Supplemental Material for Ivanova and Moss, 2023

These are figures related to the "Orthologs of the C. elegans heterochronic genes have divergent functions in C. briggsae" study.Microphotographs were acquired using DIC and fluorescence microscopy on a Zeiss Axioplan2. Plots were generated using Prism software.</p

A model for the two sequential activities of LIN-28 in specifying cell fates.

<p>Top, Genetic formalisms depicting the two <i>lin-28</i> pathways that regulate the L2-to-L3 and the L3-to-L4 fate transitions. Bottom, A schematic time course depicting the regulatory dynamics during the first three larval stages. LIN-14, LIN-28, HBL-1 and LIN-41 are expressed at the start of larval development and are eventually repressed by the microRNAs lin-4, let-7 and the three let-7 family members miR-48, miR-84, and miR-241 (3 let-7s). The approximate times of LIN-14's two activities are indicated with boxed letters. The relevant times of LIN-28's two activities that correspond to the pathways above are depicted with black lines and circled letters.</p

<i>lin-28</i> mutants can be two stages precocious due to <i>let-7</i> activity.

1<p>All strains are homozygous for null alleles of the genes indicated and carry an integrated transgene of the seam cell marker <i>wIs78(scm::GFP; ajm-1::GFP)</i>. All alleles are null.</p>2<p>Percentage of seam cells synthesizing adult alae by early L3.</p>3<p>n = number of seam cells scored.</p

The male tail tip morphogenesis is delayed in let-7 males.

<p>Nomarski images of wild type (A) and <i>let-7</i> null (B) L4 males approximately 8 hours after the L3 molt. The extracellular space between the L4 cuticle and the tail tip in the wildtype indicates the retraction of male tail tip <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Nguyen1" target="_blank">[68]</a>. Arrow head, unretracted hypodermis in the <i>let-7</i> mutant.</p

Genetic interactions of heterochronic mutants.

1<p>All animals examined were homozygous for null alleles of the genes indicated and carry an integrated transgene <i>wIs78(scm::GFP; ajm-1::GFP)</i> to mark seam cells. All alleles are null.</p>2<p>Seam cell counts were performed on L4 animals except where indicated.</p>3<p>Alae formation was assessed in the early L4 stage.</p>4<p>Strains carrying the <i>let-7</i> mutation additionally contained a linked <i>unc-3</i> mutant allele. They were grown at 15°C to limit constitutive dauer formation that results from the <i>unc-3</i> mutation at higher temperatures in these backgrounds.</p>5<p>Seam cell fusion with no alae formation was observed in the other 85% of animals.</p><p>SEM, standard error of the mean; ND, not determined.</p

<i>lin-28</i> positively regulates <i>hbl-1</i> reporter expression.

<p>Nomarski and fluorescence micrographs of <i>hbl-1::GFP::hbl-1 3′UTR</i> reporter expression. Early stages are late L1 or early L2. Late stages are L4 or age-matched post-L3 molt <i>lin-28</i> animals. A, wild type. B, <i>mir-48 mir-241; mir-84 (3 let-7s)</i>. C, <i>lin-28; mir-48 mir-241; mir-84 (lin-28; 3 let-7s)</i>. D, a <i>hbl-1::GFP::unc-54 3′UTR</i> reporter in <i>lin-28; mir-48 mir-241; mir-84 (lin-28; 3 let-7s)</i>. Se, seam nuclei. hyp, hyp7 nuclei. All fluorescence images were captured with a 2 sec. exposure time. Scale bar, 10 microns.</p

Relative contribution of <i>hbl-1</i> and <i>lin-41</i> for the <i>let-7</i> retarded phenotype.

1<p>The <i>let-7</i> mutants were identified by Unc phenotype due to the <i>unc-3</i> mutation.</p>2<p>The precocious alae were assessed at the end of L3–L4 molt or in the early L4 stage of development.</p>3<p>As previously noted, <i>hbl-1(RNAi)</i> causes a proliferation defect in the late L4 which is not interpreted as heterochronic <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Lin2" target="_blank">[53]</a>. These divisions were not scored.</p><p>ND, not determined.</p

Seam cell lineages of animals with altered <i>lin-28</i> activity.

<p>Lineage patterns characteristic of lateral hypodermal seam cells V1, V2, V3, V4 and V6. Left to right: Wild type <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Sulston1" target="_blank">[56]</a>. Animals lacking <i>mir-48</i>, <i>mir-84</i>, and <i>mir-241</i> (<i>3 let-7s</i>), or animals carrying a transgene constitutively expressing <i>lin-28</i> (<i>lin-28(gf)</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Moss3" target="_blank">[62]</a>. <i>let-7</i> null mutants, whose defect in these lineages is first visible in the late L4 stage. Two types of seam cell lineages observed in <i>lin-28</i> null mutants <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002588#pgen.1002588-Ambros1" target="_blank">[1]</a>. Seam cell lineages that skip L2 fates in <i>lin-28(low RNAi)</i> animals (see text). Three horizontal lines indicate the time of adult alae formation. Dashed lines indicate variable lineage patterns in <i>lin-28(gf)</i> animals.</p