An unconventional myosin, myosin 1d regulates Kupffer’s vesicle morphogenesis and laterality

Establishing left-right (LR) asymmetry is a fundamental process essential for arrangement of visceral organs during development. In vertebrates, motile cilia driven fluid flow in the left-right organizer (LRO) is essential for initiating symmetry breaking event. Without a definite LRO structure in invertebrates, LR asymmetry is initiated at a cellular level by actin-myosin driven chirality. In Drosophila, myosin1D drives tissue-specific chirality in hind-gut looping. Here, we show that myosin 1d (myo1d) is essential for establishing LR asymmetry in zebrafish. Using super-resolution microscopy, we show that the zebrafish LRO, Kupffer9s vesicle (KV), fails to form proper lumen size in the absence of myo1d. This process requires directed vacuolar trafficking in KV epithelial cells. Interestingly, the vacuole transporting function of zebrafish Myo1d can be substituted by myosin1C derived from an ancient eukaryote, Acanthamoeba castellanii, where it regulates the transport of contractile vacuoles. Our findings reveal an evolutionarily conserved role for an unconventional myosin in vacuole trafficking, lumen formation and determining laterality.


RESULTS
Several animal models have been used to investigate different theories for establishment of LR asymmetry 8 . This includes intracellular chirality 9 , voltage gradient flow 10 , chromatid segregation 11 and motile cilia 12 . Cilia-driven fluid flow has been reported to be essential for LR asymmetry in Mus musculus (mouse) 1 , Xenopus laevis (African Clawed Frog) 3 , Danio rerio (zebrafish) 2 and Oryctolagus cuniculus (rabbit) 13 . However, a role for cilia is not universally conserved across vertebrates. In chick embryos, symmetry breaking is regulated by asymmetric cell migration around Hensen's node and does not involve cilia based flow 14 . In addition, motile cilia are not associated with LR asymmetries that develop in invertebrates, such as the asymmetric looping of the gut and gonad in Drosophila 6,7 .
Therefore, probing common cellular events in different model systems may reveal insights into the early mechanisms of breaking symmetry that have been conserved during evolution.
Prior studies involving Drosophila myosin1D revealed a role for actin based molecular motors in establishing LR asymmetry in invertebrates 6,7 . We wondered whether myo1d has an evolutionary conserved role in specifying laterality in vertebrates. Zebrafish myo1d is expressed early and ubiquitously, including within tailbud region where the ciliated KV forms (white arrow, Supplementary Fig 1a). We generated myo1d mutants using goldy TALENs ( Supplementary Fig. 1b) 15,16,17 to reveal its role in LR patterning. Three mutant alleles of myo1d, herein named pt31a, b and c ( Supplementary Fig. 1c) were predicted to cause frame shift and amino acid truncation ( Supplementary Fig. 1d). However, zygotic homozygous mutants (myo1d pt31a/pt31a ) survived to adulthood in a Mendelian ratio suggesting that myo1d maternal expression was sufficient for embryonic development. We generated myo1d pt31a/pt31a maternal-zygotic (MZ) mutants and found that the KV had either a small or dysmorphic lumen at 8 somite stage (S) (Fig. 1 ai-iii). Consistently, injection of myo1d translation blocking antisense morpholinos also affected KV size ( Supplementary   Fig. 2a, b & Supplementary Movie 1 & 2). Next, we stained for tight junction protein, ZO-1 to assess KV morphology in myo1d MZ mutants. ZO-1 staining showed a smaller or dysmorphic KV lumen compared to controls ( Fig. 1 aiv-avi), indicating myo1d is essential for generating a spherical KV shape and forming a lumen. We crossed myo1d pt31a mutants into a transgenic line that labels membranes of KV cells, Tg(dusp6:EGFP) pt21 , and confirmed that myo1d MZ mutants have smaller or dysmorphic lumen ( Fig. 1a vii-ix, & 1b).
Next, we confirmed that Myo1D expression was detected in KV epithelial cell borders ( Supplementary Fig. 3a), whereas it was absent in myo1d MZ KVs ( Supplementary Fig   3b), suggesting that the pt31a allele is a null mutant. Also, a similar lumen defect was observed in the otic vesicle (OV) (Fig. 1c-e), suggesting that myo1d is essential for the formation of different fluid filled structures during development. A report has shown that a minimum threshold lumen size is necessary for establishing robust LR asymmetry in zebrafish 18 . Consistently, we found increased frequency of laterality defects in myo1d MZ mutants ( Fig. 1f & g) and in myo1d morphants over wildtype (Supplementary Table 1).
These experiments revealed a role for myo1d in establishing proper KV shape, lumen size and laterality in zebrafish.
Since motile cilia in the KV are required for proper laterality, we determined if loss of myo1d affected ciliogenesis. Acetylated tubulin staining showed that cilia length was normal ( Supplementary Fig. 4a & b) but motile cilia numbers per KV were less in myo1d MZ mutants ( Supplementary Fig. 4c). As KV epithelial cells are monociliated 2 , we counted KV nuclei bordering the lumen and found fewer cells in myo1d MZ embryos ( Supplementary Fig. 4d & e). Thus, the decreased cilia number was due to fewer KV cells. Together, these results indicated that myo1d loss has no effect on ciliogenesis.
At 6 hours post fertilization (hpf), KVs are formed from the dorsal forerunner cells (DFCs) near the organizer 2 . These cells proliferate and undergo a mesenchymal-to-epithelial transition to form a rosette like structure, where KV lumen forms 2 . To assess if the abnormal KV lumen phenotype in myo1d MZ mutants are a result of defects in DFC clustering, we analyzed foxj1a expression 19 . We did not observe differences in foxj1a expression in myo1d MZ and wildtype embryos ( Supplementary Fig. 5). This indicated that DFC clustering and migration was not the cause of defective KV morphogenesis.
KV lumen formation is a rapid and dynamic process during somitogenesis that spans approximately 3-hour period (1S to 8S, 10-13 hpf) when a fluid filled spherical shaped organ is aligned to the base of the notochord (see Supplementary Movie 1) 2 . Previous studies described how a symmetric KV rosette undergoes extensive cellular remodeling to become an asymmetric KV so that anterior cells retain columnar shape whereas posterior cells attain squamous or cuboidal shape 20 . On depletion of Rock2b or pharmacological inhibition of non-muscle myosin II was found to affect anterior-posterior (AP) cell shape changes in the KV 21 , suggesting that actomyosin activity is important for generating AP asymmetry. Additional fluid filling mechanisms may also be contributing to this process.
Thus, we reasoned that myo1d could contribute to the AP asymmetry through a fluid filling mechanism. In Tg(dusp6:EGFP) pt21 embryos, we observed vacuole-like structures in KV cells that were designated as such by virtue of their size (Fig. 2ai,  In ancient unicellular eukaryotes, myosin-I is involved in transporting water through contractile vacuoles (CV), which is essential for attaining amoeboid cell shape and regulating directed cell motility 23,24 . Loss of Acanthamoeba castellanii myosin-IC activity by inhibitory antibodies resulted in CV accumulation in the cell that ultimately leads to cell rupture 23 . We reasoned that a similar process may be occurring in the KV epithelial cells in myo1d MZ mutants. We found significantly higher number of fragmented nuclei in myo1d MZ embryos compared to wildtype at 1S ( Fig. 6a &b). Together, class I myosins play an evolutionary conserved role in intracellular vacuolar transport that regulates cell shape changes and drives lumen formation in the zebrafish KV.
Cystic fibrosis transmembrane conductance regulator (CFTR), which is localized in KV apical membrane, was shown to regulate water transport into the epithelial lumen 25 .
Consistently, zebrafish CFTR mutants exhibited impaired KV lumen formation 26 . Also, pharmacological treatment with Forskolin and IBMX (FIBMX) that activates CFTR expands KV lumen size 27 . We questioned whether CFTR can still function in myo1d MZ mutants.
Quantification of CFTR localization to the KV apical surface was performed using the TgBAC(cftr-GFP) embryos 26 (Supplementary Fig. 7). We observed normal CFTR localization in the KV apical membrane after Myo1d depletion (Fig. 4a, b). Interestingly, FIBMX treatment in myo1d MZ embryos showed expansion of KV lumen, implicating CFTR mediated lumen expansion was still functional in the absence of Myo1D (Fig. 4c-e).
These results support a model where myo1d and CFTR are two independent mechanisms regulating the fluid filling process in the KV (Fig. 4f). Thus, proper KV lumen formation requires multiple modes of fluid filling mechanisms such that a threshold volume is achieved in a limited developmental timeframe. It appears that KV fluid is primarily derived from posterior epithelial cells that decrease cell volume concomitant with lumen expansion. It is remarkable that this process is akin to the water expulsion mechanism found in protozoans that traffic water and other fluid filled contractile vacuoles to the plasma membrane. Similar asymmetrical fluid loss and epithelial thinning process were observed during zebrafish otic vesicle development where actomyosin interaction provides the forces necessary for expansion of lumen 28 . Consistently, this remodeling process creates new luminal space and cause a net redistribution of fluid from epithelial cells to lumen, highlighting the role of intra epithelial fluids in lumen expansion 28 . We also found lumen formation defects in the otic vesicle of myo1d MZ mutants (Figure 1c-e), suggesting that myo1d mediated epithelial cell thinning process could be a common mechanism for lumen formation during development.
In Drosophila, tissue specific and temporal expression of myosin1D in the hindgut was sufficient to drive intrinsic chirality at the cellular level that generates a consistent gut and gonad looping pattern 4 . A mechanical model suggests that anchored myosin motors walking along actin drives the filaments to turn in leftward circles 29 . With no LRO in invertebrates, these circular forces drive specific organ looping morphogenesis 4,5 . On the contrary, in zebrafish, generation of AP cell shape changes in the KV is the earliest asymmetric event. myo1d contributes to AP asymmetry in the zebrafish, prior to ciliogenesis and establishment of fluid flow 6,7 . Together, we reveal a conserved vacuolar transport function of a myosin motor protein found in primitive eukaryotes that is critical in determining left-right asymmetry in a vertebrate.

Zebrafish handling and maintenance
All the experiments using zebrafish was carried out with prior review and approval by the University of Pittsburgh Institutional Animal Care and Use Committee. Transgenic zebrafish lines used in work were: Tg(dusp6:EGFP) pt21, AB*, myo1d TALEN mutant lines generated for this work were labeled: pt31a, pt31b and pt31c alleles.

Riboprobes preparation and in situ expression analysis
Total RNA was isolated from zebrafish embryos at 24hpf.  36 .

Immunohistochemistry and microscopy
Primary antibodies used for this study: acetylated tubulin (Sigma T7451: