Picture from Huang, Xun et al. MicroPubl Biol

Figure 1. Mutants affecting RMED/V neuron specification. (A) Punc-25GFP expression in different mutants. Schematic illustrations of RME cell morphology in wild type (WT) and mutants are in the right. Scale bar: 50µm. (B) RMED cells (Arrowed) in wild type and eor-2(ju190) animals. Arrowheads point to abnormal large vesicles accumulating in the head of eor-2(ju190) animals. Scale bar: 10µm. (C) Expression of Pavr-15GFP in wild type and eor-2(ju190) animals. Pavr-15GFP is expressed in RMED/V neurons in wild type, while the expression is lost in eor-2(ju190) animals. Scale bar: 10µm. (D) Expression of Plim-4GFP in wild type and eor-2(ju190) animals. Plim-4GFP is brightly expressed in some neurons in the head region in wild type, while the expression is attenuated in eor-2(ju190) animals. Scale bar: 10µm. (A-D) All the images were taken at young adult stage.

Picture from Huang, Xun et al. MicroPubl Biol

Figure 1. EOR-1 and EOR-2 are required in RMED/V neuron specification.(A) EOR-2 is a novel protein with moderate homology in the C-terminal (boxed) to Drosophila protein CG17233. ju190 is a stop codon mutation. (B) EOR-1 contains BTB domain and nine C2H2 Zing fingers (ZnF). Alignment of nine zinc finger domains from EOR-1 and PLZF is shown. ju198 changes the histidine (H) in last zing finger to tyrosine (Y).

Picture from Huang, Xun et al. MicroPubl Biol

Table 1. RAS-ERK pathway and the canonical WNT signaling are likely not involved in RMED/V cell specification.Punc-25GFP expression in RMED/V cells in mutations in RAS or WNT signaling pathway components.

Picture from Huang, George et al. (2021) MicroPubl Biol

Figure 1. Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans: (A) Cas9 concentration affects embryonic viability, whereas DNA quality affects homology-directed repair (but not array formation). P0 worms were injected with either 50 ng/µL or 5 ng/µL Cas9/sgRNA plasmid prepared with a NEB® Monarch® and/or Invitrogen® PureLink™ HQ miniprep kit. 50 ng/µL PureLink™ HQ [n=15 (5 gene targets x 3 P0 injected worms)]; 5 ng/µL PureLink™ HQ [n=24 (8 gene targets x 3 P0 injected worms)]; 5 ng/µL NEB® Monarch® [n=9 (3 gene targets x 3 P0 injected worms)]. *p<0.0001 (2 tailed, unequal variance t-test) **p<0.000001 (2 tailed, equal variance t-test) (B) Lowering Cas9 concentration and using high-quality plasmid DNA dramatically improves fluorescent protein knock-in via CRISPR/Cas9 homology-directed repair. *We have so far been unable to isolate and genotype an N-terminal SPT-16 tag due to lethality and balancing issues. (C) Epifluorescence imaging of two-cell stage embryos derived from eight different fluorescent protein knock-in strains. The observed cellular and subcellular expression patterns were consistent with expression data archived in WormBase. Scale bar = 10 microns. (D) Confirmation of knock-in tags via PCR genotyping for the eight strains shown in C. Wild-type amplification (left) and tag amplification (right) for each gene. npp-10(utx10) and laf-1(utx16) required balancing to maintain the knock-in allele. PCR genotyping of the balanced heterozygote is shown in addition to genotyping of the wild type and homozygous knock-in. (E) Confirmation of correct insertion site for most recently targeted genes pqn-59, hum-6, T28D6.6, and ccdc-47. We have not yet excised these insertions to fully characterize the strains. Primers internal to the SEC region and the 3' flank of the target gene were used to genotype the insertion site.

Picture from Huang C et al. (2003) Development "Laminin alpha subunits and their role in C. elegans development."

Expression of the Laminin alfa gene lam-3. To observe the expression pattern of lam-3, the lam-3 promoter was used to drive GFP expression (D-F). Anterior is leftwards. (D) By the onset of elongation, lam-3::gfp is expressed in pharyngeal (p), intestinal (i) and epidermal cells (e), but not in myoblast cells. As the embryo elongates, expression in all these cells declines. (E) In larvae and adults, lam-3::gfp expression is detected in spermatheca (sp) cells. The fluorescence image is superimposed on top of the DIC image of the same animal to show the uterus (u). (F) In larvae and adults, lam-3::gfp expression is also detected in pharyngeal muscle cells (p). Scale bars: 10um in D-F.

Picture from Huang C et al. (2003) Development "Laminin alpha subunits and their role in C. elegans development."

Figure 2. Localization of laminin alfaA. Wild-type animals were stained using laminin alfaA antibodies. (A) The laminin alfaA protein is detected in the late gastrula between the rows of intestinal (i) and pharyngeal (p) precursor cells, the flanking myoblast cells (m), and the epidermoblast cells (e). Mid-plane optical section. (B) During embryo elongation, laminin alfaA is strongly detected between muscle and epidermal cells. Shown is staining associated with the two dorsal muscle quadrants. Dorsal lateral optical section. (C) In two- and threefold embryos, intense staining for laminin alfaA is observed at the basement membranes associated with the pharynx and intestine. Staining is also detected at the nerve ring (nr). (D) In larvae and adults, staining is detected at the spermatheca (s) where the protein localizes between individual endothelial cells. (E) In larvae and adults, staining is weak at pharyngeal and intestinal basement membranes but is stronger at the nerve ring. (F) Laminin alfaA is also detected in the basement membranes associated with the excretory canal (c). Anterior is towards the left, dorsal towards the top. Scale bars: 10 um.

Picture from Huang P et al. (2007) Genome Res "Identification and analysis of internal promoters in Caenorhabditis elegans ...."

(A) The klp-8 operon. Both the operon promoter (PG-L) for the leading gene C15C7.2 and the internal promoter (PG-D) for the downstream gene C15C7.1 have been extracted. (Green rectangles) Exons; (thin lines) introns; (thick red lines) intergenic regions; (gray areas) UTRs. (PO) The promoter of an operon; (PG-L) a promoter::GFP constructed from the leading gene of an operon; (PG-D) a promoter::GFP constructed from the corresponding operon downstream gene. (B) I and II show that the promoter extracted from leading gene C15C7.2 is able to drive GFP expression in the excretory cell, pharynx, pharyngeal neurons, and head neurons, while the promoter extracted from downstream gene C15C7.1 directs GFP expression in the seam cells (III and IV).

Picture from Huang P et al. (2007) Genome Res "Identification and analysis of internal promoters in Caenorhabditis elegans ...."

(C) The CEOP3332 operon. Both the operon promoter and the internal promoter for downstream gene B0336.2 are used to construct GFP fusions. (D) I and II show that the promoter extracted from leading gene B0336.3 is able to drive GFP expression in hypodermis, pharyngeal gland cell, gut, and nerve ring, while that of downstream gene B0336.2 is able to drive GFP expression in the pharynx, gut, and head neurons (III and IV).

Picture from Huang C et al. (2003) Development "Laminin alpha subunits and their role in C. elegans development."

Figure 4. Localization of Laminin alfaB. Wild-type animals were stained using Laminin alfaB antibodies. (A) The Laminin alfaB protein is detected in the late gastrula between the rows of intestinal (i) and pharyngeal (p) precursor cells, the flanking myoblast cells (m), and the epidermal cells (e). Mid-plane optical section. (B) Laminin alfaB is localized to the muscle and epidermis basement membranes in late stage embryos, larvae and adults. The protein has different distribution patterns in the pseudocoelom/epidermis (p/e) and muscle/epidermis (m/e) basement membranes. Lateral view showing two muscle quadrants, each two cells wide. The muscle cells are anchored to the epidermis through connections involving the muscle/epidermis basement membrane. The lateral epidermal ridge separates the quadrants. (C) In L3 and L4 larvae, Laminin alfaB is localized to the gonad basement membrane and is associated with the distal tip cell (dtc), a migratory cell that helps form the gonad (g). (D) In late stage embryos, larvae and adults, Laminin alfaB is localized to the basement membranes that separate the intestine (i), rectal epithelium (a; anus), epidermis and pseudocoelom. (E) Laminin antiB is localized to the basement membrane that encloses the gonad (g) primordium cells in the L1 and L2 larval stages. (F) Laminin alfaB is detected in the basement membranes associated with intestinal muscle (im) and anal depressor muscle (am). (G) Laminin alfaB is detected in vulval muscle (vm) basement membrane and somatic gonad (g; uterine epithelium and/or uterine muscle). Staining of all specialized muscles is asymmetric: stronger near their anchorage to bodywall or other epithelia. (H) Laminin alfaB is associated with coelomocytes (cc). Scale bars: 20 um for A-E.

Picture from Huang C et al. (2003) Development "Laminin alpha subunits and their role in C. elegans development."

Expression of the Laminin alfa gene epi-1. To observe epi-1 RNA expression, embryos were hybridized with antisense epi-1 probes (left panels in A-C) and stained for DNA with DAPI (right panels in A-C). Anterior is leftwards. (A) The endodermal and mesodermal precursor cells sink inwards from the ventral surface as gastrulation begins. epi-1 RNA is detected in the nuclei of the ingressing cells (arrows). (B) At midgastrulation, epi-1 RNA is detected in the cytoplasm of intestinal, pharyngeal and myoblast cells, but not epidermal (e) precursor cells. (C) At the beginning of elongation, expression is detected primarily in the body wall myoblast cells (m). Expression is weak or is not detected in intestinal (i), pharyngeal (p) and epidermal cells (e).