XB-ART-50581Int J Dev Biol 2014 Jan 01;5810-12:799-809. doi: 10.1387/ijdb.140215ml.
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Left-right patterning in Xenopus conjoined twin embryos requires serotonin signaling and gap junctions.
A number of processes operating during the first cell cleavages enable the left-right (LR) axis to be consistently oriented during Xenopus laevis development. Prior work showed that secondary organizers induced in frog embryos after cleavage stages (i.e. conjoined twins arising from ectopic induced primary axes) correctly pattern their own LR axis only when a primary (early) organizer is also present. This instructive effect confirms the unique LR patterning functions that occur during early embryogenesis, but leaves open the question: which mechanisms that operate during early stages are also involved in the orientation of later-induced organizers? We sought to distinguish the two phases of LR patterning in secondary organizers (LR patterning of the primary twin and the later transfer of this information to the secondary twin) by perturbing only the latter process. Here, we used reagents that do not affect primary LR patterning at the time secondary organizers form to inhibit each of 4 mechanisms in the induced twin. Using pharmacological, molecular-genetic, and photo-chemical tools, we show that serotonergic and gap-junctional signaling, but not proton or potassium flows, are required for the secondary organizer to appropriately pattern its LR axis in a multicellular context. We also show that consistently-asymmetric gene expression begins prior to ciliary flow. Together, our data highlight the importance of physiological signaling in the propagation of cleavage-derived LR orientation to multicellular cell fields.
PubMed ID: 25896280
PMC ID: PMC10471180
Article link: Int J Dev Biol
Species referenced: Xenopus laevis
Genes referenced: abracl actr6 adat2 adss2 agtrap aifm2 aifm3 ak6 aldh1a2 amd1 amdhd1 anapc13.2 anxa2 arg1 arpc3 atg4c atp12a atp23 atp2a2 atp6ap1 atp6v1f atp6v1g3 atxn3 banf1 bloc1s2 bloc1s5 blvrb bud31 c2hxorf38 c6h7orf57 ca10 ca2 capn9 capns2 carnmt1 cbfb ccar1 ccdc15 ccng1 ccnq cdh2 cdo1 cdx4 cebpz cep44 cep57l1 cep83 cetn2 cetn3 cetn4 cfap96 cfh cgnl1 chchd1 chmp5 chp1 chrd.1 chst2 cldn1 cmbl cmc2 cnfn.1 coa5 col2a1 col9a2 col9a3 coq5 cox20 cox6c cpt2 crcp cst3 cth ctnna1 cwc27 cxcl12 cyb5a cyth2 dab2ip dand5 dap dbx1 dctn6 denr dgcr6 dguok dhcr7 dmrta1 dnaaf4 dnajc2 dnal1 dus4l dydc1 dync1li1 dynlt1 dynlt5 ebna1bp2 ech1 eif1ad eif3k eif4a2 elf2 emilin1 eps8l3 ercc4 ercc5 erp44 ess2 etaa1 etf1 exoc8 exosc9 fam174a fermt1 fibcd1 fkbp3 foxa4 foxd1 foxi2 fubp3 fundc1 fzd8 gadd45a gby gclm gcm1 gemin2 gfod2 ggnbp2 gipc2 gkap1 glod4 gmnn gng7 gnl2 gpank1 gsc gskip gxylt1 hccs hdac1 heatr6 hesx1 hmg20b hmgb1 hmgcl hmox2 hpgd hscb hypk ifrd1 ift25 ift57 ift74 ift80 igsf3 inpp1 itm2a katna1 kcp kctd18 KIAA1143 kif15 kin kiz klhdc4 kmo krr1 krt12.2 krt7 leprotl1 lig4 llph lrrc57 lsm5 lsm6 lsm7 ltv1 lyar lyrm4 lztfl1 map9 mccc1 mcm6.2 mcur1 mdm4 meaf6 med10 med20 med8 meig1 memo1 meox2 mespa mex3c mier1 mis12 mknk1 mmp28 mnd1 morn2 morn3 mpc1 mphosph10 mpo mrps17 mtif3 mvb12a mxd3 myo19 mzt1 nedd1 nelfe net1 nkiras1 nkx3-1 nme5 nme9 nmrk2 nodal nopchap1 not nr2c1 nt5c3a ntn1 nubp1 nuf2 nutf2 nxpe2 nxt2 ofd1 olfm4 orc6 otub2 pax3 pc.1 pcnp pdcd10 pdcd5 pde3b pipox pitx1 pnhd pole4 polr1e polr1f pou4f4 ppp3ca prag1 prdx1 prkar2b prkg2 psma4 psmc3ip psmd14 psme2 psph pus7 rabl2b ralbp1 ranbp1 rap1a rassf6 rbm7 rexo1 rgs2 riok1 riok2 rmi1 rpa2 rpl31 rpl39 rpp21 rps21 rps24 rps25 rps6kb1 rps7 rsl24d1 rsph3 rufy1 sacm1l saxo2 sbds scel scgn sdk2 senp1 senp6 senp7 septin10 septin11 shh shisa1 sia2 sil1 six3 ska2 slc25a30 slc26a4.3 slc2a1 smim12 snai2 snap29 snrnp25 snx2 sox11 spam1 spata6l spdya srp19 ssb ssu72 stmn1 stx7 stxbp3 sult1e1 sult6b1.4 syap1 sycp2l sytl2 sytl5 taf9b tbca tbrg1 tdgf1.2 tdrd3 tekt1 tekt2 terf1 thap1 them4 tifa timm10 timm13 timm44 tma16 tmem167b tmem30b tmem72 tnip1 tpm1 tpm4 tpmt triqk trmt10c tsfm tsga10 tspan8 twf1 txndc17 txndc9 uba5 ube2j1 ube3a unc93a.2 upf3b uqcr10 utp11 vamp2 vamp7 wdr43 wdr82 wdr89 xpa yeats4 zbtb14 zc3h15 zcrb1 znf585b znf593 znf622 zranb2
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|Fig. 1. Organ situs in singletons and conjoined twins can be disrupted by treatments that affect the primary organizer. (A) The wildtype placement of three organs is shown: the heart with the apex on the animal’s right (red arrowhead), the stomach with the coil ending on the animal’s left (yellow arrowhead), and the gall bladder, positioned on the top of the stomach on the animal’s right (green arrowhead). Several examples of heterotaxia are also shown, with individual organs mirrored. (B) Examples of heart situs in conjoined twins. Both hearts are examined, but because the left twin is the induced one, only its situs (outlined in yellow) reveals the effects of a treatment on a late-induced organizer. The heart of the primary twin (outlined in blue) can be randomized due to leaky morphogens from the left-sided twin (Levin et al., 1996; Levin and Nascone, 1997; Nascone and Mercola, 1997). Wildtype heart situs is indicated by an orange arrowhead; inverted heart situs is indicated by a white arrowhead. All panels show tadpoles (singletons and conjoined twins) from a ventral view, with anterior pointing upward.|
|Fig. 2. Schematic for general experimental design. (A) There are two distinct phases in the orientation of the LR axis in conjoined twins: the patterning of the primary twin (starting at 1-cell, where some cells are instructed to “be left side” or “be right side”) and the transferring of information via “the big brother effect” to the induced (ectopic) organizer (starting at stage 8, where cells in the conjoined twin must receive new information about whether they are located on the left or right side). We sought reagents that would perturb only the latter process. (B) This schematic describes our experimental approach. As indicated by NEED, we sought chemical reagents that disrupt LR patterning in singleton embryos when treatments occur from 1-cell through early neurula stages (blue arrow), but not when treatments were limited to later stages (stage 8 through neurula stages, orange arrow). Results of these experiments are reported in Table 1. We then induced conjoined twins at the 8- or 16-cell stage, and treated these embryos with the identified reagents from stage 8 through neurula stages, to determine whether we could disrupt the second phase of LR axis orientation in conjoined twins. Organ situs was examined at stage 45.|
|Fig. 3. Gap junctional communication (GJC) and 5-HT, but not K+ or H+ flux, are required for LR asymmetry in conjoined twins. (A) Neither low pH, which inhibits H+ efflux pumps, nor BaCl, which blocks all K+ channels, disrupts LR patterning in late-induced twins when exposures start late. (B) Late treatment with lindane, which disrupts GJC, induces heterotaxia in conjoined twins. (C) A schematic detailing experiments with H7 mRNA, a dominant negative form of a chimeric connexin that disrupts GJC. All embryos were injected with XSiamois at the 8- or 16-cell stage. One set was injected with H7 into any two blastomeres in the top two tiers of the animal pole at the 32-cell stage. Another set was injected with H7 into any two dorsal blastomeres in the top two tiers of the animal pole at the 32-cell stage. (D) Disruption of GJC with H7 mRNA alters LR patterning in late-induced organizers, even when GJC is only disturbed in the dorsal cells; the dorsal cells contribute to the primary axis and not the induced twin. (E) Altered 5-HT signaling at late stages disrupts LR patterning in conjoined twins. In all panels, the numbers on the graphs indicate the sample size, *p<0.05 compared to untreated controls, Chi Square test. Red bars indicate inverted hearts, blue indicates inverted stomach and/ or gall bladder, and purple indicates inverted heart plus stomach and/or gall bladder.|
|Fig. 4. Caged serotonin molecules implicate 5-HT in LR patterning of a secondary organizer. (A) In singleton embryos, injection of the caged molecule (BHQ-O-5HT) at the 1-cell stage, paired with uncaging at the 32-cell stage, induces significant levels of heterotaxia. Photoactivation of 5-HT at the 8-cell stage did not significantly increase rates of LR patterning defects. (B) BHQ-O-5HT was injected at the 1-cell stage and conjoined twins were induced at the 8- or 16-cell stage. When 5-HT was uncaged at the 32-cell stage, prior to the induction of conjoined twins, LR patterning in the induced twin was randomized. When 5-HT was uncaged at stage 8, as XSiamois begins to induce the development of a conjoined twin, LR patterning defects were still observed. (C) BHQ-O-5HT and XSiamois were co-injected in the ventral vegetal left blastomere at the 8- or 16-cell stage, and 5-HT was uncaged at either 32-cell, stage 8, or stage 10. Regardless of when the 5-HT was released, heterotaxia in the induced twin was observed. In all panels, the numbers on the graphs indicate the sample size, *p<0.05 compared to embryos injected with BHQ-O-5HT but not uncaged, Chi Square test. Red bars indicate inverted hearts, blue indicates inverted stomach and/or gall bladder, and purple indicates inverted heart plus stomach and/or gall bladder.|
|Fig. 5. In situ hybridization for collagen9a2 confirms the microarray result of left sided expression prior to nodal flow. (A) Embryos processed by in situ hybridization for the marker col9a2 show expression only on the left side or (B) on both sides but with a strong left bias. Embryos were examined prior to the development of ciliary flow. (C) Control embryos probed for the marker Xslug, which was not biased in the microarray results, showed bilateral symmetric expression in all animals examined. Red arrows indicate areas of expression; white arrows indicate absence of expression. The dotted line demarcates the embryo’s midline. All panels show a dorsal view, with the anterior pointing upward.|
|Fig. 6. A model for LR asymmetry in conjoined twins. (A) The two organizers are positioned in a single blastoderm. The left and right sides of each twin are indicated. Note that the left side of the primary twin is across from the right side of the induced twin. (B) The cells of this blastoderm are oriented by polarity proteins (orange arrows) including planar cell polarity and apical basal polarity proteins. These polarity proteins are themselves oriented with respect to the anterior-posterior and dorsal-ventral axes, as described previously (Vandenberg and Levin, 2012). Within this highly organized blastoderm, both gap junctions (green cylinders) and small signaling molecules like 5-HT (purple stars) are required for proper LR patterning in the induced twin. Two roles for 5-HT are proposed. (C) One hypothesis is that 5-HT maintains subcellular asymmetries, providing an amplification of the asymmetries established by the polarity proteins. Thus, each cell in the blastoderm, or perhaps only specific cells in a late LR “organizer”, has its own physiological and biochemical LR axis. (D) The second hypothesis is that a surge of 5-HT at a specific time, and perhaps again only in specific cells in a late LR “organizer”, are sufficient to induce consistent LR gene expression. Thus, asymmetric expression is not required, but a temporal pulse is needed.|
|Supplementary Fig. 1. b-gal localization in early conjoined twins verifies RNA translation. (A) Embryos were injected with b-gal mRNA at 1-cell stage and XSiamois at 8-16 cell stage. Embryos fixed at the indicated stages (st. 10 – 12.5) express b-gal, indicating that RNA translation occurs. (B) One blastomere was injected with b-gal mRNA at the 2-, 4- or 8-cell stage, and embryos were injected with XSiamois at 8-16 cell stage. Different patterns of b-gal expression were observed in developing twins at neurula stages depending on the original location of the b-gal mRNA injection.|
|col9a2 (collagen, type IX, alpha 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior up.|