Lethal giant larvae 2 regulates development of the ciliated organ Kupffer’s vesicle

Abstract

SUMMARYMotile cilia perform crucial functions during embryonic development and throughout adult life. Development of organs containing motile cilia involves regulation of cilia formation (ciliogenesis) and formation of a luminal space (lumenogenesis) in which cilia generate fluid flows. Control of ciliogenesis and lumenogenesis is not yet fully understood, and it remains unclear whether these processes are coupled. In the zebrafish embryo, lethal giant larvae 2 (lgl2) is expressed prominently in ciliated organs. Lgl proteins are involved in establishing cell polarity and have been implicated in vesicle trafficking. Here, we identified a role for Lgl2 in development of ciliated epithelia in Kupffer's vesicle, which directs left-right asymmetry of the embryo; the otic vesicles, which give rise to the inner ear; and the pronephric ducts of the kidney. Using Kupffer's vesicle as a model ciliated organ, we found that depletion of Lgl2 disrupted lumen formation and reduced cilia number and length. Immunofluorescence and time-lapse imaging of Kupffer's vesicle morphogenesis in Lgl2-deficient embryos suggested cell adhesion defects and revealed loss of the adherens junction component E-cadherin at lateral membranes. Genetic interaction experiments indicate that Lgl2 interacts with Rab11a to regulate E-cadherin and mediate lumen formation that is uncoupled from cilia formation. These results uncover new roles and interactions for Lgl2 that are crucial for both lumenogenesis and ciliogenesis and indicate that these processes are genetically separable in zebrafish.

Functional characterization of Prickle2 and BBS7 identify overlapping phenotypes yet distinct mechanisms

“…These data suggest that KV morphogenesis can be uncoupled from KV ciliogenesis. This is consistent with published reports in which reduced KV lumen can have normal cilia length (Tay et al, 2013). Moreover, KV formation is a multistep process requiring proper migration and adhesion of precursor cells, lumenogenesis and ciliogenesis (Oteiza et al, 2010; Schneider et al, 2008; Tayeh et al, 2008; Yen et al, 2006).…”

509 Tumor suppression in basal keratinocytes via dual non-cell-autonomous functions of a Na,K-ATPase beta subunit

“…Thus, we found that atp1b1a and lgl2 not only genetically interact during epidermal cell polarity and malignancy but also during edema formation and Na/K-ATPase a-subunit localization ( Figure 7-figure supplement 1), in line with previous reports of pronephric defects and mild edema formation in lgl2 morphants (Tay et al, 2013). However, atp1b1a and lgl2 mutants also display differences.…”

Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs

“…In addition to previously described probes ( cmlc2 , foxa3 and spaw ), pitx2c and elovl6 probes were made from corresponding cDNAs amplified from a zebrafish cDNA library (see primer sequences above) and cloned using a TOPO TA cloning Kit (invitrogen). elovl6 expression was analyzed in cftr pd1048 mutants (Navis et al, 2013) or embryos injected with Lgl2 morpholino (5′-gcccatgacgcctgaacctcttcat-3′) (Tay et al, 2013). gh1 (primers: 5′-atggctagagcattggtgc-3′ and 5′-tacagggtacagttggaatc-3′) and pomca (primers: 5′-atggtgaggggagtgaggatg-3′ and tcact-catccttcctcggttg-3′) cDNA fragments were cloned using a TOPO TA cloning kit with a pCRII-TOPO vector (Invitrogen) and used as templates for antisense probes.…”