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Evolution: Was the nuclear-to-cytoplasmic ratio a key

factor in the origin of animal multicellularity?
Jeffrey Colgren and Pawel Burkhardt
Michael Sars Centre, University of Bergen, 5008 Bergen, Norway
Correspondence: [email protected] (J.C.), [email protected] (P.B.)
https://doi.org/10.1016/j.cub.2023.03.010

The ichthyosporean Sphaeroforma arctica, a protist closely related to animals, displays coenocytic
development followed by cellularization and cell release. A new study reveals that the nuclear-to-
cytoplasmic ratio drives cellularization in these fascinating organisms.

The correlation between nucleus and which is preceded by rapid division releasing daughter cells10 (Figure 1A,B).
cytoplasmic volume was documented (either nuclear or cellular) without The superficial similarity of coenocytic
over a century ago through the equivalent growth5–7. In these cases, development to cellularization in
observation of protists growing under timing of the midblastula transition some animal lineages, such as
varied nutrient conditions. Subsequent functions through the N/C ratio rising Drosophila, has brought much interest
work has shown that this appears to be above a given threshold. This general to ichthyosporeans, and previous work
a pan-eukaryotic feature, with phenomenon — developmental timing has shown an association between
convincing studies performed in yeast, being dependent on the N/C ratio — has increased number of nuclei per
plants, and animals1,2. In animals, cell been documented in a variety of coenocytic volume and timing of the
size can vary greatly within a single bilaterian animals8,9, suggesting this flip10. For these reasons, Olivetta and
organism, while the nuclear-to- mechanism was present early in animal Dudin set out to test whether N/C ratio
cytoplasmic (N/C) ratio, the ratio of evolution. is a direct regulator of development —
nucleus volume to cytoplasmic volume, Ichthyosporeans belong to the arrest of coenocytic growth,
is generally consistent within cell types3. holozoan clade, which contains both cellularization, flip and release of cell-
Within this framework, stem cells tend animals and their closest unicellular walled cells — in S. arctica11, as
to have a higher N/C ratio, suggesting relatives. Many ichthyosporeans reported in this issue of Current Biology
that this measure is correlated with undergo coenocytic development, in (Figure 1C). First, they build on previous
differentiation and cell-cycle which multinucleate cells form through work to thoroughly document the
progression3,4. sequential nuclear division without phenomenon that N/C ratio significantly
During animal development, stable accompanying cytokinesis. This increases during cellularization and flip.
multicellularity involves a transition phenomenon has been well studied Growth in low-nutrient medium
from reliance on deposited maternal in the ichthyosporean model, decreases coenocytic volume without
transcripts to utilization of the zygotic Sphaeroforma arctica, where consistent affecting nuclear division, effectively
genome, termed the maternal-to-zygotic nuclear divisions are followed by increasing the N/C ratio. This showed a
transition. In a variety of animals, this cellularization and formation of an concentration-dependent effect on flip
occurs during the midblastula transition, epithelial-like sheet before ‘flipping’ and timing. In order to decrease N/C ratio,

R298 Current Biology 33, R296–R318, April 24, 2023 ª 2023 Elsevier Inc.
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Dispatches

the authors treated coenocytes with A B Cellularization

carbendazim, an antifungal agent with
Coenocytic
anti-mitotic properties, to stop nuclear growth
division at various time points; they
found that arrest of division prior to
cellularization prevents the flip from
occurring. By washing out carbendazim
to allow N/C ratio to begin increasing,
the authors observed that the flip stage
could be rescued. Treating coenocytes
grown in low-nutrient medium with Release Detachment
carbendazim also allowed the flip to
occur at treatment times where it would C N/C D
not have in full-nutrient media. These ratio
experiments demonstrate convincingly
that there is a strong correlation
between N/C ratio and timing of the flip
in S. arctica. To take this further, the
authors mechanically altered the local
rC

density of nuclei in the coenocytes

Lowe

resulting in irregular cellularization

rN

and early flip, suggesting N/C ratio

we

is sensed locally at the cortex. This

Lo

was blocked through the addition

Both
of phosphatase inhibitors. Taken
together, these findings suggest
Ra
Block

N/C ratio is a key regulator of the

ise

multicellular development of S. arctica,

N
phos

in which nuclear division continues

phata

following arrest of coenocyte growth,

reaching a threshold ratio that triggers
ses

cellularization and flip. This work is a

wonderful example of well thought-out,
performed, and documented
experiments, using ‘classical’
Time
techniques with modern technologies. In Current Biology
a system that does not yet have the
benefit of genetic manipulation, they Figure 1. The ichthyosporean Sphaeroforma arctica displays coenocytic growth followed by
are able to artificially manipulate the cellularization and cell release.
(A) Cellularization in a S. arctica colony stained for actin (green) and DNA (blue). (B) General life cycle
N/C ratio, showing that increasing it of S. arctica: during coenocytic growth, nuclear division occurs without cytokinesis. Prior to
leads to earlier development, and that cellularization, the majority of nuclei organize around the periphery of the coenocyte and membrane
lowering or maintaining it prevents invagination forms discrete cells in an epithelial-like arrangement. These cells then detach from each
other and the mass releases individual daughter cells. (C) Workflow of the major experiments performed,
progression. showing control over N/C ratio over time. Faded cellularization or release symbolize significant
This study represents one data point disruption of these events. (D) General phylogeny of holozoans, with non-animals in orange and animals
outside of animals. N/C ratio functioning in beige. Filled circles indicate direct evidence of N/C ratio-dependent developmental timing, half-filled
circles indicate indirect evidence, and empty circles indicate these studies have not been performed.
in relation to the timing of the maternal-
Image in (A) is courtesy of Omaya Dudin, scale bar 10 mm. Silhouettes in (D) obtained from phylopic.org.
to-zygotic transition or midblastula
transition has been well established for
development in a variety of mostly explanation of experiments make them is a universal (or at least ancestral)
bilaterian animals (Figure 1D). Therefore, adoptable to a variety of systems. state for ichthyosporeans. Finally,
despite the strong similarities between Within animals, there is evidence that coenocytic development resembling
ichthyosporeans and animals, it is similar developmental timing occurs in that seen in ichthyosporeans occurs in
difficult to discern whether N/C ratio cnidarians12 and ctenophores13, though other taxa, including chytrid fungi14.
acting as a developmental timer is a these remain to be systematically Characterization of these additional
result of conservation or convergence. studied. How these mechanisms play systems will provide additional data
This is not a criticism of the current into the clonal development in points to help resolve how ancient this
study, but rather highlights that there is choanoflagellates, the sister group to developmental mechanism is. However,
an exciting opportunity for future work. animals, also remains to be studied. It since the N/C ratio is tightly regulated in
The meticulous recording and is also an open question whether this eukaryotes, its potential as a signaling

Current Biology 33, R296–R318, April 24, 2023 R299

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Dispatches

modality following a transition to transitions (though is often the case). This 8. Jukam, D., Shariati, S.A.M., and Skotheim,
J.M. (2017). Zygotic genome activation in
multicellularity is high. During the means that the potential to undergo these vertebrates. Dev. Cell 42, 316–332. https://doi.
various independent origins of transitions, and the key regulators of them, org/10.1016/j.devcel.2017.07.026.
multicellularity in eukaryotes, aspects of can persist following loss of the functional
this regulation could have been co- transition over evolutionary time. Within 9. Arata, Y., Takagi, H., Sako, Y., and Sawa, H.
(2014). Power law relationship between cell
opted for regulating organismal choanoflagellates, for example, not only is cycle duration and cell volume in the early
development through convergence. The the ability to form multicellular colonies not embryonic development of Caenorhabditis
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best way to start unraveling this is universal19, the presence of different forms
through identifying the molecular of multicellularity suggests that it has 10. Ondracka, A., Dudin, O., and Ruiz-Trillo, I.
players involved in the phenomenon. arisen multiple times20. In the animal stem (2018). Decoupling of nuclear division cycles
The authors have begun this with lineage, this potential for multicellularity and cell size during the coenocytic growth of
the Ichthyosporean Sphaeroforma arctica.
S. arctica, finding a role of would exist as a deployable unit for Curr. Biol. 28, 1964–1969.e2.
phosphatases, which have also been selection to act upon, rather than
implicated in N/C ratio-dependent something that would need to be 11. Olivetta, M., and Dudin, O. (2023). The nuclear-
to-cytoplasmic ratio drives cellularization in
development in animals15. However, the assembled prior to the advantages of the close animal relative Sphaeroforma arctica.
general molecular mechanisms stable multicellularity. In this scenario, Curr. Biol. 33, 1597–1605.
governing this switch in animals developmental timing through N/C ratio
12. Röttinger, E., Dahlin, P., and Martindale, M.Q.
continue to be debated3,16. The sensation would have been key to the (2012). A framework for the establishment of a
morphological simplicity of S. arctica origin of animal multicellularity. The work of cnidarian gene regulatory network for
may allow for the identification of the full Olivetta and Dudin11 sets the framework ‘‘endomesoderm’’ specification: The inputs of
ß-Catenin/TCF signaling. PLoS Genet. 8,
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13. Fischer, A.H., Pang, K., Henry, J.Q., and
Regardless of whether the N/C ratio Martindale, M.Q. (2014). A cleavage clock
DECLARATION OF INTERESTS
serving as a developmental timer arose in regulates features of lineage-specific
the stem holozoan lineage or much earlier differentiation in the development of a basal
The authors declare no competing interests. branching metazoan, the ctenophore
in eukaryotic evolution, its conservation Mnemiopsis leidyi. Evodevo 5, 4.
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