Single embryo gene expression analysis.

<p>Total cDNA was PCR amplified from poly-A+ RNA prepared from individual wildtype or mutant 12-cell stage embryos (indicated above each blot compilation), separated briefly on an agarose gel side-by-side with like-staged samples of the same genotype, and blotted to membrane ('pseudo Northern' blot; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106309#s4" target="_blank">Materials and Methods</a>). Replicate blots were hybridized with ³²-P labeled probes prepared from the <i>end-1</i>, <i>end-3</i>, and <i>sdz-23</i> genes, and the <i>tba-1</i> (alpha tubulin) gene as a loading control. Exposures were to x-ray film which was then scanned. All <i>end-1</i>, <i>end-3</i> and <i>sdz-23</i> exposures were for a similar length of time (18–20 hours), whereas the <i>tba-1</i> exposures were much shorter (10–15 minutes). <i>itDf2</i> is a deletion that removes the genomic region containing both <i>end-1</i> and <i>end-3</i> (along with a number of other genes). Dots below lane numbers denote samples from individual <i>mom-1(or10)</i>, <i>mom-2(or42)</i>, <i>mom-3(or78)</i> and <i>mom-4(ne19)</i> mutant embryos that express greatly reduced combined levels of <i>end-1</i> and <i>end-3</i>.</p

4D lineaging analyses of wildtype and <i>wee-1.1(ok418)</i> mutant embryos.

<p>(A) Lineaging results for the AB, MS and E lineages are shown for five wildtype embryos (left) and five <i>wee-1.1(ok418)</i> mutant embryos (strain RB669, right). The lineaging results are shown starting from the 3-cell stage to approximately the 50-cell stage. The 2AB to 4AB division is taken as time 0. The 16AB cell cycle is shown in blue, the 2MS cell cycle in green, and the 2E cell cycle in red. (B) Summary of specific cell cycle times for all wildtype and <i>wee-1.1(ok418)</i> mutant embryos examined.</p

Uncoupling Different Characteristics of the <i>C. elegans</i> E Lineage from Differentiation of Intestinal Markers

<div><p>In the 4-cell <i>C. elegans</i> embryo, a signal from P₂ to its anterior sister, EMS, specifies the posterior daughter of EMS, E, as the sole founder cell for intestine. The P₂-to-EMS signal restricts high level zygotic expression of the redundant GATA transcription factors, END-1 and END-3, to only the E lineage. Expression of END-1 or END-3 in early blastomeres is sufficient to drive intestinal differentiation. We show here that a number of E lineage characteristics, which are also regulated through P₂-EMS signaling, can be uncoupled from intestine development, and each with a different sensitivity to specific perturbations of the P₂-EMS signal. For example, we show that the extended cell cycle in Ea and Ep depends on the P₂-induced high level expression of the cell cycle regulator, WEE-1.1, in E. A mutation in <i>wee-1.1</i> results in shortened Ea and Ep cell cycles, but has no effect upon intestinal differentiation or embryogenesis. Furthermore, it has been shown previously that the total number of E lineage cell divisions is regulated by a mechanism dependent upon E being specified as the intestinal founder cell. We now show, however, that cell division counting can be uncoupled from intestine differentiation in the E lineage. Many mutations in P₂-EMS signal genes exhibit nonfully-penetrant defects in intestinal differentiation. When embryos with those mutations generate intestinal cells, they often make too many intestinal cells. In addition, at the level of individual embryos, expression of <i>end-1</i> and <i>end-3</i>, and another very early E-specific zygotic gene, <i>sdz-23</i>, exhibit stochastic and discordant defects in P₂-EMS signaling mutants. We show here that <i>sdz-23</i> is expressed close to wildtype levels in embryos deleted of both <i>end-1</i> and <i>end-3</i>. <i>sdz-23</i> does not appear to function in intestine development, raising the intriguing possibility that the P₂-EMS interaction has downstream molecular consequences within the E lineage independent of <i>end-1/3</i> and intestinal development.</p></div

Intestine development in Wnt/MAPK mutant embryos.

<p>(A) Differentiated intestinal cells stained with ICB4 antibody. The first two panels of the top row show a wildtype embryo imaged at two different focal planes. The third panel is a representative <i>mom-2(or42)</i> mutant embryo that did not develop intestinal cells. The following rows are examples of embryos with the indicated mutation that did develop ICB4-positive cells, imaged at three different focal planes (top, middle, and bottom). Note that in all the mutant embryos the number of ICB4-positive cells exceeds 20. Furthermore, the intestine is not elongated as in the wildtype, and instead appears as a clump of small cells. (B) Summary of ICB4-positive cell counts per embryo for the indicated Wnt and MAPK mutants. Note that the number spread from the wildtype number (20) is much restricted for the Wnt frizzled receptor mutant, <i>mom-5(or57)</i>, versus the other mutants, and that <i>mom-2(or9/+)</i> heterozygous embryos develop the wildtype number of intestinal cells (indicating that a single wildtype copy of <i>mom-2</i> is sufficient to determine E lineage cell-division number).</p

Model.

<p><i>end-1</i> and <i>end-3</i> are activated in the E blastomere as a result of P₂-to-EMS signaling and function redundantly to regulate all currently quantifiable characteristics of the E lineage. However, there are also genes, like <i>sdz-23</i>, that are expressed specifically in the E lineage and which also require P₂-to-EMS signaling for their activation. Intriguingly, these genes do not appear to be involved in intestine specification or development, and do not appear to be regulated by <i>end-1</i> or <i>end-3</i>.</p

<i>wee-1.1</i> expression in the early embryo is Wnt/MAPK-dependent.

<p>Top three rows show three time points of the same embryos from early 2E to late 2E stage. DIC images (left-hand column) and fluorescence micrographs (right-hand column) of embryos expressing the <i>wee-1.1</i> reporter. The bottom row shows the expression of the same transgene in 2E-staged <i>lit-1(t1512)</i>, left, and <i>mom-2(or42)</i>, right, mutant embryos. Closed arrowheads indicate the 2E nuclei. Open arrowheads indicate the 2MS nuclei. Arrows point to AB nuclei expressing the reporter GFP. Note the reduced (but not absent) expression of the <i>wee-1.1</i> reporter in the 2E nuclei in the <i>lit-1</i> and <i>mom-2</i> mutants. Bar: 10 µm.</p

E-lineage reporter expression and intestine development.

<p>In each panel, two groups of identically-staged wildtype and mutant embryos (left and right, respectively; divided by solid white line) expressing the same E-specific reporter were mounted together on a slide at around the 8-cell stage. Embryos were imaged for DIC and GFP fluorescence at the 2E stage (left 2 panels), and then imaged again 13 hours later for DIC and birefringent gut granules (right two panels). The mutation scored for each row is indicated on the left. The E-lineage reporter is indicated above the GFP fluorescence panels. Note that all <i>mom-4(or39)</i> mutant embryos express the P<i>end-1</i>:<i>gfp</i>::<i>h2b</i> reporter, albeit at lower levels than in wildtype embryos. Solid outline: examples of embryos that are GFP positive at the 2E stage but do not develop gut granules. Dashed outline: examples of embryos that did not express the reporter at the 2E stage but nonetheless went on to generate gut granules. Gut granule birefringence can appear stronger in mutants because of failed morphogenesis leading to compaction of intestinal cells, compared to the fully elongated intestine in wildtype embryos. Bar: 50 µm.</p

E lineage reporter expression in wildtype versus <i>wee-1.1(ok418)</i> embryos.

<p>A. Time course showing merged DIC and nuclear <i>end-1</i> reporter GFP in a <i>wee-1.1(ok418)</i> (left) and wildtype (right) live embryo. 4-cell stage taken as time 0. Reporter expression is shown at eight different timepoints from 41 minutes (2E) until 99 minutes (4E stage). Note that the 2E cells in the <i>wee-1.1</i> mutant embryo initiate mitosis at t = 56, 10 minutes before the wildtype 2E cells initiate mitosis. B. All three E-lineage-specific reporters exhibit wildtype expression in <i>wee-1.1(ok418)</i> mutant embryos (2E stage shown). Bar: 10 µm.</p