Picture from Poole RJ et al. (2006) Curr Biol "Early embryonic programming of neuronal left/right asymmetry in C. ...."

Picture from Poole RJ et al. (2006) Curr Biol "Early embryonic programming of neuronal left/right asymmetry in C. ...."

Figure 2. ASE Cells Generated from Ectopic Right-Sided ASEL Lineages Do Not Express ASER Markers.Each horizontal panel in this and subsequent figures illustrate from left to right the predicted lineage and then the predicted expression of gcy-7and/or gcy-5 depending on the mechanism of ASE L/R asymmetry generation or the possible lineage origins of ectopic cells. To the right of the predicted expression the actual number of cells observed expressing gcy-7prom::gfp (otIs3) and gcy-5prom::gfp (ntIs1) or gcy-5prom::mCherry(otEx2332) is indicated and can be easily compared with the different predictions on the left. In all cases, ASEL lineages and gcy-7 expression are indicated in red and ASER lineages and gcy-5 expression are indicated in blue. A crossed-out 'Notch' indicates that the genetic or cellular ablation causes a disruption of one of the several Notch interactions indicated in Figure 1B. Animals were scored as late as their viability allows.(A) Expression of gcy-7 and gcy-5 at the 3-fold to L1 stage in control embryos (upper panel), those in which the first Notch signal has been prevented (middle panel) and those in which the first and second Notch signals have been prevented (lower panel) with a temperature-sensitive allele of the Notch receptor glp-1(e2144). When embryos are grown from the one- to two-cell stage at the permissive temperature of15C, this strain shows the wild-type pattern of a maximum of two cells expressing gcy-7 and only one cell expressing gcy-5 (upper panel). When embryos grown at the non permissive temperature of 25C from the one- to two-cell to greater than 12-cell stage and then shifted to15C, no ASE lineages are formed and no expression of gcy-7 or gcy-5 is observed in the majority of embryos (lower panel). Preventing specifically the first Notch signal by growing the embryos at the non permissive temperature until the six-cell stage and then shifting the embryos to 15C generates two ectopic ASEL lineages. In these embryos, a maximum of three cells expressing gcy-7 is observed (as mentioned in Figure 1B, in the embryo gcy-7 initially marks all ASE cells because of its expression in both ASEL and ASER in the hybrid precursor state[17]), and no expression of gcy-5 is observed. (B) Expression of gcy-5 at the 3-fold to L1 stage in wild-type embryos (upper panel) and maternal and zygotic apx-1(zu183) mutants. F2 embryosfrom dpy F1 progeny of an apx-1(zu183) dpy-11 (e224)/nT1 strain carrying the gcy-5prom::mCherry (otEx2332) array were analyzed (lower panel). Abrogating the function of apx-1, the Notch ligand specific for the first Notch signal, prevents this signal and thus generates two ectopic ASEL lineages (see text for details). The gcy-5prom::mCherry (otEx2332) array (which had to be used because of the linkage of ntIs1 and apx-1) is about70% penetrant such that in wild-type embryos 70% of the embryos can be observed expressing gcy-5 in a single cell at the 3-fold to L1 stage.(C) Execution of the ASEL fate, observed upon preventing the first Notch signal with the glp-1(e2144) temperature-sensitive allele (A), is prevented in lsy-6 null mutants, leading to a switch from ASEL to ASER fate.

Picture from Poole RJ et al. (2011) PLoS Genet "A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in ...."

Figure S5 Bilaterally symmetric expression and function of hlh- 14 in the I1L/I1R lineage. A: Ventral view of the 3D-poitions of the bilaterally symmetric I1L/I1R neuroblasts (light blue) at the AB256-cell stage. The gut lineage is shown for positional information. Cell coloring after [10]. B: Lineage and hlh-14 expression analysis of the I1L/I1R lineage in wildtype (containing the ntIs1 transgene), hlh-14(tm295); ntIs1 and hlh-14(tm295); otEx4507, Ex[hlh-14FOS::yfp]; ntIs embryos. Purple lines indicate the expression of hlh-14 (assessed in hlh-14(tm295); Ex[hlh-14FOS::yfp embryos) and the numbers below indicate the number of lineages that divided as indicated and the total number of left or right lineages followed to terminal division are shown at the bottom of each lineage diagram indicate the total number of lineages followed. The mother of the I1L/I1R blastomeres divides early in hlh-14 mutants, at a time when sister lineage divides to produce one pharyngeal marginal cell (mc1) and one pharyngeal muscle cell (m3V). This defect is rescued with the hlh-14FOS::yfp transgene. Red cell names indicate neurons/glia and green cell names indicate mesoderm.

Picture from Poole RJ et al. (2011) PLoS Genet "A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in ...."

Figure 6. Asymmetric expression and function of hlh-14 in the C-lineage. A: Ventral view of the 3D-positions of certain cells of the C lineage of a representative wild-type embryo at the AB256-cell stage (shortly after the division of the ASE grandmothers), as assessed by 4D lineage analysis. The left-sided asymmetric neuroblast Caapa and the neuron PVR express hlh-14 (purple asterisks). Cells of the gut and the ASE lineages are shown for positional information. Cell coloring, except C-lineage, after [10]. B: Ventral view in both DIC and fluorescence of a representative lineaged hlh-14(tm295); Ex[hlh-14FOS::yfp]; ntIs1 embryo at the AB256-cell stage. C: Panel shows the expression (purple lines) of hlh-14 and the division pattern of part of the C-lineage as determined by 4D-lineage analysis of wild-type (containing the ntIs1 transgene, hlh- 14(gm34); otIs114, hlh-14(tm295); ntIs1 and hlh-14(tm295); otEx4507, Ex[hlh-14FOS::yfp]; ntIs1 embryos. hlh-14 expression was assessed in both gmIs20 and hlh-14(tm295); Ex[hlh-14FOS::yfp] animals. The Caapa blastomere divides early in hlh-14 mutants, at a time when closely related cells produce hypodermal cells. This defect is rescued by the hlh-14FOS::yfp transgene. The extra hyp11 cell that can be observed expressing dpy-7 (Figure S3), is indicated. The number of lineages that divided as indicated and the total number of individual left or right lineages followed to terminal division are shown at the bottom of each lineage diagram. Red cell names indicate neurons/glia and yellow cell names indicate hypodermis.

Picture from Poole RJ et al. (2011) PLoS Genet "A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in ...."

Figure 5. The achaete-scute homolog hlh-14 is required to specify neurons in the ASE lineage. A: hlh-14 is required for the terminal specification of several neuron pairs from the ABalppp/praaa lineage branch. Images of hlh-14(tm295) mutants indicating a loss of ASE (ASEL: lim- 6prom::gfp, ASER: gcy-5prom::gfp and ASEL/ASER: che-1prom::rfp) OLL (ser-2prom2:gfp) and AFD (gcy-8::gfp) cell fates. Heterozygous balanced siblings are unaffected (presence of balancer indicated by GFP in the pharynx). B: Lineage, 3D-morphogenesis and hlh-14 expression analysis in the ABalppp/praaa lineage of wild-type (containing the ntIs1 transgene), hlh-14(gm34); otIs114, hlh-14(tm295); ntIs1 and hlh-14(tm295); otEx4507, Ex[hlh-14FOS::yfp]; ntIs1 embryos. The left panel shows the division pattern and expression (purple lines) of hlh-14 in the ABalppp/praaa lineage as determined by 4Dlineage analysis. hlh-14 expression was assessed in both gmIs20 and hlh-14(tm295); Ex[hlh-14FOS::yfp] animals. In wild-type embryos the cells divided as expected. In hlh-14(gm34) mutants the 3 most posterior great-granddaughters of the ABalppp/praaa blastomere fail to divide. In hlh-14(tm295) mutants these defects extend to the 4 most posterior granddaughter. The number of lineages that divided as indicated by the lineage and the total number of individual left or right lineages followed to terminal division (and assessed for 3D cell positions) are shown at the bottom of each lineage diagram. The asterisks indicate that on one occasion the lineages still divided but aberrantly, much later than normal. N indicates neurons and G indicates glia. The right panel shows representative examples of the 3D-positions of cells from the ABalppp/ABpraaa lineage at the AB256-cell stage as assessed by 4D-lineage analysis. Both ventral and frontal views are presented. The posterior descendants of the ABalppp/praaa blastomere are dorsally and posteriorly mispositioned in hlh-14 mutants (asterisks), a position normally occupied by hypodermal cells. The fosmid array rescues the division and morphogenesis defects. The gut lineage is indicated for positional information. Cell coloring, except C-lineage, after [10]. The purple triangle under the panel indicates that hlh-14 expression is lost more rapidly in the anterior of the lineage and this correlates with more penetrant defects in posterior parts of lineage in hlh-14 mutants. C: Images of hlh-14 expression in embryos at the pre-morphogenesis (AB256-cell) stage. Top panel shows the expression of hlh-14 from an integrated translational hlh-14::gfp reporter [28]. Bottom panel shows the expression of hlh-14 from a rescuing YFP-tagged hlh-14 fosmid array. Expression in the ASE, PVQ/HSN/PHB, and DVC/PVR lineages is indicated. The full expression pattern of hlh- 14 in the ABalppp/praaa lineage is indicated by purple lines in the lineage diagram in panel C and full expression in the C-lineage is indicated in Figure 6.

Picture from Poole RJ et al. (2011) PLoS Genet "A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in ...."

Figure S4. Bilaterally symmetric expression of hlh-14 in other neuronal lineages. A: Ventral view of the 3D-positions of additional bilaterally symmetric cells that express hlh-14 at the AB256-cell stage. In the posterior of the embryo neuroblasts from the predominantly hypodermal ABplap/prap (green) and ABarpp (pink) lineages express hlh-14. Cell coloring after [10]. B: Panel shows the expression (purple lines) of hlh-14 and in certain cases the division pattern as determined by 4D-lineage analysis of the ABplap/prap lineages of wild-type embryos (containing the ntIs1 transgene). hlh-14 expression was assessed in both gmIs20 and hlh- 14(tm295); Ex[hlh-14FOS::yfp] animals. In the case of the PLM/ ALN lineage, expression was only observed in gmIs20. Red cell names indicate neurons/glia and yellow cell names indicate hypodermis. C: Panel shows the expression (purple lines) of hlh-14 in the ABarpp lineage in wild-type embryos, as determined by 4Dlineage analysis. Red cell names indicate neurons/glia and yellow cell names indicate hypodermis.