Embryonic priming of a miRNA locus predetermines postmitotic neuronal left/right asymmetry in C. elegans

Abstract

The mechanisms by which functional left/right asymmetry arises in morphologically symmetric nervous systems are poorly understood. Here, we provide a mechanistic framework for how functional asymmetry in a postmitotic neuron pair is specified in C. elegans. A key feature of this mechanism is a temporally separated, two-step activation of the lsy-6 miRNA locus. The lsy-6 locus is first "primed" by chromatin decompaction in the precursor for the left neuron, but not the right neuron, several divisions before the neurons are born. lsy-6 expression is then "boosted" to functionally relevant levels several divisions later in the mother of the left neuron, through the activity of a bilaterally expressed transcription factor that can only activate lsy-6 in the primed neuron. This study shows how cells can become committed during early developmental stages to execute a specific fate much later in development and provides a conceptual framework for understanding the generation of neuronal diversity.

Figure 1. Lineage histories and gene expression of the ASE neurons

Schematic of ASE development. Numbers on the left indicate the approximate timing of the cell divisions. The two boxes show the asymmetric gene expression in the two mature ASEs, with the two alternative configurations of the double-negative loop that result in cell-specific expression of putative guanylate cyclase receptors such as gcy-7 and gcy-5. The onset of CHE-1 expression in the mother of ASE was determined by fosmid reporter expression (Sarin et al., 2009) and by smFISH (Suppl. Figure 1).

Figure 2. lsy-6 is the first known asymmetrically expressed component of the loop in the ASE neurons

A. Schematics of fosmid reporter genes. B. Representative YFP expression in animals carrying the lsy-6::yfp^fosmid reporter. Expression is first seen around the bean stage, exclusively in ASEL and continues in ASEL until adulthood. Animals and ASEL are outlined. For quantification see Suppl. Figure 2A. * marks autofluorescence from intestinal cells. C. Expression of fosmid-based cog-1::yfp and die-1::yfp reporters. Both transcription factors start being expressed in ASE, only at the 3-fold stage, in an asymmetric manner: cog-1::yfp in ASER and die-1::yfp in ASEL. A bilaterally expressed che-1^prom::mCherry reporter was used to label the two ASEs. D. Summary of developmental expression of lsy-6, die-1 and cog-1 in the ASE neurons.

Figure 3. A downstream cis-regulatory element is necessary for exclusive lsy-6 expression in ASEL

A. Schematic of the transcriptional reporter containing the 932 bp upstream element, and representative pictures showing its expression pattern through different stages. Expression begins around the 2-fold stage in both ASE neurons and later gets restricted to ASEL. The blue colored box indicates a functional CHE-1 binding site. B. Quantification of YFP expression in two independent lines of animals carrying either the lsy-6::yfp^fosmid or the lsy-6^prom::yfp reporters, throughout different developmental stages. Around 25 animals were scored per time point, per line. C. A sequence element present downstream of the lsy-6 hairpin is sufficient to complement the lsy-6 upstream region to produce an expression pattern most similar to that from the fosmid-based reporter regarding time of onset of expression and exclusivity to ASEL. Red arrowheads show cleavage and polyadenylation sites (Gerstein et al., 2010). The downstream element can be narrowed down to 300 bp (if a 3′UTR containing a functional cleavage and polyadenylation site is included). The 300 bp element also complements expression when placed upstream of the promoter region. * marks autofluorescence from intestinal cells. For quantification see Suppl. Figure 3B.

Figure 4. The downstream regulatory element drives expression in the ABa lineage five cell divisions before the birth of ASEL

A. Expression of a transcriptional reporter containing the downstream element driving gfp begins early in the embryo and continues into larval stages in a few cells, including ASEL. Lineage analysis of gfp expression was carried out on three developing embryos from two independent lines carrying the reporter, using 4D microscopy (Schnabel et al., 1997). Different shades of green represent how many embryos show expression for any given branch. Expression is most consistent in the branch that gives rise to ASEL, while it is never observed in the ABp lineage (or in the mature ASER). B. Reporter expression driven by the downstream element is lost in tbx-37/38 double mutants. Representative pictures of a wild-type (either +/+ or +/−) and a double mutant embryo at two stages of development, showing tbx−/− animals do not express GFP at any stage. Embryos are followed from the 2-cell stage until just before hatching. At this stage tbx−/− animals are identified due to their characteristic lack of anterior pharynx (*) in addition to their obvious failed morphogenesis. GFP images are analyzed retrospectively to score wild-type and mutant animals. Quantification of this loss of expression is shown to the right (numbers on top of the bars are the number of animals with expression/number of scored animals). C. Single-molecule FISH against yfp and mCherry was performed on embryos carrying both the lsy-6::yfp^fosmid and a tbx-38^prom::mCherry reporter. Embryos were staged according to the number of nuclei and by the number of cells expressing tbx-38^prom::mCherry. See Supplemental Experimental Procedures for more detail on the procedure. Transcription from the lsy-6::yfp^fosmid is first seen around the AB32 stage (III), consistent with the timing of expression of the downstream transcriptional reporter (A) in cells that belong to the ABa lineage and thus also express tbx-38. Transcription off of tbx-38^prom begins in the 4 ABa granddaughters (I), when the transcript is still in the nucleus, and reaches its highest level in the 8 great granddaughters (II), consistent with antibody staining (Good et al., 2004). However, some mCherry mRNA from this promoter fusion seems to persist longer than endogenous TBX-38 and we can use this to trace the ABa lineage. Asterisk at the bean stage (VI) marks expression from a co-injection marker, ttx-3^prom::mCherry. The outlines of the embryos are indicated with dashed white lines as well as the outlines of the tbx-38^prom::mCherry expressing cells in I and II and the outlines of ASEL (VI) and what is very likely its mother (V). Insets in III and IV show close up views of the boxed areas. We furthermore note that smFISH that measures transcription from the endogenous che-1 locus reveals transcription at around the same time as a che-1 fosmid reporter transgene (Suppl. Figure 1), demonstrating that smFISH does not simply pick up spurious transcription. D. Semi-quantitative, real-time RT-PCR analysis confirms the early, low-level transcription of the lsy-6 locus several cell divisions before the birth of the ASE neurons (200 minutes post 2-cell stage time point; see Supplemental Experimental Procedures). E. Summary of the expression patterns of each of the two isolated cis-regulatory elements and the outcome of both acting together.

Figure 5. Early activation, or “priming”, of the lsy-6 locus is necessary to maintain the locus competent for subsequent activation

A. Schematic of the deletions generated in the lsy-6::yfp^fosmid reporter. Deletion of the downstream cis-regulatory element abolishes expression from the genomic locus. The red arrowheads show two functional cleavage and polyadenylation sites (Gerstein et al., 2010). A 3 Kb deletion that leaves the 150 bp element intact does not affect expression (for quantification see Suppl. Figure 6A; the 3′ downstream gene likely provides a cleavage and polyadenylation site in this construct). B. Schematic representation of the key regulators of lsy-6 expression and the experimental approach taken in panels C and D. tbx-37/38 are required for the downstream element-mediated, low level expression of lsy-6 (indicated with a grey box), che-1 is required for boosting lsy-6 expression in the ASEL mother cell. In panel C, the transient tbx-37/38 input into the locus is eliminated by removal of the downstream element and substituted by a transient che-1 input. In panel D, the activity of the che-1 gene is broadened to other neurons in which the lsy-6 locus may also have been primed by tbx-37/38. C. Artificial activation of the lsy-6::gfp^fos Δ150 through ectopic, heat-shock induced, expression of CHE-1 (schematized by the red arrows) restores GFP expression from this reporter (measured at the time the ASEs are born, green arrow), only when provided during a specific time window. This window coincides with the time of expression of the downstream element and the onset of transcription from the fosmid reporter seen by smFISH (Figure 4A,C). GFP expression is not only observed in ASEL but also in a few additional cells, likely including ASER. Heat-shock treatment of embryos without the heat shock inducible CHE-1 or expressing the unrelated TF HLH-1, do not result in GFP expression. N is 15–43 embryos for each time point shown. D. The lsy-6 locus is primed in multiple descendants of the ABa lineage. Ectopic expression of CHE-1 under the gpa-10 promoter (active in the ADFL/R and ASJL/R neurons) causes expression of the lsy-6::yfp^fosmid in two additional cells, only on the left side of the head, that based on position and morphology are identified as ADFL and ASJL. These two cells are closely related to ASEL by lineage and their shared precursor shows expression of the lsy-6 downstream element. Although the gpa-10 promoter also drives expression of CHE-1 in ADFR and ASJR, expression of lsy-6::yfp^fosmid is never observed in these cells.

Figure 6. Priming of the lsy-6 locus involves the establishment of an active chromatin structure, in a tbx-37/38-dependent manner

A. Schematic of the array used for visualization of the lsy-6 locus. A fragment of the lsy-6 locus containing 932 bp upstream and 1 Kb downstream was co-injected together with binding platforms containing 256 lacO repeats and bacterial genomic DNA as spacer and to increase sequence complexity. These arrays can be visualized through the binding of a GFP::LacI fusion protein. B. Representative images from one of the 4D series of images of embryos carrying chromosomally integrated, lacO labeled lsy-6 locus. The precursors of ASEL and ASER at the AB32 stage (the cell division right after tbx-37/38 expression) are boxed. The image is a maximum-intensity projection of the planes that span the nuclei of interest. Close-up, color inverted, images of their nuclei are shown to the right (Animal 1), as well as the respective nuclei from two additional embryos carrying the labeled locus. Manual traces of areas of GFP accumulation are shown in the insets. As a measure of compaction/decompaction, we assessed the area of the nucleus that has GFP intensity above background, from maximum-intensity projections obtained for both relevant nuclei. A 2-fold larger area is occupied by the lsy-6 transgene in ABalppp as compared to ABpraaa (P=0.02). N=6. Embryos from three independent integration events were analyzed. C. Quantification of the number of embryos with a de-compacted lsy-6 locus in all ABa and ABp derived branches at the AB32 stage. These numbers were obtained from 8 embryos, each carrying one of three independent lsy-6/lacO integrated transgenes and are expressed as number of decompacted nuclei/number of nuclei scored for each branch. D. Decompaction of the lsy-6 locus fails to occur in tbx-37/38 mutant embryos. A representative frame from 4D series of images of embryos from tbx-37/38 heterozygous mothers, carrying the lacI/lacO arrays is shown. The two mutant alleles are present over a balancer marked with an embryonically expressed gfp, such that homozygous mutant embryos can be easily identified by the absence of the balancer. The ASEL precursor for this embryo is boxed and a close up is shown. A comparison of the nuclear areas occupied by the lsy-6 transgene in the two ASE precursors (as calculated in panel B) is shown in the plot to the right.

Figure 7. Summary of the mechanistic framework for the establishment of ASE lateral asymmetry

Schematics of the two cis-regulatory elements in the lsy-6 locus are shown, as well as the relevant trans-acting factors for its expression and their timing of action. The function of the transient expression of TBX-37/38 exclusively in the ABa lineage is necessary to prime the lsy-6 locus 6 cell divisions before ASEL is born, producing low levels of transcription from this locus, and establishing a lineage-specific, open chromatin conformation. Priming of lsy-6 allows for a boost of expression through the action of CHE-1, which is present in both ASE neurons, producing high levels of lsy-6 only in the postmitotic ASEL. In ASER, absence of tbx-37/-38 leaves lsy-6 in a refractory state that does not respond to the presence of CHE-1. Additional repression of lsy-6 by COG-1 in ASER ensures that the miRNA will not be produced in this neuron (unpubl. data). Once the lsy-6 asymmetry is established, the rest of the asymmetric gene expression program in the ASEs is defined.