1. `
Conserved hox gene expression during larval stages in the
bat star, Patiria miniata
Alia Hidayat1, Paul Minor2, Christopher Lowe2
1University of Washington, Seattle WA; 2Hopkins Marine Station, Stanford University
Abstract
Echinoderm Patterning
In a study done by Arenas-Mena et al. (2000) the purple
sea urchin (S. purpuratus) was found to lack expression
of nearly all hox genes in early larval stages, and
expression was only detected during patterning of the
adult body plan in the mesoderm of late larval stages.
However, a recent study by Kikuchi et al. (2015)
suggests that, in contrast to what was observed in the
sea urchin, hox genes are expressed in the posterior
ectoderm of holothuroids. It is still unclear whether this
early posterior larval expression of hox genes is
ancestral to echinoderms or a trait unique to
holothuroids. Our work aims to test these two
hypotheses through investigation of hox expression in
asteroid larvae.
Left: P. miniata in bipinnaria stage. Picture credit: Lowe Lab
Methods
To analyze the spatiotemporal expression pattern of hox genes in P. miniata, we
conducted in situ hybridizations on fixed samples at multiple life stages - egg hatching
through the bipinnaria (146 hpf) and brachiolaria (45 dpf) larval forms to the formation
of the complete juvenile. In situ hybridization uses RNA probes to identify and mark
areas where a certain gene is being expressed, and when done over multiple stages,
In Situ Hybridization
Results
Hox expression was observed in larval forms in locations along the AP axis analogous
to the expression patterns observed in other animals. At early gastrula stages, much of
the expression patterns are similar between the five genes. However, as larval
development progresses, it appears that each gene has a specific expression pattern
along the AP axis. Hox expression was mainly localized to the posterior of the
embryo. This suggests that the tested hox genes are involved in constructing the
posterior portions of the larvae along the AP axis, as they do in other animals. In
addition to hox, we performed in situ hybridization against six-3, another conserved
AP axis patterning gene that is responsible for patterning the anterior in other
animals. In previous studies, six-3 expression patterns in P. miniata have been found
to be analogous to those in other bilaterians (Yankura, 2010). Our data confirms this,
as staining of six-3 appears to be limited to the anterior portion of the larvae.
Conclusions
Acknowledgements
Hox complex genes constitute an essential and ubiquitous component involved in the
construction of the animal body. Despite their wide range of body plans, every member
of the bilaterian clade utilizes these key genes to pattern the anteroposterior (AP) axis,
with one potential exception – radial echinoderms. In addition to the possession of an
adult radial body plan, Echinodermata are also mostly characterized by indirect
development, beginning their lives as bilateral larvae, which undergo radical
metamorphosis into a radial adult. The echinoderms, outside of the sea urchin,
Strongylocentrotus purpuratus, remain poorly studied, and it is unknown how
classically bilateral planning mechanisms like hox are involved in patterning these very
different life stages. To help to begin to resolve this, we present in situ hybridization
data of four hox genes present in the indirect developing bat star, Patiria miniata in its
larval stages. Hox expression was observed during the development of larval forms in
the posterior similar to the expression patterns in other bilaterian animals. These results
suggest that, despite a greatly divergent radial body plan of the adult, echinoderms use
many of the same developmental mechanisms found in bilateral organisms to pattern
their larval stages prior to metamorphosis.
The expression patterns of these genes suggest that hox is used to pattern the AP axis in
P. miniata. Expression of six-3 in the anterior and hox in the posterior in P. miniata
mirrors expression patterns throughout the bilaterian clade, and suggests that these
mechanisms are generally conserved. Though gene expression appears to be dependent
on location along the AP axis, there is not enough evidence to say that these genes are
being expressed co-linearly, and further experiments would have to be done to confirm
whether or not co-linearity of hox expression is conserved in P. miniata. That being said,
there is sufficient evidence to conclude that, unlike in S. purpuratus, hox genes are
likely being used as part of the overall AP patterning mechanism for P. miniata during
larval stages, mirroring emergent data from holothuroids (Kikuchi et al. 2015). These
results suggest that, despite a greatly divergent radial body plan, echinoderms use many
of the same developmental mechanisms found in bilateral organisms to pattern their
larval stages prior to metamorphosis.
Divergence from hox
patterning mechanisms
according to S. purpuratus
Divergence from hox according to evidence
from P. miniata and Kikuchi et al.
This research has shown that hox genes are expressed in P. miniata during the
development of bilateral larval body plan, suggesting that the mechanisms used in
early development of echinoderms may not be as divergent as previously thought. It
resolves the question of whether or not the lack of larval hox expression in sea urchins
as observed by Arenas-Mena et al. (2000) is representative of the echinoderm phylum
as a whole. Evidence from P. miniata shows that the method by which asteroids
pattern their body axis is different from that used by S. purpuratus, suggesting the
divergence from hox as represented by echinoids likely occurred after the split
between echinoids and holothuroids.
Early gastrula Mid-late gastrula Bipinnaria Brachiolaria
hox5
hox11-13c
hox9-10
hox7
six3
Anteriorpatterninggenes
allows us to determine exactly when in the
life cycle a certain gene is being expressed.
After identifying our genes of interest,
designing primers, and amplifying the
coding sequences by PCR, we cloned the
resulting PCR products into bacteria. We
then synthesized labeled antisense RNA
probes and investigated gene expression in
fixed Patiria embryos. Whole mount in situ
hybridization was performed by the methods
described in Lowe et al. (2003). P. miniata spawning. Picture credit: Lowe Lab
Posteriorpatterninggenes
A, B, C & D: Ectodermal expression of anterior patterning gene six-3 in early gastrula,
mid-late gastrula, bipinnaria, and brachiolaria stages respectively. E, F, G & H:
Expression of medial patterning gene hox5. I, J, K & L: Expression of posterior
patterning gene hox7. M, N, O & P : Expression of posterior patterning gene hox9/10.
Q, R, S & T: Expression of posterior patterning gene hox11/13c.
Icon credit: T. Ryan Gregory
I am very grateful to the members of the Lowe Lab:
Paul Minor, Paul Gonzalez, Nat Clarke, Kevin
Uhlinger, Jens Frizenwanker, and Christopher Lowe
for their invaluable guidance throughout this project.
Additional thanks for funding and support from the
National Science Foundation and REU Coordinators
Bridgette Clarkston, Megan Bassett, Pat Mulcahy,
and Corey Garza.
References
 Arenas-Mena et al. 2000. “Expression of the Hox gene complex in the indirect development of a sea urchin.” PNAS
95(22):13062-13067.
 Kikuchi et al. 2015. “Patterning of anteroposterior body axis displayed in the expression of Hox genes in sea
cucumber Apostichopus japonicas.” Development Genes and Evolution 225(5):275-86.
 Lowe et al. 2003. “Anteroposterior patterning in hemichordates and the origins of the chordate nervous system.” Cell
113:853-865.
 Yankura et al. 2010. “Uncoupling of complex regulatory patterning during evolution of larval development in
echinoderms.” BMC Biology 8:143.