Left-right symmetry breaking in mice by left-right dynein may occur via a biased chromatid segregation mechanism, without directly involving the Nodal gene

Abstract

Ever since cloning the classic iv (inversedviscerum) mutation identified the "left-right dynein" (lrd) gene in mice, most research on body laterality determination has focused on its function in motile cilia at the node embryonic organizer. This model is attractive, as it links chirality of cilia architecture to asymmetry development. However, lrd is also expressed in blastocysts and embryonic stem cells, where it was shown to bias the segregation of recombined sister chromatids away from each other in mitosis. These data suggested that lrd is part of a cellular mechanism that recognizes and selectively segregates sister chromatids based on their replication history: old "Watson" versus old "Crick" strands. We previously proposed that the mouse left-right axis is established via an asymmetric cell division prior to/or during gastrulation. In this model, left-right dynein selectively segregates epigenetically differentiated sister chromatids harboring a hypothetical "left-right axis development 1" ("lra1") gene during the left-right axis establishing cell division. Here, asymmetry development would be ultimately governed by the chirality of the cytoskeleton and the DNA molecule. Our model predicts that randomization of chromatid segregation in lrd mutants should produce embryos with 25% situs solitus, 25% situs inversus, and 50% embryonic death due to heterotaxia and isomerism. Here we confirmed this prediction by using two distinct lrd mutant alleles. Other than lrd, thus far Nodal gene is the most upstream function implicated in visceral organs laterality determination. We next tested whether the Nodal gene constitutes the lra1 gene hypothesized in the model by testing mutant's effect on 50% embryonic lethality observed in lrd mutants. Since Nodal mutation did not suppress lethality, we conclude that Nodal is not equivalent to the lra1 gene. In summary, we describe the origin of 50% lethality in lrd mutant mice not yet explained by any other laterality-generating hypothesis.

Keywords: DNA strands differentiation; asymmetric cell division; laterality development; left-right dynein; selective chromatid segregation.

FIGURE 1

Strand-specific imprinting in diploid and haploid organisms. (A) Hypothetical asymmetric cell division according to our strand-specific imprinting and selective segregation (SSIS) model. Only one pair of homologous chromosomes is illustrated. Lagging versus leading strand DNA replication epigenetically differentiates an important developmental gene on sister chromatids, ON in one and OFF in the other. A segregator, such as left-right dynein, “sorts” sister centomeres/chromatids according to their replication history in G2, causing selective segregation of older Watson template strands into specific daughter cell, and older Crick template strands into the other daughter cell (named WW:CC segregation). Hence, asymmetric DNA replication-coupled epigenetic chromatin modification and selective sister chromatid segregation in the parent cell can specify different developmental potentials to daughter cells (Klar, 1994). Symbols: W, template “Watson” strand, C, template “Crick” strand. Numbers 1–4 represent specific chromatids with respect to their strands’ constitution. (B) Illustration of how lagging strand-specific imprinting explains the “1 in 4 granddaughters switching” rule in S. pombe mating-type switching. The mat1 locus efficiently switches P and M mating-type gene information by a cell cycle controlled DNA transposition mechanism. A replication terminator ensures unidirectional DNA replication of the mat1 locus, and lagging strand DNA synthesis installs an imprint (indicated by star) in a sequence- and strand-specific manner in an unswitcable (Pu) cell. The imprint confers competence for switching at the mat1 locus only in the daughter cell inheriting the imprinted chromosome (Ps), which transposes opposite mating-type information copied from the silenced donor loci into the mat1 locus only in one of the sister chromatids (Klar, 2007).

FIGURE 2

SSIS-predictions concerning embryo situs and survival rates of lrd mutants. Proposed laterality-generating asymmetric cell division is randomized in the lrd mutant. The future L/R axis is set by cytoplasmic polarization and alignment of a single cell with respect to the anterior-posterior and dorsal-ventral body axes. Sister chromatids containing a hypothetical “leftness-encoding” left-right axis-establishing gene 1 (lra1) are epigenetically differentiated. Normally, left-right dynein would selectively segregate older Watson template strand-containing sister chromatids harboring lra1 “ON” epialleles to the left body side, and older Crick template strand-containing sister chromatids harboring lra1 “OFF” epialleles to the right body side as described in Figure 1A. Randomized segregation due to left-right dynein mutation will result in three different outcomes shown here: 25% WW:CC cell pairs, causing normal situs development, 25% CC:WW cell pairs, causing development of situs inversus, and 50% WC:WC cell pairs, causing severe developmental situs abnormalities incompatible with survival.

FIGURE 3

(A) Lra1 heterozygosity is predicted to rescue WC:WC segregants that occur in lrd mutants. As illustrated in Figure 2, SSIS predicts 50% lethality in lrd mutants due to occurrence of WC:WC segregation at 50% incidence. Lethality is due to conflicting (ON and OFF) lra1 epialleles in cells that inherited both older Watson and older Crick template strands. However, in compound lrd homozygous and lra1 heterozygous mutant embryos, WC:WC segregants are predicted to survive. This is because lra1 has only one functional allele, the lethality-causing ON/OFF combination in both sister cells described in Figure 2 cannot be generated. Therefore, a 50:50 distribution of situs solitus and situs inversus animals is expected to develop. Symbols: δ, deletion of lra1 ( = Nodal?); rest of symbols are as described in Figure 1A. (B) SSIS-predicted ratios of genotypes from an iv^+/− X iv^−/− cross (top) and an iv^+/−, lra1^+/− X iv^−/−, lra1^+/+ cross (bottom). Conventionally 50% offspring is expected to be lra1^+/−. Because WC:WC segregants (gray) are predicted not to die if they are also lra1^+/−, lra1^+/− animals should be overrepresented in the offspring by a 4:3 ratio. Moreover, iv^−/− are predicted to occur at a 3:4 ratio as opposed to 1:2 (top), and lra1^+/−, iv^−/− animals are also predicted to occur at increased rates (2/7).

FIGURE 4

A finding published by Dr. Michael Levin’s laboratory is interpreted to suggest that an SSIS-like mechanism operates during olfactory neuron asymmetry development in C. elegans. (A) An AWC precursor cell undergoes asymmetric cell division and selectively segregates epigenetically differentiated sister chromatids containing an AWC master-regulator gene in a WW:CC fashion, such that always a 1AWC^ON/1AWC^OFF olfactory cell pair develops in each worm. (B) Embryos transgenic for mutated (but not wild-type) tubulin developed either 1AWC^ON/1AWC^OFF or 2AWC^ON olfactory neuron cells at a roughly 50–50 frequency. We explain this result by the SSIS model due to randomized chromatid segregation during the critical AWC^ON/AWC^OFF neuron generating cell division due to the tubulin mutation.