Table of contents

Evolution of the hedgehog family in chordates

The hedgehog family of intercellular signaling molecules have essential functions in patterning both Drosophila and vertebrate embryos. Drosophila has a single hedgehog gene, while vertebrates have evolved at least three types of hedgehog genes (the Sonic, Desert and Indian types) by duplication and divergence of a single ancestral gene. Vertebrate Sonic-type genes typically show conserved expression in the notochord and floor plate, while Desert- and Indian-type genes have different patterns of expression in vertebrates from different classes. To determine the ancestral role of hedgehog in vertebrates, the hedgehog gene family was characterized in amphioxus. Amphioxus is the closest living relative of the vertebrates and develops a similar body plan, including a dorsal neural tube and notochord. A single amphioxus hedgehog gene, AmphiHh, was identified and is probably the only hedgehog family member in amphioxus, showing the duplication of hedgehog genes to be specific to the vertebrate lineage. AmphiHh expression was detected in the notochord and ventral neural tube, two tissues that express Sonic-type genes in vertebrates. This shows that amphioxus probably patterns its ventral neural tube using a molecular pathway conserved between amphioxus and vertebrates. AmphiHh was also expressed on the left side of the pharyngeal endoderm. This is reminiscent of the left-sided expression of Sonic hedgehog in chick embryos, which forms part of a pathway controlling left/right asymmetric development. These data show that notochord, floor plate and possibly left/right asymmetric expression are ancestral sites of hedgehog expression in vertebrates and amphioxus. In vertebrates, all these features have been retained by Sonic-type genes. This may have freed Desert-type and Indian-type hedgehog genes from selective constraint, allowing them to diverge and take on new roles in different vertebrate taxa (Shimeld, 1999).

Hedgehog in C. elegans

Components of the KDM7 family of histone demethylases are implicated in neuronal development and one member, PHF8, is also found mutated in cases of X-linked mental retardation. However, how PHF8 regulates neurodevelopmental processes and contributes to the disease is still largely missing. This study shows that the catalytic activity of a PHF8 homolog in Caenorhabditis elegans, JMJD-1.2 (see Drosophila Jarid2), is required non-cell autonomously for proper axon guidance. Loss of JMJD-1.2 deregulates the transcription of the Hedgehog-related genes wrt-8 and grl-16 whose overexpression is sufficient to induce the axonal defects. Deficiency of either wrt-8 or grl-16, or reduced expression of homologs of genes promoting Hedgehog signaling restore correct axon guidance in jmjd-1.2 mutant. Genetic and overexpression data indicate that Hedgehog-related genes act on axon guidance through actin remodelers. Thus, this study highlights a novel function of jmjd-1.2 in axon guidance that may be relevant for the onset of X-linked mental retardation and provides compelling evidences of a conserved function of the Hedgehog pathway in C. elegans axon migration (Riveiro, 2017).

Hedgehog in insects

The origin of new morphological characters is a long-standing problem in evolutionary biology. Novelties arise through changes in development, but the nature of these changes is largely unknown. In butterflies, eyespots have evolved as new pattern elements that develop from special organizers called foci. Formation of these foci is associated with novel expression patterns of the Hedgehog signaling protein, its receptor Patched, the transcription factor Cubitus interruptus, and the engrailed target gene, all of which break the conserved compartmental restrictions on this regulatory circuit in insect wings. Redeployment of preexisting regulatory circuits may be a general mechanism underlying the evolution of novelties. hh is expressed in all cells of the posterior compartment of the butterfly wing disc, as it is in Drosophila, but hh transcript levels are increased in a striking pattern in cells just outside of the subdivision midlines at specific positions along the proximodistal axis of the wing. These domains of increased hh transcription flank cells that have the potential to form foci. Higher levels of hh transcripts accumulate specifically in cells that flank the developing foci. In the presence of high levels of Hh, Patched function is inhibited, resulting in the accumulation of the activator form of Ci. Because ptc is a direct target of Ci, cells that receive and transduce the Hh signal have increased levels of ptc transcription. Activation of ptc transcription, accompanied by the accumulation of Ci protein occurs in cells that are flanked by the domains of highest hh transcription and are destined to become eyespot foci. these results indicate that the Hh signal is received and transduced by cells that will differentiate as foci. These expression patterns break the A/P compartmental restrictions on gene expression known in Drosophila. During the course of eyespot evolution, there is a relaxation of the strict En-mediated repression of ci that occurs in the posterior compartment of Drosophila. During focal establishment, en and invected are targets, rather than inducers of Hh signaling. In most species of butterflies, eyespots are found only in the posterior compartment of the wing. But in those species in which eyespots are found in the anterior compartment, both En/Inv and Ci are coexpressed in eyespot foci, including the one in the anterior compartment. Thus the expression of the Hh signaling pathway and en/inv is associated with the development of all eyespot foci and has become independent of A/P compartmental restrictions. It is suggested that during eyespot evolution, the Hh-dependent regulatory circuit that establishes foci is recruited from the circuit that acts along the A/P boundary of the wing. This recruitment of an entire regulatory circuit through changes in the regulation of a subset of components increases the facility with which new developmental functions can evolve and may be a general theme in the evolution of novelties within extant structures (Keys, 1999).

Insects can be grouped into two main categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), has been studied focusing on expression patterns of the three key signaling molecules, hedgehog, wingless and decapentaplegic, which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anterior/posterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, expression pattern of dpp is significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence is found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology (Niwa, 2000).

In the allocation phase of Drosophila 5h embryos, wg and hh are expressed in a stripe along the A/P compartment boundary and in the posterior region of each segment, respectively. However, dpp is expressed throughout the dorsal region and then in the dorsal side of the wg stripe. Later, the expression changes to give two thin stripes running anteroposteriorly along the length of the embryo. Wg, but not Dpp, is responsible for initial specification of the limb primordia with a distal identity and for induction of Dll. A model for the allocation of the limb primordium (the G-H model) is presented. A stripe of Wg induces the limb primordium expressing Dll. Repression of Dll by Dpp from the dorsal side and by Spitz (Drosophila EGF) from the ventral side limits the limb formation only in the lateral position. Then, Dpp specifies proximal cell identity in the primordium in a concentration-dependent manner. In Gryllus and Schistocerca embryos, expression of wg is detected in a stripe along the A/P compartment boundary of the body segment. In Gryllus embryos, expression of dpp is first detected along the periphery of the germ band. Similar expression patterns have been observed in Tribolium. Although the cricket and grasshopper belong to the same Orthoptera, the expression patterns of Sadpp are more complicated than those of Gbdpp. In Schistocerca embryos at early stages, Sadpp is expressed in two partial stripes in each hemisegment, intrasegmentally and intersegmentally, paralleling the D/V axis. The intrasegmental stripes extend along both dorsal and ventral sides of the presumptive leg field. Early expression patterns of Gbdpp resemble those of Dmdpp or Tcdpp more closely than those of Sadpp. Thus, the wg expression pattern appears conserved in the allocation phase, while early expression patterns of dpp seems divergent even in the Orthoptera. Thus, more data are necessary to judge whether the G-H model is also applicable as a model for initiation of limb formation in other insects (Niwa, 2000 and references therein).

In Phase 2, in the Drosophila leg imaginal disc, hh is expressed in the posterior compartment of the disc, determining the A/P pattern, and induces dpp and wg expression in the dorsal and ventral side of the A/P boundary, respectively. They act cooperatively in a concentration-dependent manner to organize the P/D axis and induce expression of Dll at the center of the disc. In Gryllus and Schistocerca limb buds, since hh and wg are expressed in the posterior and the ventral side of the A/P boundary, respectively, their functions during limb development should be conserved among the fly, cricket, beetle and grasshopper. However, expression patterns of Gbdpp are considerably different from those of Drosophila dpp: Gbdpp expression is limited to a dorsal stripe, transiently around the time of limb bud emerging, at stage 6-7. At this time, expression of Dll was found in the distal tip of the limb bud. This transient expression pattern also occurs in Schistocerca embryos. In Drosophila, removal of Dpp signaling prior to the second larval instar results in loss of Dll expression, while later removal of Dpp does not affect Dll expression, indicating that Dpp is required for the initiation but not maintenance of Dll transcription. Thus, it is reasonable to consider that transient dpp expression is enough to induce expression of Dll, which is required for the P/D leg pattern formation (Niwa, 2000 and references therein).

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. Patterns of hedgehog, wingless, and decapentaplegic expression were examined during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns are essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, it is concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM (Mito, 2002).

In the cricket leg, the single layer of surface epidermal cells forms precise patterns of structures, including bristles and spines, in the overlying cuticle. The regional specialization of the leg epidermal cells is evident along the three major axes of the leg, which include the anteroposterior (A/P), dorsoventral (D/V), and proximodistal (P/D) axes. The P/D axis relates to the distance from the body trunk, while the A/P and D/V axes unite to form the single circumferential axis. When a metathoracic leg of a Gryllus nymph in the third instar is amputated at the tibia, the distal missing part is completely recovered after about 30-35 days through four molts subsequent to the amputation. Just after the amputation, a trachea running along the P/D axis, reticulate fat bodies, and muscles are observed in sagittal sections. By 6 h after amputation, wounded muscles already start to degenerate, while hemocytes aggregate in the wound to form a scab. By day 2, epidermal cells migrate over the wound surface, and epidermal continuity is restored underneath the scab. Cell proliferation can be detected in epidermis lining the scab during this process. By day 5, the wound epidermis thickens to form a regeneration bud, or blastema, and cell proliferation is greatly activated in the blastema. Cells in the blastema lose their differentiated character and start to grow. By day 7, the blastema becomes the primordia of the tibia and tarsus concomitant with muscle recovery. By day 10, the boundary of the tibia-tarsus is visible in the blastema. Finally, all of the structures that normally lie distal to the point of amputation are restored (Mito, 2002).

In normally developing cricket leg buds, hh i expressed in the posterior (P) compartment, while wg and dpp are expressed in the ventral (V) side and dorsal (D) side of the anteroposterior (A/P) boundary, respectively. In a normal leg at the stage corresponding to the regeneration samples, hybridization signals for hh are weakly detected in epidermal cells located in the posterior region, whereas the expressions of wg and dpp are not observed. In contrast, the induced expressions of hh, wg, and dpp are observed in the blastemata of regenerating legs. The expression signals of hh are localized on the posterior side of the leg epidermis. The localization of the En protein was examined in cryosections with the monoclonal antibody mAb4D9. Signals were detected in both sagittal and transverse sections, indicating that En is localized in the posterior half of epidermis and supporting the results for hh. In the transverse sections, the En expression domain looks slightly broader than that of hh (Mito, 2002).

The expression pattern of wg is clearly observed in the ventral region of the blastema with a distal-to-proximal gradient in the signal intensity. The signals of the dpp expression are much weaker than the wg signals. Furthermore, there was variation in the expression patterns. Since such variation was not observed in the wg expression pattern, it is considered that the expression pattern of dpp is dynamically changed, as observed during leg development. The observed expression patterns of dpp were classified mainly into three types: Type I, with signals restricted in dorsodistal epithelial cells of the blastema, where intense non-specific signals appear in the trachea due to longer staining reactions; Type II, with signals observed in dorsal and distal epithelial cells, and weakly in ventral cells; and Type III, with signals so weak that no pattern is discernible (n=24). Type I expression patterns are observed in the early stages, while Type II patterns are observed even in the later stages (~4 days). Therefore, it is reasonable to consider that the expression pattern of dpp changes from Type I to Type II as the regeneration proceeds (Mito, 2002).

The expression patterns of wg and dpp in the blastema are comparable to those in the leg bud of the cricket embryo. In particular, the discrete expression of dpp (Type I) observed in the blastema is also observed in the dorsal side along the A/P boundary in the cricket leg bud, which differs from the expression of dpp in the leg imaginal disc. However, a major difference between the leg bud and blastemata is the size of the wg/dpp expression boundary: the boundary becomes a line in the blastema, similar to the apical ectodermal ridge of vertebrate limb buds, rather than a point in the insect leg bud. After wound healing, the restoration of the epidermal continuity results in the formation of a D/V boundary where dpp-expressing epidermal cells abut wg-expressing cells, which possibly initiates the formation of the P/D axis in the regeneration blastema (Mito, 2002).

Wnt/β-catenin and Hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. This study used the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. Wnt/β-catenin signaling was found to act upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. The target gene sets of the Wnt/β-catenin and Hh pathways were determined; the growth zone signaling center activates a much greater number of genes and the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal (see Drosophila caudal) and Tc-twist (see Drosophila twist). Furthermore, the Wnt pathway is required for hindgut development and Tc-senseless (see Drosophila senseless) as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function (Oberhofer, 2014).

Dorsoventral axis is coordinated with anteroposterior patterning by hedgehog signaling in the spider

The early embryo of the spider Achaearanea tepidariorum is emerging as a model for the simultaneous study of cell migration and pattern formation. A cell cluster internalized at the center of the radially symmetric germ disc expresses the evolutionarily conserved dorsal signal Decapentaplegic. This cell cluster migrates away from the germ disc center along the basal side of the epithelium to the germ disc rim. This cell migration is thought to be the symmetry-breaking event that establishes the orientation of the dorsoventral axis. In this study, knockdown of a patched homolog, At-ptc, that encodes a putative negative regulator of Hedgehog (Hh) signaling, prevents initiation of the symmetry-breaking cell migration (see The formation and migration of CM cells during early Achaearanea embryogenesis and cumulus-shift defects caused by At-ptc1 dsRNA injection). Knockdown of a smoothened homolog, At-smo, shows that Hh signaling inactivation also arrests the cells at the germ disc center, whereas moderate inactivation results in sporadic failure of cell migration termination at the germ disc rim. hh transcript expression patterns indicated that the rim and outside of the germ disc are the source of the Hh ligand. Analyses of patterning events suggests that in the germ disc, short-range Hh signal promotes anterior specification and long-range Hh signal represses caudal specification. Moreover, negative regulation of Hh signaling by At-ptc appears to be required for progressive derepression of caudal specification from the germ disc center. Cell migration defects caused by At-ptc and At-smo knockdown correlated with patterning defects in the germ disc epithelium. It is proposed that the cell migration crucial for dorsoventral axis orientation in Achaearanea is coordinated with anteroposterior patterning mediated by Hh signaling (Akiyama-Oda, 2010).

The zebrafish iguana locus encodes Dzip1, a novel zinc-finger protein required for proper regulation of Hedgehog signaling

Members of the Hedgehog (Hh) family of intercellular signaling molecules play crucial roles in animal development. Aberrant regulation of Hh signaling in humans causes developmental defects, and leads to various genetic disorders and cancers. A novel regulator of Hh signaling has been characterized through the analysis of the zebrafish midline mutant iguana (igu). Mutations in igu lead to reduced expression of Hh target genes in the ventral neural tube, similar to the phenotype seen in zebrafish mutants known to affect Hh signaling. Contradictory at first sight, igu mutations lead to expanded Hh target gene expression in somites. Genetic and pharmacological analyses reveal that the expression of Hh target genes in igu mutants requires Gli activator function but do not depend on Smoothened function. The results show that the ability of Gli proteins to activate Hh target gene expression in response to Hh signals is generally reduced in igu mutants both in the neural tube and in somites. Although this reduced Hh signaling activity leads to a loss of Hh target gene expression in the neural tube, the same low levels of Hh signaling appear to be sufficient to activate Hh target genes throughout somites because of different threshold responses to Hh signals. Hh target gene expression in igu mutants is resistant to increased protein kinase A activity that normally represses Hh signaling. Together, these data indicate that igu mutations impair both the full activation of Gli proteins in response to Hh signals, and the negative regulation of Hh signaling in tissues more distant from the source of Hh. Positional cloning reveals that the igu locus encodes Dzip1, a novel intracellular protein that contains a single zinc-finger protein-protein interaction domain. Overexpression of Igu/Dzip1 proteins suggests that Igu/Dzip1 functions in a permissive way in the Hh signaling pathway. Taken together, these studies show that Igu/Dzip1 functions as a permissive factor that is required for the proper regulation of Hh target genes in response to Hh signals (Sekimizu, 2004).

Hedgehog and axolotl tail regeneration

Tail regeneration in urodeles requires the coordinated growth and patterning of the regenerating tissues types, including the spinal cord, cartilage and muscle. The dorsoventral (DV) orientation of the spinal cord at the amputation plane determines the DV patterning of the regenerating spinal cord as well as the patterning of surrounding tissues such as cartilage. This phenomenon was investigated on a molecular level. Both the mature and regenerating axolotl spinal cord express molecular markers of DV progenitor cell domains found during embryonic neural tube development, including Pax6, Pax7 and Msx1. Furthermore, the expression of Sonic hedgehog (Shh) is localized to the ventral floor plate domain in both mature and regenerating spinal cord. Patched1 receptor expression indicates that hedgehog signaling occurs not only within the spinal cord but is also transmitted to the surrounding blastema. Cyclopamine treatment revealed that hedgehog signaling is not only required for DV patterning of the regenerating spinal cord but also has profound effects on the regeneration of surrounding, mesodermal tissues. Proliferation of tail blastema cells is severely impaired, resulting in an overall cessation of tail regeneration, and blastema cells no longer expressed the early cartilage marker Sox9. Spinal cord removal experiments reveal that hedgehog signaling, while required for blastema growth is not sufficient for tail regeneration in the absence of the spinal cord. By contrast to the cyclopamine effect on tail regeneration, cyclopamine-treated regenerating limbs achieve a normal length and contain cartilage. This study represents the first molecular localization of DV patterning information in mature tissue that controls regeneration. Interestingly, although tail regeneration does not occur through the formation of somites, the Shh-dependent pathways that control embryonic somite patterning and proliferation may be utilized within the blastema, albeit with a different topography to mediate growth and patterning of tail tissues during regeneration (Schnapp, 2005).

The Hedgehog gene family of the cnidarian Nematostella vectensis

Hedgehog signaling is an important component of cell-cell communication during bilaterian development, and abnormal Hedgehog signaling contributes to disease and birth defects. Hedgehog genes are composed of a ligand ('hedge') domain and an autocatalytic intein ('hog') domain. Hedgehog (hh) ligands bind to a conserved set of receptors and activate downstream signal transduction pathways terminating with Gli/Ci transcription factors. This study identified five intein-containing genes in the anthozoan cnidarian Nematostella vectensis, two of which (NvHh1 and NvHh2) contain definitive hedgehog ligand domains, suggesting that to date, cnidarians are the earliest branching metazoan phylum to possess definitive Hh orthologs. Expression analysis of NvHh1 and NvHh2, the receptor NvPatched, and a downstream transcription factor NvGli (a Gli3/Ci ortholog) indicate that these genes may have conserved roles in planar and trans-epithelial signaling during gut and germline development, while the three remaining intein-containing genes (NvHint1,2,3) are expressed in a cell-type-specific manner in putative neural precursors. Metazoan intein-containing genes that lack a hh ligand domain have previously only been identified within nematodes. However, this study has identified intein-containing genes from both Nematostella and in two newly annotated lophotrochozoan genomes. Phylogenetic analyses suggest that while nematode inteins may be derived from an ancestral true hedgehog gene, the newly identified cnidarian and lophotrochozoan inteins may be orthologous, suggesting that both true hedgehog and hint genes may have been present in the cnidarian-bilaterian ancestor. Genomic surveys of N. vectensis suggest that most of the components of both protostome and deuterostome Hh signaling pathways are present in anthozoans and that some appear to have been lost in ecdysozoan lineages. Cnidarians possess many bilaterian cell-cell signaling pathways (Wnt, TGFβ, FGF, and Hh) that appear to act in concert to pattern tissues along the oral-aboral axis of the polyp. Cnidarians represent a diverse group of animals with a predominantly epithelial body plan, and perhaps selective pressures to pattern epithelia resulted in the ontogeny of the hedgehog pathway in the common ancestor of the Cnidaria and Bilateria (Matus, 2008).

Hedgehog signaling regulates gene expression in planarian glia

Hedgehog signaling is critical for vertebrate central nervous system (CNS) development, but its role in CNS biology in other organisms is poorly characterized. In the planarian Schmidtea mediterranea, hedgehog (hh; see Drosophila Hedgehog) is expressed in medial cephalic ganglia neurons, suggesting a possible role in CNS maintenance or regeneration. RNA sequencing of planarian brain tissue was performed following RNAi of hh and patched (ptc; see Drosophila Patched), which encodes the Hh receptor. Two misregulated genes, intermediate filament-1 (if-1) and calamari (cali), were expressed in a previously unidentified non-neural CNS cell type. These cells expressed orthologs of astrocyte-associated genes involved in neurotransmitter uptake and metabolism, and extended processes enveloping regions of high synapse concentration. It is proposed that these cells are planarian glia. Planarian glia were distributed broadly, but only expressed if-1 and cali in the neuropil near hh+ neurons. Planarian glia and their regulation by Hedgehog signaling present a novel tractable system for dissection of glia biology (Wang, 2016).

Table of contents

hedgehog continued: Biological Overview | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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