004).

Once mesoderm spreading is complete, the leading edge of the mesoderm comes into contact with Dpp-expressing cells in the dorsal ectoderm. Dpp signaling might be sufficient for the activation of some of the target genes required for the patterning of the visceral mesoderm, such as tin and bap during stage 10. However, Dpp is insufficient for other inductive events such as the activation of tin and eve in different heart precursors. The loss of eve expression in ths;pyr and htl mutants does not appear to be due to a breakdown in mesoderm spreading. Although this spreading is delayed in the mutants, it does ultimately occur. The late activation of the Htl receptor may be essential for the induction of eve expression and the specification of pericardial tissues. Previous studies suggest that Dpp works together with another signal that may be localized in the dorsal ectoderm. This second signal appears to trigger Ras signaling because the expression of a constitutively activated form of Ras causes expanded expression of eve. Evidence that the second signal might be FGF stems from the analysis of a dominant-negative Htl receptor, which blocks the full expression of cardiac and pericardial gene markers after the mesoderm has spread. The present study considerably strengthens the case that FGF is the second signal that patterns the dorsal mesoderm. The misexpression of Ths in the mesoderm causes a substantial expansion in the dorsal mesoderm and the number of Eve-expressing cells. Moreover, ths and pyr are expressed in specific 'spots' within the dorsal ectoderm that are adjacent to the internal mesoderm where eve is activated. Thus, the simplest interpretation of the results is that FGF signaling controls both the spreading and patterning of the dorsal mesoderm (Stathopoulos, 2004).

The spreading and subsequent subdivision of the mesoderm into distinct dorsal and ventral lineages can be viewed as direct readouts of the Dorsal gradient. The identification of mesoderm enhancers for htl and dof/hbr/smsf based on clustering of Dorsal-binding sites (and associated sequence motifs) suggests that these genes are directly activated by high levels of the Dorsal gradient. Htl-dependent signaling is triggered by Ths and Pyr, which are selectively expressed in the neurogenic ectoderm in response to low levels of the Dorsal gradient. After spreading, dorsal mesoderm cells comes into contact with Dpp-expressing cells in the dorsal ectoderm, and are thereby induced to form dorsal lineages such as cardiac tissues. The same low levels of the Dorsal gradient that activate ths and pyr also activate sog expression and repress dpp. The Sog inhibitor ensures that Dpp signaling is restricted to the dorsal ectoderm. Thus, the differential regulation of Htl and its ligands determines the precise limits of mesoderm-ectoderm germ-layer interactions during gastrulation (Stathopoulos, 2004).


GENE STRUCTURE

Two species of DFR1 cDNAs have been isolated that differed with respect to their 5'-UTR. Analysis of the genomic organization reveals that DFR1 is composed of three exons. The entire coding region is contained within the third exon. Two distinct DFR1 transcripts possessing either the first or the second exon in combination with the third exon are generated by alternative splicing (Ito, 1994).

Exons - 3

Bases in 3' UTR - 523


PROTEIN STRUCTURE

Amino Acids - 714

Structural Domains

DFR1 and Breathless proteins contain, respectively, two and five immunoglobulin-like domains in the extracellular region (DFR1), and both receptors have a split tyrosine kinase domain in the intracellular region. The N-terminal portion of the protein contains a signal sequence involved in protein secretion. The extracellular and intracellular domain of the Fr1 protein are assigned to amino acid residues 20-294 and 319-714 respectively. The overall sequence of the kinase domain between FR1 and vertebrate FGF receptors is about 60%. The C-terminal tail of FR1 includes a tripeptide sequence, Tyr-Leu-Asp, which may provide the tyrosine phosophorylation site supposedly required for binding of SH2 of phosphoinositide-specific phospholipase C-gamma (Shishido, 1993).


FGF receptor 1 continued: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 June 2004  

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