Phosphatidylinositol 4-kinase β is required for the ciliogenesis of zebrafish otic vesicle

The primary cilium, an important microtubule-based organelle, protrudes from nearly all the vertebrate cells. The motility of cilia is necessary for various developmental and physiological processes. Phosphoinositides (PIs) and its metabolite, PtdIns(4,5)P2, have been revealed to contribute to cilia assembly and disassembly. As an important kinase of the PI pathway and signaling, phosphatidylinositol 4-kinase β (PI4KB) is the one of the most extensively studied phosphatidylinositol 4-kinase isoform. However, its potential roles in organ development remain to be characterized. To investigate the developmental role of Pi4kb, especially its function on zebrafish ciliogenesis, we generated pi4kb deletion mutants using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technique. The homozygous pi4kb mutants exhibit an absence of primary cilia in the inner ear, neuromasts, and pronephric ducts accompanied by severe edema in the eyes and other organs. Moreover, smaller otic vesicle, malformed semicircular canals, and the insensitivity on sound stimulation were characteristics of pi4kb mutants. At the protein level, both in vivo and in vitro analyses revealed that synthesis of Pi4p was greatly reduced owing to the loss of Pi4kb. In addition, the expression of the Pi4kb-binding partner of neuronal calcium sensor-1, as well as the phosphorylation of phosphatidylinositol-4-phosphate downstream effecter of Akt, was significantly inhibited in pi4kb mutants. Taken together, our work uncovers a novel role of Pi4kb in zebrafish inner ear development and the functional formation of hearing ability by determining hair cell ciliogenesis.     

Hedgehog signalling is required for perichondral osteoblast differentiation in zebrafish

In tetrapod long bones, Hedgehog signalling is required for osteoblast differentiation in the perichondrium. In this work we analyse skeletogenesis in zebrafish larvae treated with the Hedgehog signalling inhibitor cyclopamine. We show that cyclopamine treatment leads to the loss of perichondral ossification of two bones in the head. We find that the Hedgehog co-receptors patched1 and patched2 are expressed in regions of the perichondrium that will form bone before the onset of ossification. We also show that cyclopamine treatment strongly reduces the expression of osteoblast markers in the perichondrium and that perichondral ossification is enhanced in patched1 mutant fish. This data suggests a conserved role for Hedgehog signalling in promoting perichondral osteoblast differentiation during vertebrate skeletal development. However, unlike what is seen during long bone development, we did not observe ectopic chondrocytes in the perichondrium when Hedgehog signalling is blocked. This result may point to subtle differences between the development of the skeleton in the skull and limb.     

BMP signalling regulates anteroposterior endoderm patterning in zebrafish

In vertebrates, the embryonic dorsoventral asymmetry is regulated by the bone morphogenetic proteins (Bmp) activity gradient. In the present study, we have used dorsalized swirl (bmp2b) and ventralized chordino (chordin) zebrafish mutants to investigate the effects of dorsoventral signalling on endoderm patterning and on the differentiation and positioning of its derivatives. Alterations of dorsoventral Bmp signalling do not perturb the induction of endodermal precursors, as shown by normal amounts of cells expressing cas and sox17 in swirl and chordino gastrulae, but affect dramatically the expression pattern of her5, a regulator of endoderm anteroposterior patterning in zebrafish. In particular, increased levels of Bmp signalling in chordino gastrulae are associated with a markedly reduced her5 expression domain, that may be abolished by injecting bmp2b mRNA. Conversely, in swirl mutants, lacking Bmp2b, the her5 expression domain is expanded. Thus, a gradient of Bmp2b signalling defines the extension of the her5 expression domain at gastrulation and the allocation of anterior endodermal precursors. A balanced Bmp2b signalling is also required for the normal development of the pancreas, as shown by the sharp reduction of the pancreatic primordium in swirl embryos and its expansion in chordino mutants. In the latter, at 3 days post-fertilization, the increased Bmp signalling does not compromise the endocrine/exocrine pancreas compartmentalization, but the right/left positioning of the pancreas and liver is randomized. Our results suggest that by regulating the expression of her5, the Bmp2b/Chordin gradient directs the anteroposterior patterning of endoderm in zebrafish embryos.     

Gene miles-apart is required for formation of otic vesicle and hair cells in zebrafish

Hearing loss is a serious burden to physical and mental health worldwide. Aberrant development and damage of hearing organs are recognized as the causes of hearing loss, the molecular mechanisms underlining these pathological processes remain elusive. Investigation of new molecular mechanisms involved in proliferation, differentiation, migration and maintenance of neuromast primordium and hair cells will contribute to better understanding of hearing loss pathology. This knowledge will enable the development of protective agents and mechanism study of drug ototoxicity. In this study, we demonstrate that the zebrafish gene miles-apart, a homolog of sphingosine-1-phosphate receptor 2 (s1pr2) in mammals, has an important role in the development of otic vesicle, neuromasts and survival of hair cells. Whole-mount in situ hybridization of embryos showed that miles-apart expression occurred mainly in the encephalic region and the somites at 24 h.p.f. (hour post fertilization), in the midbrain/hindbrain boundary, the brainstem and the pre-neuromast of lateral line at 48 h.p.f. in a strict spatiotemporal regulation. Both up- and downregulation of miles-apart led to abnormal otoliths and semicircular canals, excess or few hair cells and neuromasts, and their disarranged depositions in the lateral lines. Miles-apart (Mil) dysregulation also caused abnormal expression of hearing-associated genes, including hmx2, fgf3, fgf8a, foxi1, otop1, pax2.1 and tmieb during zebrafish organogenesis. Moreover, in larvae miles-apart gene knockdown significantly upregulated proapoptotic gene zBax2 and downregulated prosurvival gene zMcl1b; in contrast, the level of zBax2 was decreased and of zMcl1b enhanced by miles-apart overexpression. Collectively, Mil activity is linked to organization and number decision of hair cells within a neuromast, also to deposition of neuromasts and formation of otic vesicle during zebrafish organogenesis. At the larva stage, Mil as an upstream regulator of bcl-2 gene family has a role in protection of hair cells against apoptosis by promoting expression of prosurvival gene zMcl1b and suppressing proapoptotic gene zBax2.     

Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon

Midway between the anterior neural border and the midbrain-hindbrain boundary, two well-known local signalling centres in the early developing brain, is a further transverse boundary with putative signalling properties -- the zona limitans intrathalamica (ZLI). Here, we describe formation of the ZLI in zebrafish in relation to expression of sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), and to development of the forebrain regions that flank the ZLI: the prethalamus and thalamus. We find that enhanced Hh signalling increases the size of prethalamic and thalamic gene expression domains, whereas lack of Hh signalling leads to absence of these domains. In addition, we show that shh and twhh display both unique and redundant functions during diencephalic patterning. Genetic ablation of the basal plate shows that Hh expression in the ZLI alone is sufficient for diencephalic differentiation. Furthermore, acquisition of correct prethalamic and thalamic gene expression is dependent on direct Hh signalling. We conclude that proper maturation of the diencephalon requires ZLI-derived Hh signalling.     

Geminin is required for left-right patterning through regulating Kupffer's vesicle formation and ciliogenesis in zebrafish

Geminin plays an important role in coordinating the cell cycle with anterior–posterior patterning during embryonic development. However, whether it is involved in the regulation of left–right (LR) patterning remains unknown. Here, we reported that geminin is required for setting up heart and visceral laterality during zebrafish development. Defective heart and visceral laterality was observed in geminin morphants. Further study demonstrated that the left-sided nodal/spaw in the lateral plate mesoderm (LPM) as well as the sideness of its downstream targets lefty2 and lefty1 was perturbed in geminin morphants. Upstream of the left-sided Nodal signal along the regulatory cascade of LR asymmetry, knock down of geminin resulted in defective Kupffer’s vesicle (KV) formation and ciliogenesis rather than middle line defects. Predominant distribution of an antisense morpholino against geminin in dorsal forerunner cells (DFCs) led to defective KV morphogenesis and perturbed LR asymmetry, similar to those of geminin morphants, indicating a cell-autonomous role of geminin in regulating KV formation and ciliogenesis. Our results demonstrate that geminin is required for proper KV formation and ciliogenesis, thus playing an important part in setting up LR asymmetry.     

Expression of zebrafish hip: Response to Hedgehog signalling,comparison with ptc1 expression,and possible role in otic patterning

In zebrafish, Hedgehog (Hh) signalling is required to specify posterior otic identity. This presents a conundrum, as the nearest source of Hh to the developing inner ear is the ventral midline, in the notochord and floorplate. How can a source of Hh that is ostensibly constant with respect to the anteroposterior axis of the otic vesicle specify posterior otic identity? One possibility is that localised inhibition of Hh signalling is involved. Here we show that genes coding for three inhibitors of Hh signalling, su(fu), dzip1 and hip, are expressed in and around the developing otic vesicle. su(fu) and dzip1 are ubiquitously expressed and unaffected by Hh levels. The expression of hip, however, is positively regulated by Hh signalling and has a complex, dynamic pattern. It is detectable in the neural tube, otic vesicle, statoacoustic ganglion, brain, fin buds, mouth, somites, pronephros and branchial arches. These expression domains bear some similarity, but are not identical, to those of ptc1, a Hh receptor gene that is also positively regulated by Hh signalling. In the neural tube, for instance, hip is expressed in a subset of the ptc1 expression domain, while in other regions, including the otic vesicle, hip and ptc1 expression domains differ. Significantly, we find that initial expression of hip is higher in and adjacent to anterior otic regions, while ptc1 expression becomes progressively restricted to the posterior of the ear. Hip-mediated inhibition of Hh signalling may therefore be important in restricting the effects of Hh to posterior regions of the developing inner ear.     

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.     

Hedgehog signaling is required for primary motoneuron induction in zebrafish

Sonic hedgehog (Shh) is crucial for motoneuron development in chick and mouse. However, zebrafish embryos homozygous for a deletion of the shh locus have normal numbers of motoneurons, raising the possibility that zebrafish motoneurons may be specified differently. Unlike other vertebrates, zebrafish express three hh genes in the embryonic midline: shh, echidna hedgehog (ehh) and tiggywinkle hedgehog (twhh). Therefore, it is possible that Twhh and Ehh are sufficient for motoneuron formation in the absence of Shh. To test this hypothesis we have eliminated, or severely reduced, all three Hh signals using mutations that directly or indirectly reduce Hh signaling and antisense morpholinos. Our analysis shows that Hh signals are required for zebrafish motoneuron induction. However, each of the three zebrafish Hhs is individually dispensable for motoneuron development because the other two can compensate for its loss. Our results also suggest that Twhh and Shh are more important for motoneuron development than Ehh.     

Hedgehog signaling is required for differentiation of endocardial progenitors in zebrafish

Endocardial cells form the inner endothelial layer of the heart tube, surrounded by the myocardium. Signaling pathways that regulate endocardial cell specification and differentiation are largely unknown and the origin of endocardial progenitors is still being debated. To study pathways that regulate endocardial differentiation in a zebrafish model system, we isolated zebrafish NFATc1 homolog which is expressed in endocardial but not vascular endothelial cells. We further demonstrate that Hedgehog (Hh) but not VegfA or Notch signaling is required for early endocardial morphogenesis. Pharmacological inhibition of Hh signaling with cyclopamine treatment resulted in nearly complete loss of the endocardial marker expression. Simultaneous knockdown of the two zebrafish sonic hedgehog homologs, shh and twhh or Hh co-receptor smoothened (smo) resulted in similar defects in endocardial morphogenesis. Inhibition of Hh signaling resulted in the loss of fibronectin (fn1) expression in the presumptive endocardial progenitors as early as the 10-somite stage which suggests that Hh signaling is required for the earliest stages of endocardial specification. We further show that the endoderm plays a critical role in migration but not specification or differentiation of the endocardial progenitors while notochord-derived Hh is a likely source for the specification and differentiation signal. Mosaic analysis using cell transplantation shows that Smo function is required cell-autonomously within endocardial progenitor cells. Our results argue that Hh provides a critical signal to induce the specification and differentiation of endocardial progenitors.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

Retinoic acid in the anteroposterior patterning of the zebrafish trunk

Retinoic acid has been linked to pattern formation in the vertebrate anteroposterior axis. This report describes the spatial and temporal distributions of both endogenous retinoic acid and retinoic acid synthase activity along the anteroposterior axis of neurulating zebrafish embryos, as detected by a transient transgenic assay and by a zymography bioassay. Both retinoic acid levels and synthase activity were found to be highest in anterior regions of the trunk at all of the stages which were analysed. The drug disulfiram inhibited retinoic acid synthase activity in the zebrafish trunk both in vitro and in vivo, and reduced retinoic acid levels in vivo. Disulfiram treatment of neurulating embryos resulted in larvae with hypertrophic wavy notochords, shortened spinal cords and deformed pectoral fins. The results support the hypothesis that retinoic acid plays a role in the coordination of axial patterning at the developing node/zone of involution, as well as in the subsequent development of anterior trunk structures such as the fins.     

MAPK/ERK signalling is required for zebrafish cardiac regeneration

Objectives

To better understand the molecular mechanisms of regeneration and explore the potential signalling pathways as therapeutic targets for heart attacks.

Results

After treatment with the MEK inhibitor AZD6244 upon cardiac injury, the core members in MAPK/ERK signalling—mek and erk—demonstrate elevated expression, and these proteins are deposited at the injury site in zebrafish. pERK is also induced in non-cardiomyocytes near the injury site. Furthermore, the induced expression of a dominant-negative form of MEK1 inhibits zebrafish cardiac regeneration, characterized by increased cardiac fibrosis (a hallmark of regenerative failure), reduced or delayed production of regenerative myocardium, and migration of FLI1⁺ endothelial cells, without direct inhibition of cardiomyocyte proliferation.

Conclusion

Appropriate activation of MAPK/ERK signalling is essential for zebrafish cardiac regeneration.

     

A restrictive role for Hedgehog signalling during otic specification in Xenopus

Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Here, we report that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus, making use of both gain- and loss-of-function approaches. Ectopic activation of Hedgehog signaling resulted in the development of ectopic vesicular structures expressing the otic marker genes XPax-2, Xdll-3, and Xwnt-3A, thus revealing otic identity. Induction of ectopic otic vesicles was also achieved by misexpression of two different inhibitors of Hh signaling: the putative Hh antagonist mHIP and XPtc1deltaLoop2, a dominant-negative form of the Hh receptor Patched. In addition, misexpression of XPtc1deltaLoop2 as well as treatment of Xenopus embryos with the specific Hh signaling antagonist cyclopamine resulted in the formation of enlarged otic vesicles. In summary, our observations suggest that a defined level of Hh signaling provides a restrictive environment for otic fate in Xenopus embryos.     

The developing lamprey ear closely resembles the zebrafish otic vesicle: otx1 expression can account for all major patterning differences

The inner ear of adult agnathan vertebrates is relatively symmetric about the anteroposterior axis, with only two semicircular canals and a single sensory macula. This contrasts with the highly asymmetric gnathostome arrangement of three canals and several separate maculae. Symmetric ears can be obtained experimentally in gnathostomes in several ways, including by manipulation of zebrafish Hedgehog signalling, and it has been suggested that these phenotypes might represent an atavistic condition. We have found, however, that the symmetry of the adult lamprey inner ear is not reflected in its early development; the lamprey otic vesicle is highly asymmetric about the anteroposterior axis, both morphologically and molecularly, and bears a striking resemblance to the zebrafish otic vesicle. The single sensory macula originates as two foci of hair cells, and later shows regions of homology to the zebrafish utricular and saccular maculae. It is likely, therefore, that the last common ancestor of lampreys and gnathostomes already had well-defined otic anteroposterior asymmetries. Both lamprey and zebrafish otic vesicles express a target of Hedgehog signalling, patched, indicating that both are responsive to Hedgehog signalling. One significant distinction between agnathans and gnathostomes, however, is the acquisition of otic Otx1 expression in the gnathostome lineage. We show that Otx1 knockdown in zebrafish, as in Otx1(-/-) mice, gives rise to lamprey-like inner ears. The role of Otx1 in the gnathostome ear is therefore highly conserved; otic Otx1 expression is likely to account not only for the gain of a third semicircular canal and crista in gnathostomes, but also for the separation of the zones of the single macula into distinct regions.     

Hedgehog signaling is directly required for the development of zebrafish dorsal root ganglia neurons

Hedgehog (Hh) signal transduction is directly required in zebrafish DRG precursors for proper development of DRG neurons. Zebrafish mutations in the Hh signaling pathway result in the absence of DRG neurons and the loss of expression of neurogenin1 (ngn1), a gene required for determination of DRG precursors. Cell transplantation experiments demonstrate that Hh acts directly on DRG neuron precursors. Blocking Hh pathway activation at later stages of embryogenesis with the steroidal alkaloid, cyclopamine, further reveals that the requirement for a Hh signal response in DRG precursors correlates with the onset of ngn1 expression. These results suggest that Hh signaling may normally promote DRG development by regulating expression of ngn1 in DRG precursors.     

The glypican Dally-like is required for Hedgehog signalling in the embryonic epidermis of Drosophila

The Drosophila genes dally and dally-like encode glypicans, which are heparan sulphate proteoglycans anchored to the cell membrane by a glycosylphosphatidylinositol link. Genetic studies have implicated Dally and Dally-like in Wingless signalling in embryos and imaginal discs. Here, we test the signalling properties of these molecules in the embryonic epidermis. We demonstrate that RNA interference silencing of dally-like, but not dally, gives a segment polarity phenotype identical to that of null mutations in wingless or hedgehog. Using heterologous expression in embryos, we uncoupled the Hedgehog and Wingless signalling pathways and found that Dally-like and Dally, separately or together, are not necessary for Wingless signalling. Dally-like, however, is strictly necessary for Hedgehog signal transduction. Epistatic experiments show that Dally-like is required for the reception of the Hedgehog signal, upstream or at the level of the Patched receptor.