BIOLOGICAL OVERVIEW

FGF8-like1 and FGF8-like2 encode putative ligands of the FGF receptor Htl and are required for mesoderm migration in the Drosophila gastrula

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).

pyramus and thisbe: FGF genes that pattern the mesoderm of Drosophila embryos

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).

Multiple lineages enable robust development of the neuropil-glia architecture in adult Drosophila

Neural remodeling is essential for the development of a functional nervous system and has been extensively studied in the metamorphosis of Drosophila. Despite the crucial roles of glial cells in brain functions, including learning and behavior, little is known of how adult glial cells develop in the context of neural remodeling. This study shows that the architecture of neuropil-glia in the adult Drosophila brain, which is composed of astrocyte-like glia (ALG) and ensheathing glia (EG), robustly develops from two different populations in the larva: the larval EG and glial cell missing-positive (gcm⁺) cells. Whereas gcm⁺ cells proliferate and generate adult ALG and EG, larval EG dedifferentiate, proliferate and redifferentiate into the same glial subtypes. Each glial lineage occupies a certain brain area complementary to the other, and together they form the adult neuropil-glia architecture. Both lineages require the FGF receptor Heartless to proliferate, and the homeoprotein Prospero to differentiate into ALG. Lineage-specific inhibition of gliogenesis revealed that each lineage compensates for deficiency in the proliferation of the other. Together, the lineages ensure the robust development of adult neuropil-glia, thereby ensuring a functional brain (Kato, 2020).

The neuropil-glial architectures in larval ventral nerve cords (VNC) and brains are composed of a small number of neuropil-glia, generated during the embryonic stage, whereas a large number of neuropil-glia form the glial architecture in the adult VNC (Enriquez, 2018) and brain (Awasaki, 2008; Kremer, 2017). The neuropil-glial architecture appears to change in concert with the remodeling of the brain. One group proposed a model for the generation of the adult neuropil-glia architecture, in which both larval ALG and EG undergo programmed cell death. Others reported that the cell bodies of larval ALG persist during pupal life, and they re-infiltrate their fine process into the neuropil at the late pupal stage. Neuropil-glia for an adult brain are generated from a small number of specific larval neuroblasts, termed as type II neuroblasts. However, they are not accountable for the entire architecture of neuropil-glia in an adult brain; the superiormost and the inferior regions of a brain remain uncovered by neuropil-glia generated by type II neuroblasts. Thus, a broad conceptual view of how the architecture of neuropil-glia undergoes remodeling remains to be elucidated (Kato, 2020).

This study has investigated the fate of larval ALG, EG and glial cell missing-positive (gcm⁺) cells, and found that the larval EG dedifferentiate, proliferate and redifferentiate into adult ALG and EG. Together with the gcm⁺ lineage, the larval EG lineage generates the adult neuropil-glia architecture. Finally, to investigate the interaction between the lineages in the development of the neuropil-glial architecture, this study evaluated whether one lineage compensates for the failure of gliogenesis in the other lineage (Kato, 2020).

The adult architecture of neuropil-glia is formed from two lineages: the differentiated larval EG lineage and the gcm⁺ lineage. Both lineages require htl for proliferation and Pros for differentiation of ALG. Each lineage compensates for the failure of the other to proliferate. Thus, the architecture of adult neuropil-glia develops robustly to ensure a functional adult brain (Kato, 2020).

Previous studies have suggested that the adult neuropil-glia are derived from larval neuroblasts, and larval neuropil-glia (both L-EG and L-ALG) undergo programmed cell death during metamorphosis. Given that neuroblasts give rise to gcm⁺ cells, which then generate mature glial cells, the fate of gcm⁺ cells was traced and were shown to generate adult neuropil-glia. Although this result is mostly consistent with previous reports, the area occupied by adult neuropil-glia derived from gcm⁺ cells was larger than that occupied by type II neuroblast-derived glia, as reported by Ren (2018). The results suggest that type II neuroblasts are not the sole origin of gcm⁺ cells that generate adult neuropil-glia. Furthermore, this study demonstrates that L-EG also participates in the genesis of adult neuropil-glia. Collectively, this study demonstrates that adult neuropil-glia are generated from gcm⁺ cells and L-EG (Kato, 2020).

The L-EG and gcm⁺ lineages undergo proliferation at the early pupal stage to generate the architecture of neuropil-glia in the adult, which is more complex and has 100-fold more glial cells than the larva. Adult flies process a vast amount of sensory information and exhibit complex behaviors, such as courtship, aggression, flight and walking. Accordingly, the structure of the adult brain is more elaborate, with more subdivided neuropils and 20-fold more neurons than the larval brain. Thus, the cell proliferation of both lineages leads to an increase in the number of glial cells, which likely occurs in coordination with the elaboration of adult neural circuits (Kato, 2020).

Neuron-glia interactions underlie the adjustment of glial cell numbers to neuronal structure through cell survival or cell proliferation in flies and vertebrates. This study shows that the FGF receptor htl is required for cell proliferation in both L-EG and gcm⁺ lineages in early pupal life. In flies, the htl ligand Pyramus, which is secreted from neurons, regulates the proliferation of htl-positive cortex glia during the larval stage (Avet-Rochex, 2012). Similarly, it is possible that htl ligands from neurons non-cell autonomously regulate the proliferation of lineage cells. Consistent with this notion, the data show that the total number of neuropil-glia in an area is limited, unless htl is constitutively activated. Thus, such non-cell autonomous regulation may adjust the numbers of neuropil-glia in adult neural circuits, thereby enabling the complex behavior of adult animals (Kato, 2020).

ALG and EG were present in both larval and adult brains, and each cell type shares morphological features and the expression of certain markers between stages. The data show the similarities in the developmental program of neuropil-glia for embryos/larvae and adults. It was ascertain that Pros is required for the differentiation of adult ALG, as it is for the development of embryonic/larval ALG. In embryos and larvae, Pros is also required to maintain the proliferative ability of ALG. The KD of pros in the lineages results in fewer GFP+ neuropil-glia at the 50% pupa stage in some areas of the brain. This implies that Pros is involved in the regulation of cell number in the development of adult neuropil-glia. The KD of pros in the cell lineages results in the appearance of Naz+ cells. The exact identity of these cells remains unknown. Rather than differentiating into adult ALG, the persisting weak Naz+ cells in adult brains may have acquired EG-like characteristics. However, it is difficult to assess this possibility because of the lack of markers that can clearly identify adult EG. Alternatively, they may be undifferentiated cells that have failed to differentiate into adult ALG. This notion is consistent with the fact that EG are Naz- and the undifferentiated cells at the 25% pupa stage are weak/faint Naz+ cells (Kato, 2020).

This study has established that htl is required for the proliferation of the L-EG and gcm⁺ lineages. In the development of embryonic/larval neuropil-glia, the number of ALG in htl^AB42 null mutants is similar to that in the control, suggesting that htl is not involved in cell proliferation in embryos. Instead, htl is required for the proper organization of IG/ALG during embryogenesis, and for extending the fine projections of larval ALG into the neuropil. In the current analysis, the cell-proliferation phenotype of htl KD emerged in the early pupal stage; thus, the role of htl in later stages was not specifically investigated. htl is also expressed in neuropil-glia at the 50% pupa stage, when their maturation is initiated. Thus, these results do not rule out the involvement of htl in the maturation of adult ALG in later pupal stages. Nevertheless, they show that the developmental programs for larva and adult neuropil-glia partially differ (Kato, 2020).

The plasticity of glial cells in terms of their differentiation is well established in vertebrates. Radial glial progenitors generate neurons and, subsequently, some of them generate oligodendrocytes and astrocytes during development. Radial glia progenitors persist in adults and transform into neural stem cells. Both astrocytes and NG2-glia [oligodendrocyte precursor cells (OPCs)] in adult brains proliferate after injury, and generate astrocytes and oligodendrocytes, respectively. In some cases, astrocytes may even transdifferentiate into neurons after injury. NG2-glia/OPCs also generate astrocytes in cell culture, in development and after injury. Some studies also reported that NG2 glia generate neurons in adult mice. Drosophila ALG also proliferate in response to injury in larval ventral nerve cords. However, whether they differentiate into different glial subtypes or neurons is currently unexplored. Foo (2017) reported the presence of adult neural progenitor cells in Drosophila that can generate glial cells and neurons in response to a defect in glial cells. In contrast, the changes of L-EG into the progenitor state, in which cells proliferate and then differentiate, are developmentally regulated. Thus, it may serve as an excellent model for the investigation of glial cell plasticity (Kato, 2020).

This study showed that adult neuropil-glia are derived from two lineages: L-EG and gcm⁺. What is the significance of having two lineages to establish the architecture of adult neuropil-glia? The peculiar distribution patterns of the lineages may relate to the evolution of insect brains. In numerous hemimetabolous insects (e.g. locusts and cockroaches), the mandibular, maxillary and labial ganglia, which are mostly occupied by L-EG lineage cells in flies, are detached from the protocerebrum, deutocerebrum and tritocerebrum, and located more inferiorly to (i.e., below) the esophagus. In these insects, L-EG may generate neuropil-glia for the mandibular, maxillary and labial ganglia, whereas gcm⁺ cells may generate neuropil-glia for the protocerebrum, deutocerebrum and tritocerebrum. In contrast, in flies, the two different populations appear to generate neuropil-glia for one structure (i.e. a brain) as all of the areas are fused together. This notion is consistent with the idea that the segmental distribution pattern of specific embryonic neuroblasts is evolutionarily conserved between Drosophila and hemimetabolous insects (Kato, 2020).

This study demonstrates that inhibition of glial proliferation in one lineage is rescued by the other lineage. This indicates that, regardless of evolutionary relevance, the multiple lineages (i.e., L-EG and gcm⁺ cells) ensure robust development of the adult neuropil-glia architecture. Such robust development of glial architecture has been reported in several contexts. In the thorax and brain, neuroblasts generate adult neuropil-glia and compensate for the failure of gliogenesis from other neuroblasts. This study reveals that the ability to compensate for deficiencies in a lineage is not restricted to neuroblast-derived glia and is greater in scope. Each lineage (L-EG or gcm⁺) rescues the entire loss of the other lineage. A similar mechanism is involved in the development of mouse oligodendrocytes. Two lineages of oligodendrocyte precursor cells (a ventral and a dorsal population) generate oligodendrocytes in embryos and in postnatal animals. One lineage completely takes over the brain when the other fails to develop, preventing any locomotor defect. Therefore, multiple lineages with glial ability to adjust to the surroundings guarantee the robust development of glial architecture. Thus, it is suggested that glial plasticity may be a widespread strategy for ensuring the robust development of functional brains (Kato, 2020).

Receptor tyrosine kinases CAD96CA and FGFR1 function as the cell membrane receptors of insect juvenile hormone

Juvenile hormone (JH) is important to maintain insect larval status; however, its cell membrane receptor has not been identified. Using the lepidopteran insect Helicoverpa armigera (cotton bollworm), a serious agricultural pest, as a model, this study determined that receptor tyrosine kinases (RTKs) cadherin 96ca (Cad96Ca), a protein with one n-terminal extracellular cadherin domain, and fibroblast growth factor receptor homologue (FGFR1) function as JH cell membrane receptors by their roles in JH-regulated gene expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of JH intracellular receptor MET1 and cofactor Taiman, and high affinity to JH III (see Juvenile Hormone III). Gene knockout of Cad96Ca and Fgfr1 by CRISPR/Cas9 in embryo and knockdown in various insect cells, and overexpression of CAD96CA and FGFR1 in mammalian HEK-293T cells all supported CAD96CA and FGFR1 transmitting JH signal as JH cell membrane receptors (Li, 2025).

Juvenile hormone (JH) plays a vital role in insect development and maintaining insect larval status. JH is an acyclic sesquiterpenoid known to enter cells freely via diffusion because of its lipid-soluble character. JH binds its intracellular receptor methoprene-tolerant protein (MET), a basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) family protein. MET forms a transcription complex with the transcription factor Taiman (TAI, also known as FISC, p160/SRC, and is a steroid receptor coactivator) to initiate gene transcription. An important gene in the JH pathway is Kruppel homologue 1 (Kr-h1), which encodes the zinc-finger transcription factor Kr-h1. Kr-h1 acts downstream of MET and is induced rapidly by JH to regulate larval growth and development. Other genes, for example, the early trypsin gene of Aedes aegypti (AaEt), JH-inducible 21 kDa protein (Jhp21), JH esterase (Jhe), vitellogenin (Vg), Drosophila JH-inducible gene 1 (Jhi-1), and JH-inducible gene 26 (Jhi-26) are regulated by JH (Li, 2025).

However, some studies suggest that cell membrane receptors also play essential roles in JH signaling. For example, in A. aegypti, receptor tyrosine kinases (RTKs) are involved in JH-induced rapid increases in inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation of calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate MET and TAI, resulting in Kr-h1 gene transcription in response to JH. JH III, also via RTKs, leads to rapid calcium release and influx in Helicoverpa armigera epidermal cells. JH induces MET phosphorylation to increase MET interacting with TAI, which enhances Kr-h1 transcription in H. armigera. In Drosophila melanogaster, JH through RTK and PKC protein kinase C (PKC) induces phosphorylation of Ultraspiracle (USP). The phenomenon that RTK transmits JH signal has long been predicted; however, the RTKs critical for JH signaling have yet to be identified from numerous RTKs in vivo (Li, 2025).

RTKs constitute a class of cell surface transmembrane proteins that play important roles in mediating extracellular to intracellular signaling. Humans carry approximately 60 RTKs, the Drosophila genome encodes 21 RTK genes, Bombyx mori has 20 RTKs, and the German cockroach genome identifies 16 RTKs. H. armigera has 20 RTK candidates with gene codes in the H. armigera genome. The cotton bollworm is a well-known and worldwide distributed agricultural pest in Lepidoptera, which threatens cotton and many other vegetable crops by rapidly producing resistance to various chemical insecticides and Bt-transgenic cotton. Using H. armigera as a model, this study focuses on identifying the RTKs functioning as the JH receptors and demonstrating the mechanism. The RTKs were screened sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH-regulated gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH-induced rapid increase of intracellular calcium levels, JH-induced phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH-regulated gene expression, maintaining larval status, JH-induced rapid increase of intracellular calcium levels, JH-induced phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously. These data not only improve knowledge of JH signaling and open the door to studying insect development but also present new targets to explore the new growth regulators to control the pest (Li, 2025).

JH regulates insect development through intracellular and membrane signaling; however, the cell membrane receptors and the mechanism are unclear. In this study, CAD96CA and FGFR1 were screened out from the total 20 RTKs in the H. armigera genome and identified as JH III cell membrane receptors, which transmit JH signal for gene expression to prevent pupation and have a high affinity to JH III (Li, 2025).

JH induces a set of gene expression, such as Kr-h1, Vg, Jhi-1, and Jhi-26 , a rapid intracellular calcium increase, phosphorylation of MET and TAI, and prevents pupation. Several RTKs are involved in JH III-induced gene expression and calcium increase; however, only Cad96ca, Nrk, Fgfr1, and Wsck are involved in the JH III-induced pupation delay, in which, only CAD96CA, NRK, and FGFR1 are involved in the JH-induced phosphorylation of MET1 and TAI, and only CAD96CA and FGFR1 can bind JH III. Therefore, CAD96CA and FGFR1 are finally determined as JH III receptors (Li, 2025).

CAD96CA (also known as Stitcher, Ret-like receptor tyrosine kinase) activates upon epidermal wounding in Drosophila and promotes growth and suppresses autophagy in the Drosophila epithelial imaginal wing discs. There is a CAD96CA in the genome of the H. armigera, which is without function study. This study reports that CAD96CA prevents pupation by transmitting JH signal as a JH cell membrane receptor. CAD96CA of other insects has a universal function of transmitting JH signal to trigger Ca2+ mobilization, as demonstrated by the study in Sf9 cell lines of S. frugiperda and S2 cell lines of D. melanogaster (Li, 2025).

FGFRs control cell migration and differentiation in the developing embryo of D. melanogaster. The ligand of FGFR is FGF in D. melanogaster. FGF binds FGFR and triggers cell proliferation, differentiation, migration, and survival. Three FGF ligands and two FGF receptors (FGFRs) are identified in Drosophila. The Drosophila FGF-FGFR interaction is specific. Different ligands have different functions. The activation of FGFRs by specific ligands can affect specific biological processes. The FGFR in the membrane of Sf9 cells can bind to Vip3Aa toxin. One FGF and one FGFR are in the H. armigera genome, which have yet to be studied functionally. The study found that FGFR prevents insect pupation by transmitting JH signal as a JH cell membrane receptor. Exploring the molecular mechanism and output by which multiple ligands transmit signals through the same receptor is exciting and challenging in future work (Li, 2025).

CAD96CA and FGFR1 have similar functions in JH signaling, including transmitting JH signal for Kr-h1 expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of transcription factors MET1 and TAI, and high affinity to JH III. CAD96CA and FGFR1 are essential in the JH signal pathway, and the loss-of-function of each is sufficient to trigger strong effects on pupation, suggesting they can transmit JH signal individually. The difference is that CAD96CA expression has no tissue specificity, and the Fgfr1 gene is highly expressed in the midgut. A possibility is that CAD96CA and FGFR1 play roles by forming homodimer or heterodimer with each other or with other RTKs in tissues, which needs to be addressed in future studies. CAD96CA and FGFR1 transmit JH III signals in three different insect cell lines, suggesting their conserved roles in other insects (Li, 2025).

Homozygous Cad96ca null Drosophila die at late pupal stagee, suggesting that CAD96CA is critical to insect pupation. This study further revealed that Cad96ca mosaic mutation by CRISPR/Cas9 caused precocious pupation in H. armigera, suggesting that CAD96CA plays roles to prevent pupation. Similarly, null mutant of Fgfr1 or Fgfr2 in mouse is embryonic lethal. Htl (Fgfr) homozygous mutant in D. melanogaster die during late embryogenesis, too, suggesting FGFR1 is important to embryogenesis. However, in H. armigera, Fgfr1 mosaic mutation mainly caused precocious pupation, suggesting FGFR1 is necessary to prevent pupation. The double mutation of Cad96ca and Fgfr1 caused earlier pupation and death compared to the single mutation of Cad96ca or Fgfr1. These data suggested that both CAD96CA and FGFR1 can transmit JH signal to prevent pupation independently and cooperatively. MET1 is the intracellular receptor of JH. Knockout of Met1 in the B. mori leads to precocious metamorphosis and death in the penultimate instar. In the Spodoptera exigua, knockout of Met1 also results in precocious metamorphosis and death in the penultimate and final instars. In D. melanogaster, both Met and Gce null mutants die during the larval-pupal transition. In the H. armigera, most of the Met1 mutants died during the transformation from the final instar larva to the pupa, too. These data suggest that JH via MET1 prevents pupation. In the triple mutants of Met1, Cad96ca, and Fgfr1, most larvae died at the 5th and 6th instar larval stages, which is much more serious than the Met1 mutation, or Cad96ca and Fgfr1 double mutation, because of the mutation both intracellular receptor MET1 and two membrane receptors CAD96CA and FGFR1 of JH. These data suggest that JH exerts a complete regulatory role through cell membrane receptors and intracellular receptor, because the cell membrane receptors regulate the phosphorylation of the intracellular receptor MET1 and the interacting transcription factor TAI. The results from different insects suggest that JH via MET1, CAD96CA, and FGFR1 play roles in preventing metamorphosis. In B. mori, after the knockout of JH acid methyltransferase (Jhamt) using TALENs, the larvae died during L1 or L2 larval stages. The knockdown of Jhamt in Drosophila by RNAi does not exhibit a visible effect on development. Knockdown of Jhamt by RNAi in the Tribolium larval stage results in precocious larval-pupal metamorphosis. Homozygous Jhamt -/- larvae in mosquitoes hatch normally and live to the final-instar larvae (L4), and die prior to pupation. The results from different insects suggest that the insect development in the early larval instars is less dependent on JH. The phenotypes from H. armigera CAD96CA and FGFR1 editing are consistent with the general knowledge that the primary role of JH is to maintain larval status and antagonize 20E-induced metamorphosis. The insulin/insulin-like growth factor signaling (IIS) plays a major role in promoting cell proliferation and determining the larval growth rate, and 20E promotes metamorphosis. These hormones cross talk to regulate insect growth and metamorphic development (Li, 2025).

The phenotypes of gene mutation in H. armigera are somehow different from those obtained by homozygous mutation in other animals, due to the mosaic mutation by CRISPR/Cas9. In addition, RNAi of Cad96ca and Fgfr1 was observed precocious pupation as was the case in CRISPR/Cas9, suggesting the RNAi can be used for the study of gene function in insects, especially when the gene editing is embryonic lethal (Li, 2025).

The knockdown of Cad96ca, Nrk, Fgfr1, and Wsck showed phenotypes resistant to JH III induction and the decrease of Kr-h1 and increase of Br-z7 expression, but knockdown of Vegfr and Drl only decreased Kr-h1, without increase of Br-z7. Br-z7 is involved in 20E-induced metamorphosis in H. armigera, whereas, Kr-h1 is a JH early response gene that mediates JH action and represses Br expression. The high expression of Br-z7 is possible due to the down-regulation of Kr-h1 in Cad96ca, Nrk, Fgfr1 and Wsck knockdown larvae. The different expression profiles of Br-z7 in Vegfr and Drl knockdown larvae suggest other roles of Vegfr and Drl in JH signaling, which need further study (Li, 2025).

This study found six RTKs that respond to JH induction by participating in JH-induced gene expression and intracellular calcium increase; however, they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH-induced phosphorylation of MET1 and TAI and binding to JH III. The RTKs transmitting JH signal were screened primarily by examining some of JH-induced gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential (Li, 2025).

In addition, GPCRs also play a role in JH signaling. JH triggers GPCR, RTK, PLC, IP3R, and PKC to phosphorylate Na+/K+-ATPase-subunit, consequently activating Na+/K+-ATPase for the induction of patency in L. migratoria vitellogenin follicular epithelium; JH activates a signaling cascade including GPCR, PLC, extracellular Ca2+, and PKC, which induces vitellogenin receptor (VgR) phosphorylation and promotes vitellogenin (Vg) endocytosis in Locusta migratoria. JH activates a signaling cascade including GPCR, Cdc42, Par6, and aPKC, leading to an enlarged opening of patency for Vg transport. In Tribolium castaneum, the dopamine D2-like receptor-mediated JH signaling promotes the accumulation of vitellogenin and increases the level of cAMP in oocytes. In H. armigera, GPCRs are involved in JH III-induced broad isoform 7 (Br-Z7) phosphorylation. In summary, these published results indicate that RTKs and GPCRs contribute to JH signaling on the cell membrane; however, the GPCR functions as JH receptor needs to be addressed in the future study. The RNAi of RTKs does not affect JH-induced Jhi-1 expression, which implies that other receptors exist, presenting a target for future study of the new JH III receptor (Li, 2025).

RTKs are high-affinity cell surface receptors for many cytokines, polypeptide growth factors, and peptide hormones. Up to now, there is no report that RTK binds lipid hormone. This study determined that CAD96CA and FGFR1 have a high affinity to JH III by MST and ITC methods after they were isolated from the cell membrane (Li, 2025).

The [3H]JH III detection method is used to determine Drosophila MET in vitro translation product binding JH III, and Tribolium MET binding JH III. However, the commercial production of [3H]JH III has ceased, whereas the microscale thermophoresis (MST) method is a widely used method to detect protein binding of small molecules. Therefore, MST was used in this study as the alternative method to measure the binding strengths of RTKs with JH III. Using the MST method, this study determined that the saturable specific binding of Helicoverpa MET1 to JH III is Kd of 6.38 nM, which is comparable to that report for Drosophila MET and Tribolium MET determined using [3H]JH III, confirming MST method can be used to detect protein binding JH III. The CAD96CA exhibited saturable specific binding to JH III with a Kd of 11.96 nM, and FGFR1 showed a Kd of 23.61 nM, which is higher than that of MET1 for JH III, suggesting lower binding affinity of RTKs than the intracellular receptor MET1 for JH III. A similar phenomenon is reported in another study, the binding affinities of steroid membrane receptors are orders of magnitude lower than those of nuclear receptors. NRK did not bind JH III. One possible explanation is that NRK has a low affinity to JH III and thus transmits JH signal without binding JH, or NRK requires association with other proteins to play roles. This study provides new evidence for the binding of lipid hormones by RTK and a new method to study the binding of ligands to receptors (Li, 2025).

This study also verified the affinity of CAD96CA and FGFR1 with JH III through the ITC method, determining their respective Kd values as 79.6 and 88.5 nanomolar. ITC is a versatile analytical method for the character of molecular interactions. ITC is applied in the membrane protein family, containing GPCRs, ion channels, and transporters. The ITC method requires relatively high ligand and receptor concentrations for better saturation curves. However, when a protein solution of 1000 nM, protein aggregation occurred, thus a protein solution was used with a concentration of 700 nM. The Kd value detected by ITC is slightly higher than the result of the MST method; the results are sufficient to confirm the high affinity of CAD96CA and FGFR1 binding to JH III (Li, 2025).

Although JH I and JH II are natural hormones for lepidopteran larvae, H. armigera and B. mori also respond to JH III. In B. mori Bm-aff3 cells, the effective concentrations (EC50) of JHs (JH I, JH II, JH III, JHA, or methyl farnesoate) to induce Kr-h1 transcription are 1.6x¹⁰10, 1.2x¹⁰10, 2.6x¹⁰10, 6.0x¹⁰8, and 1.1x¹⁰7 M, respectively. In cultures of wing imaginal discs from B. mori, 1-2 μM JH III promotes cuticle protein 4 gene expression (Deng et al., 2011). The effective concentration of JH III to induce rapid calcium increase in HaEpi cells is ≥1 &mi;M and 500 ng of 6th instar larva. JH III is a commercially available reagent; therefore, JH III was used to carry out the experiments in this study, and the results hypothesize the possibility of CAD96CA and FGFR1 binding other JHs in addition to JH III, which should be addressed in future study (Li, 2025).

MET is determined as JH’s intracellular receptor by its characters binding to JH and regulating Kr-h1 expression. In this study, cell membrane receptors CAD96CA and FGFR1 are also able to bind JH III and transmit JH III signal to regulate a set of JH III-induced gene expression including Kr-h1. Obviously, both intracellular receptor MET and cell membrane receptor CAD96CA and FGFR1 are involved in JH III signaling as receptors. JH III transmits signal by cell membrane receptor and intracellular receptor at different signaling stages, with cell membrane receptor CAD96CA and FGFR1 inducing rapid Ca2+ signaling, which regulates the phosphorylation of MET and TAI to enhance the function of MET for gene transcription, and the intracellular receptor MET regulates gene transcription by diffusion into cells based on its lipid characteristics (Li, 2025).

The study in human cell line HEK293 shows that overexpression of B. mori JH intracellular receptor MET2 and its cofactor SRC together in HEK293 cells may induce JH-dependent luciferase reporter expression through a plasmid that contains the JH specific kJHRE (JH response element containing the E-box core sequence for JH binding), suggesting JH can diffuse into cells to initiate a gene expression when the insect MET2 and SRC and kJHRE exist. This study showed that overexpressing CAD96CA or FGFR1 in HEK-293T cells elicits Ca2+ elevation under JH III induction, suggesting CAD96CA or FGFR1 transmit a rapid signal of JH III in HEK-293T cells, which might trigger further cellular responses of HEK-293T to JH III. These data suggest that both cell membrane receptors CAD96CA and FGFR1 and intracellular receptor MET of JH can respond to JH. These proteins might be used as switches to induce a gene expression or regulate cell fate in heterogeneous cells by JH induction when the side effects are determined (Li, 2025).

CAD96CA and FGFR1 were involved in JH III signaling, including maintaining larval status, JH III-induced rapid intracellular calcium increase, gene expression, and phosphorylation of MET and TAI. CAD96CA and FGFR1 had high affinity to JH III and were possible cell membrane receptors of JH III and other JHs. CAD96CA and FGFR1 had a general role in transmitting the JH III signal for gene expression in various insect cells, suggesting their conserved roles in other insects. JH III transmits the signal by either cell membrane receptor or intracellular receptor at different stages in the signaling, with JH III transmitting the signal by cell membrane receptor CAD96CA and FGFR1 to induce rapid Ca2+ signaling, which regulates the phosphorylation of MET and TAI to enhance the function of MET for gene transcription, and intracellular receptor MET regulates gene transcription by diffusion into cells based on its lipid characteristics. CAD96CA and FGFR1 can transmit JH signal to prevent pupation independently and cooperatively. This study presents a platform to identify the agonist or inhibitor of JH cell membrane receptors to develop an environmental-friendly insect growth regulator (Li, 2025).

Earlier Studies of Heartless

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?

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).

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).

date revised: 12 June 2023  

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