Characterization Of A Maternal Effect Allele Affecting Behavior And The Formation Of Left-Right Axis In Zebrafish (Danio rerio)

Abstract

In vertebrates, the bilateral symmetry of the external body plan conceals consistent left-right (LR) asymmetries in the disposition, morphology and function of internal organs. Genetics and molecular mechanisms that establish LR identities in the two halves of the developing embryo act between late gastrulation and early somitogenesis and are known to be conserved amongst the different classes of vertebrates (Levin, 2005). In the vertebrate embryo the event responsible for breaking of the initial body symmetry occurs during late gastrulation at the posterior end of the notochord (the node of mammalian embryos) where a transient ciliated structure becomes evident. In zebrafish, this structure is identified as the Kupffer’s vesicle (Essner et al., 2005; Kupffer, 1868; Okada et al., 2005). Cilia in this structure posses a rotating beating movement that generates a leftward flow of extracellular fluid that triggers the asymmetrical transcription of Nodal and Lefty genes in the left lateral plate mesoderm, and the transcription factor Pitx2 during early somitogenesis (Blum et al., 2009). Disturbance or absence of nodal leftward flow results in laterality defects and randomization of left-right asymmetries (Essner et al., 2005). Recently it has been reported first evidence of nodal and Pitx orthologues isolated in two species of snails with opposite body handedness and direction of shell coiling. Authors found that nodal and Pitx are both expressed in the right side of the embryo in the dextral species and in the left side in the sinistral species. These results suggest that the asymmetrical expression of nodal and Pitx may represent an ancestral conserved feature in the evolution of Bilateria (Grande and Patel, 2009). Furthermore, from previous studies it is known that in snails, body handedness is controlled by a maternal effect gene that determines the direction of shell coiling in the offspring (Boycott and Diver, 1923). Here in this study we report the first evidence of a maternal effect gene controlling the establishment of L-R body asymmetries in a vertebrate embryo suggesting that other mechanisms are evolutionary conserved from snails to vertebrates. In a recent study Facchin et al. (2009a) showed that the progeny of lines of zebrafish artificially selected for the right eye preference in scrutiny a mirror had a significant increase in the frequency of reversed left-right asymmetry in the epithalamus. At the beginning of the present study it has been proposed that Facchin’s selection for behavioral lateralization could have lead to the isolation of a spontaneous mutant allele responsible for the alteration of normal left-right patterning in zebrafish neuroanatomical structures. Based on a preliminary mendelian analysis of this trait, we performed selective crosses on females identified as mutant carriers to test the hypothesis of a recessive maternal effect mutation. We identified three different classes in the group of analyzed females according to the percentage of reversed brain asymmetries in their offspring. Females generating a frequency of 0-5%, between 5 and 16% and females generating more than 16% of progeny with reversed asymmetries. Females from the last group were considered as putative mutants, according to Facchin’s data (2009a). We then decided to investigate in this strain the expression of members of signaling pathways responsible for the establishment of visceral and diencephalic left-right asymmetries. Single probe in situ hybridization analyses with lefty1 and ndr2 (cyclops) revealed that approximately 50% of embryos from putative mutant females showed altered -bilateral or right sided- expression of these markers that are normally expressed on the left side of the dorsal diencephalon and in the left lateral plate mesoderm (LPM). A third Nodal-related gene, southpaw (ndr3), is asymmetrically expressed upstream cyclops and lefty1 in the left LPM at early somitogenesis stages. Also southpaw is expressed bilaterally or right-sided in approximately 50% of putative mutant’s progeny, suggesting that the mutation could act upstream the activation of the asymmetrical Nodal pathway. We therefore decided to investigate the effect of the analyzed gene on the Kupffer’s vesicle (KV) structure and morphogenesis. In vivo observations revealed that putative mutants’ offspring develop a smaller KV. We measured AP diameter and LR diameter and area of KV in embryos derived from the three groups of females. Progeny from females that generate 5-16% offspring with right parapineal display an intermediate phenotype in the size of KV. Our hypothesis suggests that smaller size of KV can reduce the amount of morphogens accumulated by the leftward flow, thus leading to a randomization of the expression of genes of the Nodal pathway. In vivo observation are going to be supported by in situ hybridization data performed using probes specific for genes expressed in the cluster of cells (dorsal forerunner cells) that aggregate to form KV. Furthermore it was posited that this initial symmetry-breaking event determines the development of lateralized brain functions and early differences in epithalamic left–right asymmetry give rise to individual variation in coping styles and personality. We tested these two hypotheses by sorting zebrafish with left or right parapineal at birth using a foxD3:GFP marker and by measuring visual and motor laterality and three personality dimensions as they become adults. Significant differences between fish with opposite parapineal position were found in all laterality tests while the influence of asymmetry of the habenulae on personality was more complex. Fish with atypical right parapineal position, tended to be bolder when inspecting a predator, spent less time in the peripheral portion of an open field and covered a shorter distance when released in the dark. Activity in the open field was not associated to anatomical asymmetry but correlated with laterality of predator inspection that in turn was influenced by parapineal position. One personality dimension, sociality, appeared uncorrelated to both anatomical and functional asymmetries and was instead influenced by the sex of the fish, thus suggesting that other actors, i.e. hormonal, may be implicated in its development.