Conserved hox gene expression during larval stages in the

bat star, Patiria miniata

Alia Hidayat1, Paul Minor2, Christopher Lowe2
1
University of Washington, Seattle WA; 2Hopkins Marine Station, Stanford University

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. In
the absence of studies on AP patterning in other
echinoderms, it has been assumed that this departure
from traditional usage of hox patterning genes occurred
at the base of the echinoderm clade, and that the
observations made in S. purpuratus would be consistent
with other members of the echinoderm clade. Our
observations aim to evaluate this assumption by
investigating hox expression in a different echinoderm
group, the asteroids.
Left: P. miniata in bipinnaria stage. Picture credit: Lowe Lab

Early
gastrula

Mid-late
gastrula

Bipinnaria

Conclusions
Brachiola
ria

six3
hox5

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 on the whole are 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. 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.

hox7
hox9-10
Posterior patterning genes

Hox complex genes constitute an essential and ubiquitous mechanism in the

construction of the animal body. Despite possessing a multitude of phenotypes, 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 in larval forms in a laterally specific
fashion analogous to the expression patterns in other 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.

Anterior patterning genes

In Situ Hybridization

Abstract

Divergence from hox

according to asteroids

Divergence from hox

patterning mechanisms
according to echinoids

hox11-13c

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.

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, and that this
divergence occurred somewhere within the echinoderm clade.
Icon credit: T. Ryan Gregory

Methods

Results

Acknowledgements

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,
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

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 the larva
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 ubiquitous AP axis patterning
gene which 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.

Members of the Lowe Lab

Paul Minor
Paul Gonzalez
Nat Clarke
Kevin Uhlinger
Jens Fritzenwanker
Christopher Lowe
NSF REU Coordinators
Bridgette Clarkston
Megan Bassett
Pat Mulcahy
Corey Garza