Presence of midline cilia supersedes the expression of Lefty1 in forming the midline barrier during the establishment of left-right asymmetry
Preprint posted on May 17, 2018 https://www.biorxiv.org/content/early/2018/05/17/324533
The breakage of left-right symmetry during development is a process that has both fascinated and frustrated developmental biologists for decades. During early development, cells in an initially symmetrical embryo must acquire positional identities along the left/right (L-R) axis to ensure that organs later develop in the correct position, and then if required, go on to develop asymmetrically themselves.
In vertebrates, the initial breakage in symmetry is thought to originate from the beating of polarised cilia, which produce a leftward flow of extraembryonic fluid within the left-right organiser region (the node in mammals). Another type of cilia sense this flow, and activate the left-determinant gene Nodal in the left lateral plate mesoderm (LPM) only. Importantly, expression of the Nodal inhibitor Lefty1 forms a barrier along the midline to prevent activation of Nodal on the right-hand side. Nodal then propagates an asymmetric signalling cascade to correctly pattern the developing embryo along the L-R axis.
Graphical abstract, kindly provided by the Weatherbee lab: In a wild-type embryo, Nodal and Pitx2 are restricted to the left-hand side due to the midline barrier, which is established and maintained by midline cilia and Lefty1-mediated Nodal inhibition. However, in transition zone (TZ) mutants, midline cilia are greatly reduced in number and Lefty1 expression is not sufficient to inhibit Nodal. Consequently, Nodal diffuses across the midline barrier and the its signalling cascade is activated on both sides. Ultimately, this leads to numerous laterality defects later in development.
Many questions how the Nodal signalling cascade is initiated, maintained and transduced remain to be answered. Even the exact roles and importance of cilia to the initial breakage of L-R patterning are somewhat controversial, as the iv mouse mutant for example has immotile cilia, but 25% of embryos develop normally. Discrepancies like these between theories that explain phenotypes of some mutants very well, but not of others, suggest that there is still much to understand about the establishment and maintenance of L-R asymmetry.
In this preprint, Shylo and colleagues investigated L-R patterning in three different mouse ciliary mutants. At first, their phenotypes seemed contradictory to each other, and also differed from current models. After investigating these inconsistencies further, the authors unexpectedly discovered a novel requirement for midline cilia to establish and maintain the midline barrier, independent of Lefty1 expression.
- The authors first studied mouse embryos containing a null mutation in Tmem107 (Tmem107null), a gene which encodes a component of the transition zone of primary cilia. Tmem107null embryos developed L-R asymmetry defects such as randomised heart looping, stomach positioning and left pulmonary isomerism. Earlier in development, the nodes of Tmem107null embryos were found to contain fewer cilia, although those present appeared normal. In terms of gene expression, in situ hybridisation showed that Nodal and Pitx2 were expressed bilaterally in the LPM of Tmem107null embryos, in contrast to wild type controls, where Nodal and its target Pitx2 were confined to the left-hand side.
- The formation of the midline barrier, and Lefty1 expression, is thought to be dependent on Sonic Hedgehog (Shh) signalling. In Tmem107null embryos, the Shh targets FoxA2 and Ptch1 were reduced at the midline compared to controls, and Lefty1 was absent. From this the authors concluded that without functional Tmem107, Shh signalling is not properly transduced, leading to loss of Lefty1 and a defective midline barrier.
- The authors next investigated another ciliary transition zone mutant Mks1 (Mks1krc), known to have the same laterality defects as Tmem107null embryos. Similar to Tmem107null embryos, Nodal and Pitx2 were bilaterally expressed, and Shh targets absent. Surprisingly, Lefty1 expression was present in the midline of Mks1krc. This contradicts previous work suggesting that firstly Lefty1 in the midline barrier is Shh-dependent, and secondly that Lefty1 is sufficient for establishing the midline barrier.
- Further questions arose after authors investigated the hypomorphic Tmem107 mutant (Tmem107schlei) mouse line, which despite containing fewer and abnormally-shaped nodal cilia, exhibit no laterality defects. Interestingly however, a population of cilia were spotted in the midlines of wild-type and Tmem107schlei embryos, which were absent in the midlines of both Tmem107null and Mks1krc embryos.
From Figure 8 – used with permission from the authors. Midline cilia are absent in transition zone ciliary mutants that later develop laterality defects: Confocal Z-stacks show the node and/or midlines of Tmem107schlei, Tmem107null and Mks1krc embryos, after immunostaining with an antibody against the cilia marker ARL13B. Cilia are almost absent in Tmemnull and Mks1krc midlines, but can be observed in Tmem107schlei midlines and wild-type controls.
Why I chose this paper
The subject of how cells acquire and process positional information has always been fascinating to me, and the heated debates about cilia and L-R asymmetry were used as literature review exercises multiple times during my university days. Although this paper does not go into the function of the midline cilia, it provides another piece of the L-R asymmetry puzzle, which might eventually show us how L-R patterning is defined, maintained, and then propagated in later development.
As a general reason, I think there is a temptation to put experimental findings to one side when they contradict current theories, or to cherry pick data until they fit, instead of exploring confusing results further. In this preprint, Shylo and colleagues show a series of simple yet directed experiments to investigate unexpected results, which lead them to discover something novel which will hopefully be very interesting for follow-up research.
- For me, the biggest question from this work is what the function of midline cilia might be– are they involved in Shh signalling or sensing to maintain the midline barrier? I wonder whether midline cilia are present in other ciliary mutants which have unexpected laterality phenotypes.
- Connected to the above point, it might be interesting to know if the few cilia that are present in the midline or nodes of Tmem107null and Mks1krc mutants are phenotypically normal. Even though they might be positive for cilia markers, do the nodal cilia still beat, or are any midline cilia present the same length and shape as in wild type?
- As well as Shh signalling, other developmental pathways such as Wnt and FGF signalling are important for nodal cilia function in L-R asymmetry– I wonder if any of these developmental signalling pathway components are also disrupted in Tmem107null, Tmem107schlei and Mks1krc mutants, and if they also contribute to midline establishment and function.
- Schweikert, A., et al. (2018). Vertebrate Left-Right Asymmetry: What can Nodal Cascade Gene Expression Patterns Tell Us? J. Cardiovasc. Dev. Dis 5, 1.
- Tsiairis, C.D. and McMahon, A.P. (2009) An Hh-dependent pathway in lateral plate mesoderm enables the generation of left/right asymmetry. Curr. Biol. CB, 19,1912-1917.
- Weatherbee, S.D., Niswander, L.A. and Anderson, K.V. (2009) A mouse model for Meckel syndrome reveals Mks1 is required for ciliogenesis and Hedgehog signalling. Human molecular genetics. 18, 4565-4575.
- Christopher, K.J., Wang, B., Kong, Y, et al. (2012). Forward genetics uncovers Transmembrane protein 107 as a novel factor required for ciliogenesis and Sonic hedgehog signalin. Dev. Bio. 368, 382-392
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