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Choosing Sides: Two steps in establishing the polarisation vector in algal zygotes

Posted by , on 14 March 2017

Comment on “Two-step cell polarization in algal zygotes”, Nature Plants, 3, 16221, (2017).

Department of Biology, Ghent University, Krijgslaan 281 S8, 9000 Ghent, Belgium

VIB-UGent Center for Plant Systems Biology, Technologiepark 927, B-9052 Ghent, Belgium

Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium

 

Complex multicellular life has evolved from unicellular organisms along at least five independent paths, giving rise animals, plants, fungi, red algae and brown algae, respectively. Asymmetric divisions are key in this process as a means to create diverse cell types. At the molecular level, the uneven distribution of molecular components such as mRNA, proteins or organelles determines a polarisation vector. A subsequent cell division along this polarization vector results in two daughters cells which are no longer equivalent and receive different cell fates.

In recent years, insights in the molecular mechanisms determining cell polarisation and asymmetric cell division have mainly emerged from investigations on animals. With exception of yeasts, the mechanisms of polarity establishment are much less well established in other groups of eukaryotes. While many model cell polarity systems from land plants lend them to the research of polarity signalling, polarity is often either predetermined (e.g. pollen) or the cells are enclosed in the surrounding tissue (e.g. zygotes), which renders them less suitable for studies examining polarity establishment. Conversely, eggs of brown algae are released in the surrounding seawater as radial symmetric spheres and polarity is established after fertilization. Since the middle of the 19th century, there has been interest in the gametes, zygotes and embryos of fucoid brown algae (Fucus and Silvetia) and a considerable effort has been invested in the study of their gamete recognition and fertilisation, cell wall biosynthesis and polarity acquisition. One asset of these organisms is their broadcast spawning nature. Gamete releases can be experimentally induced resulting in large populations of synchronously developing embryos. In addition, the zygotes are also large (60–100 µm) and therefore easy to (micro-)manipulate.

In fucoids the mechanism of cell polarisation has been well established. Upon fertilization the apolar egg cell develops a cell wall and starts the polarization process. The sperm entry site and subsequently the light direction provide cues for establishing the polarization vector de novo and both determine the direction and sense of the polarisation axis simultaneously. Unfortunately, it is very hard to culture fucoid brown algae. Hence, they are less amenable to genetic and molecular studies. Similarly the development of the cultivable, but isogamous, brown alga Ectocarpus siliculosus as a successful model for genomic research has been accompanied by a shift towards the developmental research questions related to life cycle and fertilisation biology, but away from polarity establishment.

 

[Scheme - Fucus cell polarisation]
Figure 1. The polarization process in fucoid zygotes. A. Fertilized zygote, showing a sperm induced F-actin patch. B. Zygote polarizing according to the light direction. C. Polarized zygote showing a photopolarized F-actin patch, polarized endomembrane cytoskeleton and polarized MT cytoskeleton. D. Asymmetrically divided embryo with a thallus and rhizoid cell. Adapted from Bogaert et al. 2013.
 

 

Since many years, the Phycology Group of Prof. Dr. Olivier De Clerck is studying diverse biological aspects of the brown alga Dictyota for which laboratory culture are more easy to maintain. Fascinated by plant developmental biology I approached Prof. Dr. Olivier De Clerck to inquire for a master dissertation. Coincidentally while studying the mechanism of gamete recognition in Dictyota in the summer of 2007, Prof. Dr. Olivier De Clerck observed that the spherical egg cells deform into elongated rugby ball shaped spheroids after fertilization. Propelled by this finding a master dissertation subject was discussed over some Belgian beers and we teamed up with Prof. Dr. Tom Beeckman of the Root Development group VIB – PSB in Ghent. Early development of the Dictyota embryo quickly turned out to be different from Fucus and the collaboration ultimately matured in a PhD fellowship funded by the FWO (Research Foundation – Flanders).

 

[habitus Dictyota]
Figure 2. Female gametophyte of Dictyota dichotoma in the field (credits: H. Verbruggen).

I vividly remember the excitement, observing that the egg cells of Dictyota elongate minutes after addition of the male gametes. Within three minutes a population of spherical egg cells was transformed into a homogenous population of rugby ball shaped cells, with the elongation observable under the stereomicroscope. From that moment, it was clear that the direction of the elongation must have been predetermined; no process could be so fast to determine an elongation direction after fertilisation in those mere seconds. This was later corroborated by TEM sections showing a heterogenous organisation of the organelles suggesting the presence of a preformed axis in the unfertilized eggs. During a research stay in the lab of Dr. Susana Coelho and Dr. Mark Cock at the SBR Roscoff I managed to visualize the autofluorescence of the chloroplasts of eggs being fertilized and provided in vivo evidence that the preformed axis indeed represents the elongation axis.

It is peculiar that this notion has been left unnoticed for so many years given the many efforts of John Lloyd Williams in the late 19th century describing the periodicity of gamete release and the parthenogenetic development of Dictyota dichotoma. Prof. Dr. Dieter G. Müller, best known for his ground breaking work on Ectocarpus siliculosus, his early work also concentrated on Dictyota dichotoma describing the release periodicity of the female gametophytes in laboratory conditions. Only later we heard that Prof. DG Müller observed in the 60s the very same elongation of the eggs, while working for the developmental biologist Prof. Dr. Lionel F. Jaffe. The observation was received with much excitement and Jaffe suspected fundamental differences with the fertilisation response of fucoids, but a technical difficulty in the culturing withheld them from following up this observation. Most probably priorities must have laid in detangling the fertilisation and polarisation of the zygote of Fucus at that time.

 

[chlorooplast distribution - elongation]
Figure 3. Chloroplast autofluorescence showing the preformed axis predicts the elongation axis. Adapted from Bogaert et al. 2017.

The notion to walk in the footsteps of two of the most iconic early developmental biologists working on brown algae was most exciting to us. Quickly we realized that the elongation had implications for the way the cell polarize. While the egg cell has a predetermined elongation axis the direction of the polarisation vector, (aka ‘the sense of the polarization vector’) is not predetermined. In other words, it is not decided which side is going to develop into the leaf-like upper part or the lower root-like part of the alga. This was best illustrated by the fact that while the elongation direction was unresponsive to the direction of the light, which of the two poles will develop into the rhizoid is determined by the direction of the unilateral light. Moreover, toluidine blue O staining – a marker for permanent fixation of the rhizoid pole in fucoids – was only observed hours after fertilization. Therefore determination of the polarisation vector in Dictyota is inherently a two-phased process where initially the direction of the polarisation vector is determined and only later also the sense of the polarization vector.

 

[scheme Dictyota, Fucus, Arabidopsis]
Figure 4. A scheme comparing zygote polarization in Dictyota, Fucus and Arabidopsis. Timing of the two phases of polarisation are denoted in blue. Red dashed line represents direction of the polarisation vector (without sense). Red line with arrow denotes a polarisation vector with both direction and sense. Adapted from Bogaert et al. 2017.

These two phases are not only completed with a different timing, they also rely on at least two different mechanisms because they depend on two different cues. While the direction is maternally determined, the sense of the polarisation axis is determined by the light direction. Due to the different timing, the two phases even occur in different life stages: the direction in the polarisation vector in the oogonium and the sense is determined hours after fertilization in the diploid sporophyte. In animal systems it is however well known that developmental processes during the first cell divisions are under control of the transcriptome transcribed before egg arrest because de novo transcription (zygotic genome activation) is postponed. This is termed the maternal-to-zygotic transition. The broadcast spawning nature makes it relatively easy to yield large populations of cells, because we did not have to rely on microdissection techniques and FACS techniques. Therefore with some moderate effort we could obtain three biological replicate libraries of mRNA of gametes, zygotes and asymmetrical divided embryos. This allowed us to show that there is a large degree of zygotic genome activation taking place already during the first cell cycle, showing that the next generation is indeed acquiring developmental control over the cell polarisation.

I am thrilled with this publication, because it once more shows that research on oogamous brown algae is not from the past. The availability of an oogamous broadcast spawning brown alga that can be easily cultured and releases its gametes into the external medium opened up the opportunity for developing it into a research model complementary fucoids. This publication however shows that different and complementary insight can be gained from Dictyota early development.

References

Bogaert KA, Arun A, Coelho SM, De Clerck O (2013) Brown algae as model for plant organogenesis. In Methods in Molecular Biology: Plant Organogenesis (De Smet I ed). Humana Press. Springer Protocols, Heidelberg, 959: 97-125.

Bogaert KA, Beeckman T, De Clerck O (2017) Two-step cell polarisation in algal zygotes. Nature Plants 3: doi:10.1038/nplants.2016.221.




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