Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependant pigment progenitors in zebrafish

<div><p>Skin pigment patterns are important, being under strong selection for multiple roles including camouflage and UV protection. Pigment cells underlying these patterns form from adult pigment stem cells (APSCs). In zebrafish, APSCs derive from embryonic neural crest cells, but sit dormant until activated to produce pigment cells during metamorphosis. The APSCs are set-aside in an ErbB signaling dependent manner, but the mechanism maintaining quiescence until metamorphosis remains unknown. Mutants for a pigment pattern gene, <i>parade</i>, exhibit ectopic pigment cells localised to the ventral trunk, but also supernumerary cells restricted to the Ventral Stripe. Contrary to expectations, these melanocytes and iridophores are discrete cells, but closely apposed. We show that <i>parade</i> encodes Endothelin receptor Aa, expressed in the blood vessels, most prominently in the medial blood vessels, consistent with the ventral trunk phenotype. We provide evidence that neuronal fates are not affected in <i>parade</i> mutants, arguing against transdifferentiation of sympathetic neurons to pigment cells. We show that inhibition of BMP signaling prevents specification of sympathetic neurons, indicating conservation of this molecular mechanism with chick and mouse. However, inhibition of sympathetic neuron differentiation does not enhance the <i>parade</i> phenotype. Instead, we pinpoint ventral trunk-restricted proliferation of neural crest cells as an early feature of the <i>parade</i> phenotype. Importantly, using a chemical genetic screen for rescue of the ectopic pigment cell phenotype of <i>parade</i> mutants (whilst leaving the embryonic pattern untouched), we identify ErbB inhibitors as a key hit. The time-window of sensitivity to these inhibitors mirrors precisely the window defined previously as crucial for the setting aside of APSCs in the embryo, strongly implicating adult pigment stem cells as the source of the ectopic pigment cells. We propose that a novel population of APSCs exists in association with medial blood vessels, and that their quiescence is dependent upon Endothelin-dependent factors expressed by the blood vessels.</p></div

Sox10 contributes to the balance of fate choice in dorsal root ganglion progenitors

The development of functional peripheral ganglia requires a balance of specification of both neuronal and glial components. In the developing dorsal root ganglia (DRGs), these compo- nents form from partially-restricted bipotent neuroglial precursors derived from the neural crest. Work in mouse and chick has identified several factors, including Delta/Notch signal- ing, required for specification of a balance of these components. We have previously shown in zebrafish that the Sry-related HMG domain transcription factor, Sox10, plays an unex- pected, but crucial, role in sensory neuron fate specification in vivo. In the same study we described a novel Sox10 mutant allele, sox10baz1, in which sensory neuron numbers are elevated above those of wild-types. Here we investigate the origin of this neurogenic pheno- type. We demonstrate that the supernumerary neurons are sensory neurons, and that enteric and sympathetic neurons are almost absent just as in classical sox10 null alleles; peripheral glial development is also severely abrogated in a manner similar to other sox10 mutant alleles. Examination of proliferation and apoptosis in the developing DRG reveals very low levels of both processes in wild-type and sox10baz1, excluding changes in the bal- ance of these as an explanation for the overproduction of sensory neurons. Using chemical inhibition of Delta-Notch-Notch signaling we demonstrate that in embryonic zebrafish, as in mouse and chick, lateral inhibition during the phase of trunk DRG development is required to achieve a balance between glial and neuronal numbers. Importantly, however, we show that this mechanism is insufficient to explain quantitative aspects of the baz1 phenotype. The Sox10(baz1) protein shows a single amino acid substitution in the DNA binding HMG domain; structural analysis indicates that this change is likely to result in reduced flexibility in the HMG domain, consistent with sequence-specific modification of Sox10 binding to DNA. Unlike other Sox10 mutant proteins, Sox10(baz1) retains an ability to drive neurogenin1 transcription. We show that overexpression of neurogenin1 is sufficient to produce supernu- merary DRG sensory neurons in a wild-type background, and can rescue the sensory neu- ron phenotype of sox10 morphants in a manner closely resembling the baz1 phenotype. We conclude that an imbalance of neuronal and glial fate specification results from the Sox10 (baz1) protein\u2019s unique ability to drive sensory neuron specification whilst failing to drive glial development. The sox10baz1 phenotype reveals for the first time that a Notch-dependent lat- eral inhibition mechanism is not sufficient to fully explain the balance of neurons and glia in the developing DRGs, and that a second Sox10-dependent mechanism is necessary. Sox10 is thus a key transcription factor in achieving the balance of sensory neuronal and glial fates

Dataset for "Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependant pigment progenitors in zebrafish"

This dataset contains the data underlying the paper "Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependant pigment progenitors in zebrafish" that were not included directly in Results or Supplementary Information. It contains the cell counts from figures 2, 4, 5, and 6.Various methods were used to collect the data. For details, see the Materials and Methods section of the associated paper

Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds

Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35–40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosome-associated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and protein-sorting mechanisms in the secretory pathway

Precocious and supernumerary sensory neuron specification in <i>sox10</i>^<i>baz1</i> mutants.

<p>A,B) <i>neurod1</i> expression is seen in more cells (close-ups in left panels; arrowheads indicate a subset of <i>neurod1</i>^<i>+</i> cells) and extending more posteriorly (right panels; arrowhead marks posteriormost <i>neurod1</i>^<i>+</i> DRG) in <i>baz1</i> mutants compared with WT siblings at both 36 and 45 hpf. C) Counts of <i>neurod1</i>^<i>+</i> cells on one side of embryo at 36 and 45 hpf embryos (N = 11 for all conditions except 36 hpf <i>baz1</i>, where N = 13). <i>baz1</i> mutants significantly different to WT siblings (Student’s <i>t</i> test; ***, p<0.0001. In this and all subsequent images, embryos are shown in lateral view with dorsal to the top and anterior to the left, unless otherwise stated. Scale bar, 100 μm.</p

Medial pathway neural precursors undergo precocious and supernumerary differentiation into neurons in baz1 mutants.

<p>Confocal images of developing trunk DRGs of WT (A, D, G, J), <i>baz1</i> (B, E, H, K) and <i>m618</i> mutants (C, F, I, L) showing Elav1/Hu (red) and <i>sox10</i>:<i>GFP</i> (green) at each of 36 (A-C), 42 (D-F), 48 hpf (G-I) and 5 dpf (J-L). Arrowheads indicate subset of Elav1/Hu⁺ DRG sensory neurons. M-P) Counts (mean±s.d.) of trunk (Tr) and tail (Ta) and total (TOT) Elav1⁺ cells in DRGs of <i>baz1</i> (yellow) and <i>m618</i> (blue) mutants and their respective WT siblings. Significantly elevated numbers of neurons are indicated (two-tailed Student’s <i>t</i> test; **, p<0.01; ***, p<0.001). Note in panels J-L) that variable prominence of Elav1/Hu detection in spinal cord is an artefact of antibody penetration into CNS at this late stage. Scale bar, 50 μm.</p

Quantification of Notch reporter differences in m618 and baz1 mutants at 48 hpf.

<p>A-F) confocal acquisitions of WT control (A,D) and mutant (B, <i>m618</i>; E, <i>baz1</i>) trunk regions, followed by Notch reporter (NRE:EGFP) fluorescence analysis (C,F). A slight increase of reporter signal is detected in the neural tube (nt) and dorsal root ganglia (drg, arrowhead) of <i>m618</i> mutants (B) compared to controls (A), while a decrease of signal is detected in the same regions of <i>baz1</i> mutants (compare E with D). Relative fluorescence intensity (RFI) in aorta (a) and intersomitic vessels (v) appears unmodified in all conditions. n = 6 measurements per condition. n.s. = not significant; ** = p<0.01; *** = p<0.001.</p

deltaA and deltaD gene expression overlaps with neurog1 in the nascent DRGs.

<p>A-C) <i>deltaA</i> expression (red) clearly overlaps with <i>neurog1</i> (green) in the nascent DRG (arrows) at 30 hpf. D-F) At 38 hpf, <i>deltaA</i> expression is clearly seen in the DRGs, but weaker signals make it difficult to discern if expression is in the same cells as express <i>neurog1</i> or simply in other cells of the ganglia. G-I) <i>deltaD</i> expression (red) clearly overlaps with <i>neurog1</i> (green) in the nascent DRG (arrows) at 38 hpf. All main panels are confocal images of fluorescent dual-color <i>in situ</i> hybridisations in lateral view, with insets showing <i>y-z</i> planes (left) and <i>x-z</i> planes (above) for each. Insets in the bottom right of panels C, F and I show enlargements of the double-labeled cells indicated by the arrows. nc, notochord; sc, spinal cord.</p

Proliferation in the DRGs is negligible in <i>baz1</i> mutants and their WT siblings.

<p>Proliferation in the DRGs is negligible in <i>baz1</i> mutants and their WT siblings.</p