Quantitative effects of hedgehog and decapentaplegic activity on the patterning of the Drosophila wing

AbstractBackground: Members of the hedgehog (hh) gene family encode a novel class of proteins implicated in positional signalling in both invertebrates and vertebrates. In Drosophila, the hh gene has been shown to regulate patterning of the imaginal discs, the precursors of the insect limbs. In a remarkably similar fashion, the function and expression of the sonic hedgehog (shh) gene is closely associated with the ‘zone of polarizing activity’ (ZPA) that controls antero-posterior patterning of the vertebrate limb. Both of these functions suggest a role for hedgehog family proteins as morphogens. An alternative possibility, however, is that hh and its homologues act to control the expression of other instructive signalling molecules.Results We have explored this issue by examining the effects on Drosophila wing patterning of ectopically expressing varying levels of hh and shh, as well as of the putative hh target gene, decapentaplegic (dpp), a member of the transforming growth factor-β family of signalling molecules. We find that different levels of hh activity can induce graded changes in the patterning of the wing, and that zebrafish shh acts in a similar though attenuated fashion. Varying levels of ectopic hh and shh activity can differentially activate transcription of the patched and dpp genes. Furthermore, ectopic expression of dpp alone is sufficient to induce the pattern alterations caused by ectopic hh or shh activity.Conclusion Thus, hh family proteins can elicit different responses in a dose-dependent manner in the imaginal disc. The principal function of hh, however, is to activate transcription of dpp at the compartment boundary, thereby establishing a source of dpp activity that is the primary determinant of antero-posterior patterning

Gli2a protein localization reveals a role for Iguana/DZIP1 in primary ciliogenesis and a dependence of Hedgehog signal transduction on primary cilia in the zebrafish

Background: In mammalian cells, the integrity of the primary cilium is critical for proper regulation of the Hedgehog (Hh) signal transduction pathway. Whether or not this dependence on the primary cilium is a universal feature of vertebrate Hedgehog signalling has remained contentious due, in part, to the apparent divergence of the intracellular transduction pathway between mammals and teleost fish. Results: Here, using a functional Gli2-GFP fusion protein, we show that, as in mammals, the Gli2 transcription factor localizes to the primary cilia of cells in the zebrafish embryo and that this localization is modulated by the activity of the Hh pathway. Moreover, we show that the Igu/DZIP1 protein, previously implicated in the modulation of Gli activity in zebrafish, also localizes to the primary cilium and is required for its proper formation. Conclusion: Our findings demonstrate a conserved role of the primary cilium in mediating Hedgehog signalling activity across the vertebrate phylum and validate the use of the zebrafish as a representative model for the in vivo analysis of vertebrate Hedgehog signalling

The role of Sox6 in zebrafish muscle fiber type specification

Background The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification. Methods The GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres. Results Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine. Conclusions The Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation in the zebrafish, a role similar to that ascribed to its murine ortholog

Inactivation of dispatched 1 by the chameleon mutation disrupts Hedgehog signalling in the zebrafish embryo

AbstractSearches of zebrafish EST and whole genome shotgun sequence databases for sequences encoding the sterol-sensing domain (SSD) protein motif identified two sets of DNA sequences with significant homology to the Drosophila dispatched gene required for release of secreted Hedgehog protein. Using morpholino antisense oligonucleotides, we found that inhibition of one of these genes, designated Disp1, results in a phenotype similar to that of the “you-type” mutants, previously implicated in signalling by Hedgehog proteins in the zebrafish embryo. Injection of disp1 mRNA into embryos homozygous for one such mutation, chameleon (con) results in rescue of the mutant phenotype. Radiation hybrid mapping localised disp1 to the same region of LG20 to which the con mutation was mapped by meiotic recombination analysis. Sequence analysis of disp1 cDNA derived from homozygous con mutant embryos revealed that both mutant alleles are associated with premature termination codons in the disp1 coding sequence. By analysing the expression of markers of specific cell types in the neural tube, pancreas and myotome of con mutant and Disp1 morphant embryos, we conclude that Disp1 activity is essential for the secretion of lipid-modified Hh proteins from midline structures

Expression of patched, prdm1 and engrailed in the lamprey somite reveals conserved responses to Hedgehog signaling

In the zebrafish embryo, expression of the prdm1 and patched1 genes in adaxial cells is indicative of their specification to give rise to slow twitch muscle fibers in response to Hedgehog (Hh) signaling. Subsets of these slow twitch muscle progenitors activate engrailed (eng) strongly in response to high-level Hh signaling, and differentiate into muscle pioneer cells, which are important for subsequent development of the horizontal myoseptum. In addition, eng is expressed more weakly in medial fast fibers in response to lower Hh levels. Somite morphology in the lamprey, an agnathan (jawless) vertebrate, differs significantly from that of teleosts. In particular, the lamprey does not have clear epaxial/hypaxial domains, lacks a horizontal myoseptum, and does not appear to possess distinct populations of fast and slow fibers in the embryonic somite. Nevertheless, Hh is expressed in the midline of the lamprey embryo, and we report here that, as in zebrafish, homologues of patched and prdm1 are expressed in adaxial regions of the lamprey somite, and an eng homologue is also expressed in the somite. However, the lamprey adaxial region does not exhibit the same distinct adaxial cell morphology as in the zebrafish. In addition, the expression of follistatin is not excluded from the adaxial region, and eng is not detected in discrete muscle pioneer-like cells. These data suggest the presence of conserved responses to Hh signaling in lamprey somites, although the full range of effects elicited by Hh in the zebrafish somite is not recapitulated

Tardbpl splicing rescues motor neuron and axonal development in a mutant tardbp zebrafish

Mutations in the transactive response DNA binding protein-43 (TARDBP/TDP-43) gene, which regulates transcription and splicing, causes a familial form of amyotrophic lateral sclerosis (ALS). Here, we characterize and report the first tardbp mutation in zebrafish, which introduces a premature stop codon (Y220X), eliminating expression of the Tardbp protein. Another TARDBP ortholog, tardbpl, in zebrafish is shown to encode a Tardbp-like protein which is truncated compared with Tardbp itself and lacks part of the C-terminal glycine-rich domain (GRD). Here, we show that tardbp mutation leads to the generation of a novel tardbpl splice form (tardbpl-FL) capable of making a full-length Tardbp protein (Tardbpl-FL), which compensates for the loss of Tardbp. This finding provides a novel in vivo model to study TDP-43-mediated splicing regulation. Additionally, we show that elimination of both zebrafish TARDBP orthologs results in a severe motor phenotype with shortened motor axons, locomotion defects and death at around 10 days post fertilization. The Tardbp/Tardbpl knockout model generated in this study provides an excellent in vivo system to study the role of the functional loss of Tardbp and its involvement in ALS pathogenesis

Blood vessel occlusion by Cryptococcus neoformans is a mechanism for haemorrhagic dissemination of infection

Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis

Neutrophils use selective autophagy receptor Sqstm1/p62 to target Staphylococcus aureus for degradation in vivo in zebrafish

Macroautophagy/autophagy functions to degrade cellular components and intracellular pathogens. Autophagy receptors, including SQSTM1/p62, target intracellular pathogens. Staphylococcus aureus is a significant pathogen of humans, especially in immunocompromise. S. aureus may use neutrophils as a proliferative niche, but their intracellular fate following phagocytosis has not been analyzed in vivo. In vitro, SQSTM1 can colocalize with intracellular Staphylococcus aureus, but whether SQSTM1 is beneficial or detrimental in host defense against S. aureus in vivo is unknown. Here we determine the fate and location of S. aureus within neutrophils throughout zebrafish infection. We show Lc3 and Sqstm1 recruitment to phagocytosed S. aureus is altered depending on the bacterial location within the neutrophil and that Lc3 marking of bacterial phagosomes within neutrophils may precede bacterial degradation. Finally, we show Sqstm1 is important for controlling cytosolic bacteria, demonstrating for the first time a key role of Sqstm1 in autophagic control of S. aureus in neutrophils

SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination

BACKGROUND: Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. METHODS: In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. FINDINGS: Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p < 0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p < 0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p < 0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p = 0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R = 0.05, p = 0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. INTERPRETATION: The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. FUNDING: This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript

Large-scale phenotyping of patients with long COVID post-hospitalization reveals mechanistic subtypes of disease

One in ten severe acute respiratory syndrome coronavirus 2 infections result in prolonged symptoms termed long coronavirus disease (COVID), yet disease phenotypes and mechanisms are poorly understood1. Here we profiled 368 plasma proteins in 657 participants ≥3 months following hospitalization. Of these, 426 had at least one long COVID symptom and 233 had fully recovered. Elevated markers of myeloid inflammation and complement activation were associated with long COVID. IL-1R2, MATN2 and COLEC12 were associated with cardiorespiratory symptoms, fatigue and anxiety/depression; MATN2, CSF3 and C1QA were elevated in gastrointestinal symptoms and C1QA was elevated in cognitive impairment. Additional markers of alterations in nerve tissue repair (SPON-1 and NFASC) were elevated in those with cognitive impairment and SCG3, suggestive of brain–gut axis disturbance, was elevated in gastrointestinal symptoms. Severe acute respiratory syndrome coronavirus 2-specific immunoglobulin G (IgG) was persistently elevated in some individuals with long COVID, but virus was not detected in sputum. Analysis of inflammatory markers in nasal fluids showed no association with symptoms. Our study aimed to understand inflammatory processes that underlie long COVID and was not designed for biomarker discovery. Our findings suggest that specific inflammatory pathways related to tissue damage are implicated in subtypes of long COVID, which might be targeted in future therapeutic trials