Spatio-temporal regulation of concurrent developmental processes by generic signaling downstream of chemokine receptors

Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration.</jats:p

Six3 Represses Nodal Activity to Establish Early Brain Asymmetry in Zebrafish

SummaryThe vertebrate brain is anatomically and functionally asymmetric; however, the molecular mechanisms that establish left-right brain patterning are largely unknown. In zebrafish, asymmetric left-sided Nodal signaling within the developing dorsal diencephalon is required for determining the direction of epithalamic asymmetries. Here, we show that Six3, a transcription factor essential for forebrain formation and associated with holoprosencephaly in humans, regulates diencephalic Nodal activity during initial establishment of brain asymmetry. Reduction of Six3 function causes brain-specific deregulation of Nodal pathway activity, resulting in epithalamic laterality defects. Based on misexpression and genetic epistasis experiments, we propose that Six3 acts in the neuroectoderm to establish a prepattern of bilateral repression of Nodal activity. Subsequently, Nodal signaling from the left lateral plate mesoderm alleviates this repression ipsilaterally. Our data reveal a Six3-dependent mechanism for establishment of correct brain laterality and provide an entry point to understanding the genetic regulation of Nodal signaling in the brain

Chemokine GPCR signaling inhibits beta-catenin during Zebrafish axis formation

Embryonic axis formation in vertebrates is initiated by the establishment of the dorsal Nieuwkoop blastula organizer, marked by the nuclear accumulation of maternal β-catenin, a transcriptional effector of canonical Wnt signaling. Known regulators of axis specification include the canonical Wnt pathway components that positively or negatively affect β-catenin. An involvement of G-protein coupled receptors (GPCRs) was hypothesized from experiments implicating G proteins and intracellular calcium in axis formation, but such GPCRs have not been identified. Mobilization of intracellular Ca(2+) stores generates Ca(2+) transients in the superficial blastomeres of zebrafish blastulae when the nuclear accumulation of maternal β-catenin marks the formation of the Nieuwkoop organizer. Moreover, intracellular Ca(2+) downstream of non-canonical Wnt ligands was proposed to inhibit β-catenin and axis formation, but mechanisms remain unclear. Here we report a novel function of Ccr7 GPCR and its chemokine ligand Ccl19.1, previously implicated in chemotaxis and other responses of dendritic cells in mammals, as negative regulators of β-catenin and axis formation in zebrafish. We show that interference with the maternally and ubiquitously expressed zebrafish Ccr7 or Ccl19.1 expands the blastula organizer and the dorsoanterior tissues at the expense of the ventroposterior ones. Conversely, Ccr7 or Ccl19.1 overexpression limits axis formation. Epistatic analyses demonstrate that Ccr7 acts downstream of Ccl19.1 ligand and upstream of β-catenin transcriptional targets. Moreover, Ccl19/Ccr7 signaling reduces the level and nuclear accumulation of maternal β-catenin and its axis-inducing activity and can also inhibit the Gsk3β -insensitive form of β-catenin. Mutational and pharmacologic experiments reveal that Ccr7 functions during axis formation as a GPCR to inhibit β-catenin, likely by promoting Ca(2+) transients throughout the blastula. Our study delineates a novel negative, Gsk3β-independent control mechanism of β-catenin and implicates Ccr7 as a long-hypothesized GPCR regulating vertebrate axis formation

Model organisms contribute to diagnosis and discovery in the Undiagnosed Diseases Network: Current state and a future vision

Decreased sequencing costs have led to an explosion of genetic and genomic data. These data have revealed thousands of candidate human disease variants. Establishing which variants cause phenotypes and diseases, however, has remained challenging. Significant progress has been made, including advances by the National Institutes of Health (NIH)-funded Undiagnosed Diseases Network (UDN). However, 6000-13,000 additional disease genes remain to be identified. The continued discovery of rare diseases and their genetic underpinnings provides benefits to affected patients, of whom there are more than 400 million worldwide, and also advances understanding the mechanisms of more common diseases. Platforms employing model organisms enable discovery of novel gene-disease relationships, help establish variant pathogenicity, and often lead to the exploration of underlying mechanisms of pathophysiology that suggest new therapies. The Model Organism Screening Center (MOSC) of the UDN is a unique resource dedicated to utilizing informatics and functional studies in model organisms, including worm (Caenorhabditis elegans), fly (Drosophila melanogaster), and zebrafish (Danio rerio), to aid in diagnosis. The MOSC has directly contributed to the diagnosis of challenging cases, including multiple patients with complex, multi-organ phenotypes. In addition, the MOSC provides a framework for how basic scientists and clinicians can collaborate to drive diagnoses. Customized experimental plans take into account patient presentations, specific genes and variant(s), and appropriateness of each model organism for analysis. The MOSC also generates bioinformatic and experimental tools and reagents for the wider scientific community. Two elements of the MOSC that have been instrumental in its success are (1) multidisciplinary teams with expertise in variant bioinformatics and in human and model organism genetics, and (2) mechanisms for ongoing communication with clinical teams. Here we provide a position statement regarding the central role of model organisms for continued discovery of disease genes, and we advocate for the continuation and expansion of MOSC-type research entities as a Model Organisms Network (MON) to be funded through grant applications submitted to the NIH, family groups focused on specific rare diseases, other philanthropic organizations, industry partnerships, and other sources of support

Chemokine GPCR Signaling Inhibits β-Catenin during Zebrafish Axis Formation

<div><p>Embryonic axis formation in vertebrates is initiated by the establishment of the dorsal Nieuwkoop blastula organizer, marked by the nuclear accumulation of maternal β-catenin, a transcriptional effector of canonical Wnt signaling. Known regulators of axis specification include the canonical Wnt pathway components that positively or negatively affect β-catenin. An involvement of G-protein coupled receptors (GPCRs) was hypothesized from experiments implicating G proteins and intracellular calcium in axis formation, but such GPCRs have not been identified. Mobilization of intracellular Ca²⁺ stores generates Ca²⁺ transients in the superficial blastomeres of zebrafish blastulae when the nuclear accumulation of maternal β-catenin marks the formation of the Nieuwkoop organizer. Moreover, intracellular Ca²⁺ downstream of non-canonical Wnt ligands was proposed to inhibit β-catenin and axis formation, but mechanisms remain unclear. Here we report a novel function of Ccr7 GPCR and its chemokine ligand Ccl19.1, previously implicated in chemotaxis and other responses of dendritic cells in mammals, as negative regulators of β-catenin and axis formation in zebrafish. We show that interference with the maternally and ubiquitously expressed zebrafish Ccr7 or Ccl19.1 expands the blastula organizer and the dorsoanterior tissues at the expense of the ventroposterior ones. Conversely, Ccr7 or Ccl19.1 overexpression limits axis formation. Epistatic analyses demonstrate that Ccr7 acts downstream of Ccl19.1 ligand and upstream of β-catenin transcriptional targets. Moreover, Ccl19/Ccr7 signaling reduces the level and nuclear accumulation of maternal β-catenin and its axis-inducing activity and can also inhibit the Gsk3β -insensitive form of β-catenin. Mutational and pharmacologic experiments reveal that Ccr7 functions during axis formation as a GPCR to inhibit β-catenin, likely by promoting Ca²⁺ transients throughout the blastula. Our study delineates a novel negative, Gsk3β-independent control mechanism of β-catenin and implicates Ccr7 as a long-hypothesized GPCR regulating vertebrate axis formation.</p> </div

Ccr7 is required for proper AP and DV embryo patterning.

<p>(A) The spectrum of dorsalized phenotypes in embryos injected with MO1-<i>ccr7</i> at 27 hpf, ranging from highly dorsalized (C4–5; b) with truncated tails and trunks to C3 with tail deficiencies (b′) (total <i>n</i> = 190, seven independent experiments for 12 ng MO1-<i>ccr7</i> and <i>n</i> = 35, one experiment for 20 ng MO1-<i>ccr7</i>). The frequency of each phenotypic category is indicated in the right panel (c). The scale bars represent 200 µm in all figures. (B) Ccr7 overexpression (150–200 pg RNA) caused a spectrum of ventralized phenotypes, ranging from V3 to V1 (arrows show anterior and notochord deficiencies) to WT-like (a). The frequency of each phenotypic category is indicated in the right panel (b; <i>n</i> = 96, two experiments). (C) Expression of dorsal/ventral markers in Ccr7 morphants compared to control embryos revealed by WISH. (a–e′) Expression of dorsal genes was upregulated or expanded: at high-oblong stage (3.3–3.7 hpf), <i>boz</i>, <i>n</i> = 8/8; at sphere stage (4 hpf), <i>boz</i>, <i>n</i> = 16/28; <i>mkp3</i>, <i>n</i> = 9/12; <i>chd</i>, <i>n</i> = 11/15; at shield stage (6 hpf), <i>gsc</i>, <i>n</i> = 18/37. (f–j′) Expression of ventral genes was reduced: at dome (4.3 hpf) stage, <i>bmp2b</i>, <i>n</i> = 19/24; at 30% epiboly stage (4.7 hpf), <i>ved</i>, <i>n</i> = 12/14; at shield stage, <i>bmp4</i>, <i>n</i> = 9/10; <i>szl</i>, <i>n</i> = 11/12; <i>vox</i>, <i>n</i> = 16/22. Animal views, dorsal to the right. (D) Expression of dorsal/ventral markers in Ccr7-overexpressing embryos revealed by WISH. Expression of ventral markers was expanded (a–c′), while dorsal markers were decreased (d–e′): at sphere stage, <i>boz</i>, <i>n</i> = 8/8; <i>chd</i>, <i>n</i> = 10/13; at 40% epiboly stage (5 hpf), <i>bmp2b</i>, <i>n</i> = 12/12; <i>bmp4</i>, <i>n</i> = 14/14; <i>szl</i>, <i>n</i> = 13/13. Animal views, dorsal to the right.</p

Ccr7 inhibits β-catenin activity via a Gsk3β-indepenedent mechanism.

<p>(A) Hyper-dorsalized phenotypes caused by β-catenin overexpression (b,b′, 25 pg, <i>n</i> = 22/25), compared to control WT embryos (a), were suppressed by Ccr7 overexpression (c; 150 pg, <i>n</i> = 8/12). (d–f) Expansion of <i>gsc</i> expression domain in β-catenin overexpressing embryos (e), relative to control WT embryos (d), was suppressed by co-injection of <i>ccr7</i> RNA (f). Animal views, dorsal to the right. (g) Frequency of embryos with <i>gsc</i> expression domain encompassing more (>180°) or less (<180°) than half of the embryo equator. (B) (a–c) LiCl-treated embryos (b; <i>n</i> = 16/20) show dorsalized phenotypes at 30 hpf compared to control embryos (a). LiCl-dependent dorsalization was suppressed by injection of <i>ccr7</i> RNA (c; <i>n</i> = 8/20, two experiments). (d–f) <i>gsc</i> expression at shield stage (6 hpf) in control (d), LiCl-treated (e; <i>n</i> = 13/14), and LiCl-treated and <i>ccr7</i> RNA-injected embryos (f; <i>n</i> = 9/12). Animal views, dorsal to the right. (g–i) β-catenin immunostaining at 256-cell stage in control (g), LiCl-treated (h, <i>n</i> = 9/10), and LiCl-treated embryos overexpressing Ccr7 (i; <i>n</i> = 9/11). Arrows point to a few β-catenin-positive nuclei in control embryos (g) and LiCl-treated embryos overexpressing Ccr7 (i). (C) Ccr7 antagonizes the ability of ΔNβ-catenin to rescue the ventralized <i>ich</i> mutant phenotype. (a) V1–V4 phenotypic classes, with V4 corresponding to the strongest <i>ich</i> phenotype. (b) Frequencies of the V1–V4 phenotypic classes of <i>ich</i> mutants injected with synthetic <i>ΔNβ-catenin</i> RNA alone or co-injected with <i>ccr7</i> RNA. Injected amounts of RNAs in pg are shown below the graph, and the number of embryos in each group above each bar. (D) (a–c) Co-injections of <i>ΔNβ-catenin-gfp</i> RNA and MO1-<i>ccr7</i> or <i>ccr7</i> RNA showed that Ccr7 can downregulate β-catenin, shown at higher-magnification (d–f). Compared to control (a, d), <i>ccr7</i> RNA overexpressing blastulae showed strongly decreased (b, e), while MO1-<i>ccr7</i> injected blastulae showed increased, ΔNβ-catenin-GFP signal (c, f). <i>H2B-RFP</i> RNA was injected as nuclear background control (a′–c′ and higher magnification in d′–f′). (E) Western blot analysis of co-injection of <i>ΔNβ-catenin-gfp</i> RNA and <i>ccr7</i> RNA or MO1-<i>ccr7</i>. Quantification of the relative protein level (signal intensity) from three independent immunoblots (bottom panel). * <i>p</i><0.05.</p

Ccl19.1 chemokine functions as a Ccr7 ligand in axis formation.

<p>(A) Injection of <i>ccl19.1</i> RNA (100–120 pg) resulted in ventralized embryo morphology at 27 hpf (a, a′; <i>n</i> = 30/45; lateral views with anterior to the left) and expansion of <i>szl</i> expression domain (b′) compared to control (b; <i>n</i> = 12/15). Shield stage, animal views with ventral to the left. (B) Ccl19.1 antagonizes the ability of ΔNβ-catenin to rescue the ventralized <i>ich</i> mutant phenotype. (a) V1–V4 phenotypic classes, with V4 corresponding to the strongest <i>ich</i> phenotype (also shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001403#pbio-1001403-g003" target="_blank">Figure 3Ca</a>). (b) Frequencies of the V1–V4 phenotypic classes of <i>ich</i> mutant embryos injected with synthetic <i>ΔNβ-catenin</i> RNA alone or co-injected with <i>ccl19.1</i> RNA. Injected amounts of RNAs in pg are shown below the graph, and the number of embryos in each group, above each bar. (C) (a) The spectrum of dorsalized phenotypes at 27 hpf in embryos injected with 4 ng MO1-<i>ccl19.1</i> classified into five categories, ranging from C4–C5 (the most severe class) to WT-like. (b) Frequency of each phenotypic category (<i>n</i> = 104, three experiments). (c) WISH analysis of dorsal/ventral markers in <i>ccr19.1</i> morphants compared to control blastulae. (a′–c″) Expression of dorsal genes was upregulated or expanded: sphere (4 hpf), <i>boz</i>, <i>n</i> = 22/31; <i>mkp3</i>, <i>n</i> = 11/11; shield (6 hpf), <i>gsc</i>, <i>n</i> = 9/10. (d′–e″) Expression domains of ventral genes were reduced: 30% epiboly (4.7 hpf), <i>ved</i>, <i>n</i> = 10/12; shield (6 hpf), <i>szl</i>, <i>n</i> = 11/12. Animal pole views, dorsal to the right. (D) Co-injection of MO1-<i>ccr7</i> and MO1-<i>ccl19.1</i> leads to dorsalization in a synergistic fashion. Injection of low doses of MO1-<i>ccr7</i> (b; <i>n</i> = 24) and MO1-<i>ccl19.1</i> (c; <i>n</i> = 26) alone did not cause dorsalized phenotypes, as observed for uninjected control embryos (a; 11 hpf, <i>n</i> = 36). (d) Embryos co-injected with the same doses of both MOs resulted in dorsalization (<i>n</i> = 12/23). (e) Frequency of dorsalized embryos in a–d. (E) <i>szl</i> expression was expanded in <i>ccl19.1</i> RNA-injected (b; 100 pg), compared to uninjected, control embryos (a) and was reduced in MO1-<i>ccr7</i>-injected (c; 12 ng) and <i>ccl19.1</i> RNA (100 pg) and MO1-<i>ccr7</i> (12 ng) co-injected embryos (d; two experiments). See text for details. Animal views of shield stage embryos, dorsal to the right.</p

Depletion of Ccr7 activity partially rescues axis formation in <i>ichabod</i> mutants.

<p>(A) Penetrance of strongly ventralized phenotypes displayed by maternal β-catenin-2, <i>ich</i>, mutant embryos (a, 92%, <i>n</i> = 110) was reduced by injection of MO1-<i>ccr7</i> (b–h, 10 ng; 40%, <i>n</i> = 98, five experiments). Arrows indicate partial axes and arrowheads indicate rudimentary head structures. (B) <i>myod1</i> expression in uninjected (a) and MO1-<i>ccr7-</i>injected <i>ich</i> embryos (b,b′) revealed somitic tissue. Arrows indicate partial double axes. (C) In contrast to uninjected <i>ich</i> mutants (a,b,c), in MO1-<i>ccr7</i>-injected <i>ich</i> embryos, the organizer genes <i>gsc</i> (a′, <i>n</i> = 11/12) and <i>chd</i> (b′, <i>n</i> = 8/8) were expressed; while expression of the ventral gene, <i>eve1,</i> was significantly reduced (c′, <i>n</i> = 30/40). Animal views. (D) Lack of β-catenin nuclear accumulation, detected by immunostaining at 256-cell stage, in <i>ich</i> mutants (a), was suppressed in MO1-<i>ccr7</i>-injected <i>ich</i> embryos (b,c; <i>n</i> = 9/10, two experiments). (a′–c′) Higher magnification of boxed areas in a–c. (E) Dorsal domain of nuclear accumulation of β-catenin, detected by immunostaining at 256-cell stage in WT embryos (a), was expanded in <i>ccr7</i> morphants (b; <i>n</i> = 6/13), while it was diminished in Ccr7 overexpressing embryos (c; <i>n</i> = 8/11). (a′–c′) Higher magnification of boxed areas in a–c. (F) Ectopic β-catenin nuclear accumulation, detected by immunostaining at 256–512-cell stage, in <i>ich</i> mutants injected with <i>β-catenin</i> RNA (a; 25 pg, <i>n</i> = 9/10), was suppressed by co-injecting <i>ccr7</i> RNA (b; 150 pg, <i>n</i> = 7/10). (a′–b′) Higher magnification of boxed areas in a–b. (G) Ccr7 gain-of-function decreased both levels of endogenous β-catenin and ectopic β-catenin-GFP. Western blotting of β-catenin and GFP protein from uninjected control, <i>β-catenin-GFP</i> RNA (10 pg) injected, or <i>β-catenin-GFP</i> RNA (10 pg)/<i>ccr7</i> RNA (200 pg) co-injected embryos (all at 3–3.3 hpf). Graphs below show the relative protein level (signal intensity) quantified from three separate immunoblots. * <i>p</i><0.05.</p