438 paper

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AUBIO438: Evolutionary Developmental Biology
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Seahorse Hippocampus hippocampus Model System
Research Proposal ( Phylum Chordata).
Hadil Elsayed
Evo-devo termed as evolutionary developmental biology that is considered to be an enormous and
expressive term in terms of embryonic development and many evolutionary events that occur in terms of
development and genetic information held whether switched on or off during the embryonic development
(Benazeraf and Pourquie, 2013). Evolutionary developmental biology according to my opinion is a huge
world of researches, genetic databases, unstable world of evolution and natural selection of genes and
traits, many new findings and many occurring species with different patterns, and morphological features.
The closer and closer we get to know all the similarities and the more deeper we get to know the more
complicated and complex world of biodiversity and changes throughout many years of evolution and
adaptation. Many interesting model systems have been studied and are used as standard models for the
embryonic developments of their closely related species and therefore they are also related to their extant
ancestors. My model system has not been studied and not many researches have been implied on it,
Hippocampus hippocampus. Sea horses have been the center of debate and many interesting features in
one species, the anterior-posterior development axis formation mainly is a curious core of the
development of both body structure along with the genes that are involved in their axis formation, tail
prehensibility and the male pouch adaptation. In my proposal I will demonstrate the interesting traits
about the anterior-posterior body axis formation in Hippocampus hippocampus, including the
evolutionary novelty of the male brooding structures that makes them unique throughout their
Sygnathidae family. Moreover, I will explore their tail prehensibility and what is unique about their tail
growth and muscle interactions. There is less knowledge to these three Evo-devo questions about sea
horses and less experiment done on them to reveal the answers for these questions. However, these
questions should be explored and experimented to lead us to understand more about their evolution and
the reasons behind it. Furthermore, I will clarify the connection between the genomic duplication and the
developing new gene functions and the convergent evolutions that led to the new traits and new
morphological and physiological features.
Hippocampus hippocampus is referred to as the short snout sea horses as that is characteristic that
differentiates them from other sea horses species. They are bilateral metazoans bony vertebrates that

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belong to phylum chordate and are members of Sygnathidae family that include closely related species,
such as pipefishes and sea horses.
Both species look closely related morphologically and having the same male brooding structure but
different body posture and therefore different gene functions due to many evolutionary events that might
have occurred that will be discussed later in my proposal. They inhabit small and shallow home ranges
and coastal waters and sometime they migrate to deeper waters due to change in seasons. They breed
from April to October and the gestation period remains for a one month (Boisseau, 1967). They mature at
an early age, and they have rapid growth rates and short generation time which basically explains that
facture that they can recover rapidly. Sea horse appear morphologically as an upright body posture like
homo sapiens, they have a horse-like head, monkey-like tail and a kangaroo like pouch, their eyes can
move in all directions independently; furthermore, they lack scales as they have thin skin stretched over a
series of bony plates that are visible as rings around the trunk (Project Seahorse). Their skin is stretched
over a series of bony plates that are visible as rings around the trunk and tail and they don’t have scales.
Some have spines and bony bumps and they have a coronet on their head that looks like a crown.
Moreover, they have an exoskeleton unlike other fishes from the same family, as their bodies are made of
hard, and bony external surface that provide protection for them as they don’t have scales. They belong to
the Sygnathidae family as they look alike but different from the upright body posture and seahorses
specifically belong to the genus Hippocampus from the Greek word horse (hippos) and sea monster
(campus). Their snouts enable them to probe into nooks and crannies for their prey. Seahorses have heads
at right angles of the body and prehensile tails that allow them to attach to any surrounding and grasp
food and preys. They have a unique pouch in males that is considered to be unique for the Hippocampus
species. Moreover, they don’t have stomach or teeth, and therefore they suck the prey through the tubular
snout and pass it through an inefficient digestive system. Their gills are small and compacted and they
swim by the propulsive force of the oscillating dorsal fin and the use of the pectoral fins on either side of
the body for steering and stability (Breder and Edgerton, 1942).
Figure1: This figure show the significant difference in body
structures of both pipefish and seahorses, however they are
related to each other in terms in other characteristics such as
food pivoting and there role in the ecosystem.

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Reproduction and lifecycle
Males become pregnant instead of females, this is considered to be an unusual mode of
reproduction. The sexual maturity of the male seahorses is determined by the presence of the male
brooding pouch. Male pregnancy is dependent on the water temperature. Most sea horses are
monogamous, forming pair bonds that last the entire breeding season. They perform a courtship dance
and they separate the rest of the day. The female inserts her ovipositor into the male’s brood pouch and
that is where she deposits her eggs and the male fertilizes it. The eggs then get embedded in the male
pouch wall and become enveloped in tissues. The pouch helps with the placental fluid that bathes the
eggs, and provides the eggs with nutrients, and oxygen developing embryos while removing the waste
products (Stolting and Wilson, 2007). However, the pouch fluid is altered at one stage during pregnancy
as that helps to being more like the surrounding sea water, and this therefore help reduce the stress for
offspring at birth. The pregnancy duration remains between two and four weeks, and can sometimes
affect by the surrounding temperature, and the duration can be decreased with the increase in temperature.
When the egg is deposited in the male’s pouch and fertilized, at that point the embryo is developing
starting from the body characteristics such as snouts, eyes, and body tail. The third stage resembles the
features of the sea horse that the eyes gets detailed eyes, and the development of the short snout, the tail is
beginning to form and appear at the base of the body of the embryo. The fourth stage indicates the snout
cleavage from the beat of the body and the internal organs begin to development, as the fifth stage starts
to emphasize the appearance of the snout, and their body size is getting bigger in size and the tail is
defined and their dorsal fin. The sixth stage indicates that the snout is fully cleaved and the spinal column
is evident in this stage (A seahorse life cycle, 2009). The male sea horses can release up to 100 to 200 and
some other species can release as high as 1,500. The young sea horses are independent right after birth
and receive no more parental care. They are from seven to 12 millimeters.
Consequently, considering Hippocampus species as the central focus to determine many aspects of its
appearance and functions, in my paper I will mainly focus on three Evo-devo questions that are
interesting for such model system; what are the candidate genes that are involved in anterior posterior axis
body formation and what are the reasons and the possible evolutionary explanations behinds the fates of
these genes? What is unique and distinctive feature of the prehensile tail of sea horses and there related
explanation behind the unique formations and unique movement of it amongst other ray finfishes? And
finally, what is the evolutionary explanation behind the presence of the male brooding structures in

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Hippocampus species? These questions will be mentioned on the basis of evolutionary differentiation
amongst organisms used as model system to help with sea horses model system and perform the basics
that can help as starting points for experimentation of sea horses model system.
Breeding Hippocampus hippocampus in the laboratory
Experimenting seahorses at the lab is not an easy process. Sea horses are considered to be sensitive
species that should be reared and bred in sustainable environment for their behavior and life style. The
most important aspect of breeding sea horses in the laboratory is providing the suitable tanks/ aquariums
that are of suitable height and temperatures and surroundings. There are many aspects that should be
controlled in a tank/aquarium such as the height of the tank, the temperature that can be maintained for
sea horses’ reproductive purposes and the filtration process that are crucial to be maintained in the tank
for various reasons that will be mentioned in my proposal. The mating system of seahorses needs a deep
height water system habitat for them to involved in the courtship dance before mating and this increase
the efficiency of mating more than experimenting them in an aquarium. A good rule is the height of the
aquarium should be suitable enough for sea horses, as they swim vertically and they need more space for
courtship, they are sometime aggressive when healthy, therefore not a good idea to put a lot of species in
one aquarium. Therefore, it is best to experiment them at the coast. Tanks that can carry them can be a bit
too harsh for them to inhabit such environment, as they need deeper height and warmer temperature for
reproductive purposes (Ruiz, 2010).
Second aspect, would be the temperature aspect as it is considered to be important for sea horses
reproduction and newly born fries, and the suitable temperature known is 80F. Temperatures with less
than 80 F (26.5°C) can be insufficient as it affects their breeding and reproduction instinct and delay the
pregnancy duration and therefore affecting the experiment timings. Water quality should be maintained
and should be relative to the sea water, as they can be stable environment, as the male brooding structure
provide the same water quality for the newly born before giving birth to them so they can be adaptable to
the surrounding water. The aquarium should have many open areas as they can swim freely in and many
structures that they can hold on to with their prehensile functional tail. Moreover and third, filtration is
another important aspect as newly born fries can be infected and the parasites and bacteria can stick to
their bodies and therefore affecting the research and the experiment as a whole, therefore some kind of
detergent should be added to get rid of the parasites in the aquarium.
Fourth aspect that should be put into consideration while breeding sea horses in the laboratory and is
considered to be the most important and crucial element to take care of during breeding sea horses is their

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eggs and their compatibility and enhancement for the research. The eggs that gets hatched in the pouch of
the male, are pink and are slightly transparent and therefore this in turn increase the ability to undergo
some tests such as antibody staining and in-situ hybridization to follow the growth patterns and the
anterior and posterior axis formation in the lab. Moreover, there is an advantage of seahorses’ eggs, and
that there will be plenty of them after one mating process and therefore this can enhance the research
more widely and efficiently. The incubation time can be a little bit long that can vary from 3 weeks to 4
weeks for the eggs to hatch and dissociate from the male pouch. One more advantage to my own
perspective is that the eggs are attached to the pouch of the male and the male in turn have the ability to
handle parental care for the eggs as it offers all the nutrients and oxygen needed for the embryo
development. The pouch also can offer protection for the eggs. Consequently, this can important for
determining their life cycle after being hatched from the male brooding structure and monitoring how
long it takes them to be sexually mature does this and how they grow in size by themselves and this can
also enhance to determine their behavior after being hatched (Ruiz, 2010).
Moreover, the nutrient system of the sea horses can be easily bred in the laboratory. Sea horses have
little time to absorb nutrient before the food is eliminated, sea horses need to constantly search and ingest
food, and it is preferred to feed them twice a day and preferably three times a day. Sea horses from the sea
can be used to prey on living food, while bred species can be easily adapted to frozen food, as they have
been living on them since being bred in the aquarium during laboratory experiments. However, when sea
horses are kept without feeding that can feed on their own tissues and eventually die gradually and this
can impact negatively on the breeding population in the laboratory. Therefore, the feeding system of sea
horses must be handled efficiently and in an organized manner. Furthermore, the aspect of fry newly born
sea horses, that have a different condition while breeding in the laboratory, they cannot be fed for the 18
to 20 hours, and then they can eventually can search and see food after this duration; moreover, they
cannot handle starvation and they can die easily (Olivotto et al. 2008; Ruiz, 2010).
Anterior-posterior axis body formation of Hippocampus hippocampus
As mentioned earlier, sea horses have a unique body axis and posture in which implies many
evolutionary and developmental questions about the axis formation during the embryonic development in
the Syngnathidae family. This can lead to imply an important question of what are the types of genes that
underlies the specific morphological changes in this family and the how are they different from the
conserved genes from the same family, what could have happened in evolution that led sea horses to be
morphologically compared to other ray fin fishes. This can imply the information to understand and

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emphasize more on the relationship between the genomic variation and the fate of phenotypic complexity
in ray finfishes.
Sequenced animal genomes
Zebrafish, Danio rerio is a standard model organism in the study of somitogenesis. Many other
sequenced body fishes has been used as models to help determine the candidate and divergent genes that
can be present in sea horses and has different functions and fates in embryonic development (Campanelli
and Gedeon, 2010). Some other bony fishes that helped to support the hypothesis of hox gene clusters and
genomic divergence and concluding some factors that led to such body axis in sea horses, such as
Takifugu nigroviridis. They are considered to be including the genes that could be somehow involved in
sea horses body axis formation and can help investigate in more broader ways of what could have been
involved as candidate genes (Amores et al. 2004). These species are considered to be excellent model
systems for studying both the developmental and genetic basis of convergent skeletal evolution
throughout species. Based on the experiments and researches that have been done mostly on zebrafish as
a model system for fishes and their embryonic development, some genes have concluded the clarification
that they can be a leading point to reach the goal of sea horses model system research proposal and act as
starting point to answer Evo-devo questions for sea horses model system.
Gene duplication, Divergence of regulatory Cis-elements and Hox gene clusters
In order to fully understand the main factors that can be they key of the adaptation and the unique
morphological features of sea horses; the demand of apprehending the meaning of gene duplication,
genomic divergence, the evolution of regulatory cis-elements and hox gene clusters should be understood
and be considered the main elements for such change due to genomic reasons and evolutionary purposes
that can be the possible explanation of such appearance. First impression and possible explanation to such
change is, gene duplication this term plays an important and major role in changing and implicating new
gene functions and expressions that lead to new patterning formation. Amores and other researchers have
implemented a research that emphasizes that morphological features that have changed throughout
evolution is due to the genome duplication and the increase in developmental diversification of duplicate
genes and their related functions (Amore et al. 2004). Genomic duplication and convergent evolution
have led to many biodiversity changes expansion. These causes have changed many fates in some species
and led to many morphological changes that can be obvious to indicate that there are many gene
duplications that can lead to new variable gene functions in gene families (William A Cresko , 2003).
Moreover and secondly, Sequence divergence in cis-regulatory elements is an important aspect

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contributing to functional diversity of genes during evolution. During evolution, many paralogous genes
can diverge, resulting in a gain or loss of function due to changes in the coding sequences or regulatory
elements (Class notes). As a consequence, these duplicated genes may eventually subdivide the functions
of the original ancestral gene or evolve new activities that can lead to evolutionary novelties or different
unique morphological features
throughout evolution (Tumpel 2006).
Furthermore and finally, hox genes are
also an important factor that should be
indicted and included in the field of
research for Hippocmpus genus. The
regulation of hox gene clusters are
important in determining the active ones
that are responsible for the body axis
formation and elongation, they can help
in determining the different fates and the adaptations in them that led to such morphological features.
(Young et al. 2009). Another method can be done through the altering of the body elongation that can
also help determine the main key in body elongation (Benazeraf and Pourquie, 2013). Amores et al. 2004
performed a research between two members of the Actinoptergygii class, the zebrafish and the fugufish as
seen in Figure 1, and determined the differences between them in terms of the present and absent/lost hox
genes clusters and how did that involved evolutionary changes in both species. This additional
information can be relatively helpful in determining the lost
and present clusters of hox genes that might have been lost
in sea horses and therefore different body axis is revealed
that differentiate it from other ray-finned fishes.
The experiment concluded that the change in body plans is
due to changes and reductions in Hox clusters and therefore
differentiating between species body plans (Amores et al.
2004). Moreover, some indicated evidence concluded that
the jawless vertebrate, Lamprey, that the hox genes clusters
duplicated once before lamprey lineage and duplicated again
after the divergence (Amores et al.2004).

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The drosophila model system has been always the main key for the embryonic development
indicating the conserved steps throughout evolution in segmentation formation and development.
Patterning information helped determine many answers that can lead to develop the major features and
reasons leading to anterior and posterior formation basis and background. The segmentation process is
only about gene interactions initiated and expressed by the maternal factors that pattern and process the
embryo. The steps are as follows; first the maternal factors act as regulators and controllers of gap genes
that constitute the probable segments. Second, gap genes gradually regulate and control the expression of
the third step which the pair-rule genes that can be expressed and transcribed in overlapping regions
corresponding to exchanging segments. In turn, pair-rule
genes control the expression of segment polarity genes
that generate anterior and posterior regions within each
segment. Finally segments acquire individual identities
through the function of hox genes. There are some hox
genes that are highly conserved and have regulation duties
in segmental identities in both vertebrate and invertebrates. Studies have
shown that fgf8 is important in ensuring tight coordination of the segmentation process and temperal Hox
gene activation (Dubrulle 2001). This research can help emphasize the tracking of fgf8 mRNA that might
provide the possible explanation of the axial formation in Hippocampus species, in the posterior end of
the body and therefore help establish the understanding of the concentration gradients in the cells.
Moreover, some other researches done on somite formation indicating that both HOXB9 and HOXA10
should be also examined in order to determine their expression and activation sites in the anterior part of
the body to determine the changes and their regulations (Dubrelle 2001). The evolution of regulatory cis-
elements in the different expressionS of Hoxa2 as a coparalogous in Takifugu rubripes (pufferfish) can be
another turning point to be put into consideration of sea horses model system (Tumpel et al.2006).
Furthermore, This information can emphasize the hox genes regulation in such activation of the identities
of body’s anterior and posterior regions. The events in the anterior-posterior regionalization and mesp-a
implicates that each segment acquires anteroposterior regionalization are located in the anterior
presomatic mesoderm (Durbin et al. 2000). Additionally, the association of Wnt/beta-catenin signals has
been shown to regulate the size and the specification during the anterior-posterior formation (Benazeraf
and Pourquie 2013). Wnt3 is also one of the genes that are expressed in the ectoderm embryonic
development and can also be expressed in the posterior region and not being expressed in any other
location in the embryo; Wnt5a is functioning in the convergence and the extension of the embryo during
axis formation (Yamaguchi 2001).
http://www.learner.org/courses/biology
/images/archive/fullsize/1985_fs.jpg

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The research will be mainly dependent on the main conserved pathways in body and axis formation,
and specifically in the somite formation (Stulberg et al. 2012). Those pathways include Wnt pathway,
FGF pathway (fibroblast growth factor), and BMP signaling and the notch signaling pathway (Schroter et
al. 2012; Bajard et al. 2014; Dubrulle et al. 2001; Pourquie, 2011). Those pathways are the fate
determining mechanism in the body axis formation as they act as morphogenes that organizes the
activated and inactivated genes involved in the body formation during the embryonic development. They
can be referred to as the switch on and off regulators/ maternal factors that can help determine the growth
fate during the somite formation (Bajard et al. 2014). The formation of the trunk is mainly dependent on
the morphogenetic mechanisms that are considered be conserved throughout evolution and not being part
of the convergent evolution. The clock and wavefront model, the clock is considered to be the specific
time that a certain pathway and related genes can work and functions at that certain, however, that can
only be regulated and activated by the wavefront which send the signals from the clock to perform in
periodic series of somites (Dubrulle 2001; Baker et al. 2006). The oscillation is controlled by the
segmentation clock (Campanelli and Gedeon, 2010). Consequently, every species have a specific
segmentation clock regulation that must have adapted according to the genomic divergence and the gene
duplication that altered new functions and due to the context of evolution and new morphological features
(Krol et al. 2011).
Further experiments should be done on sea horses by examining the work of the these pathways and
how do they inactivate the transcription repressors in order to remain oscillating and continue the somite
formation and what are the genes that have duplicated and adapted its function in order to give different
body axis formation posture in sea horses Hippocampus species? This can be done be experimenting the
related genes that can be responsible during the formation process, by examining the mRNA expression
during formation process by the method of in-situ hybridization1
; examining the oscillation somite
formation that can be monitored by time-lapse imagining of a clock reporter for the activation and
regulation genes such as hox homeotic genes that provide the identity of different pars of the embryo
during formation and development (Benazeraf and Pourquie, 2013). The experiments can lead to use to
figure our gradients of some pathways and to what extinct are they important in somite formation
depending on their time to be switched ON and OFF. The second question that can be applied to clock
1 Definition: In situ hybridization, is defined as the localization method and the detection of specific mRNA
sequences expression in morphologically preserved tissues sections or cell preparations by the hybridization of the
complementary strand of a nucleotide probe to sequence that will be experimented of a certain location.

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and wavefront model, is how such movements can be highly regulated in the clock oscillations in sea
horses that can therefore determine the tissue gradients in formation and growth using the time-lapse
imaging such it has been used in zebrafish somite formation model system by Delaune et al. 2012.
Prehensile tail
The interesting and questionable morphological feature about Hippocampus hippocampus is there
prehensile tail that gives them a unique and exceptional feature and function. Sea horses are able to bend
their tail ventrally and the their role in important in grasping and holding appendages and structures while
swimming and waiting for their prey or camouflage to their surroundings while running away from the
predator. Melina Hale conducted a study about the features of the prehensile tail and determined the main
structures of it that gave it its important and unique feature and stability (Hale, 1996). The part of the tail
that has been tested is the dermal plates, vertebrae and the axial muscles that are considered to be part of
the mechanical system. The bending of the tail laterally and ventrally are due to the forces transmitted
from the hypaxial myomeres that bend the tail in such ways, this transmission force is part of the dermal
plates system. This morphological feature evolved from a complex mechanical system of muscles,
tendons and bones used for lateral bending during undulatory locomotion and the basic organization of
this system is conserved all throughout the fish evolution.
Hale in her research mentioned the relationship between the body heavy plates and how that
hinders and affects the tail bending as a result (Hale, 1996). The modifications to the tail muscoskeletal
apparatus have given seahorses its ventral bending and prehensile abilities can be tested by applying a
series of experiment of embryonic development to determine the changes and what has led to the change
and evolution of the mechanical forces in the tail by
antibody staining to track the movements and in-
situ hybridization to track the development during
the embryonic development. Another question to be
applied is the role of the dermal plates and their
association with the mechanical force of the tail. To
examine the difference in evolution of tail bending
and grasping in both pipefishes and seahorses, the
difference between their tail muscles and
movements can be determined and emphasized through series of experimentation and therefore help
(Hale, 1996)

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determine the real reason of the evolution and what has been changed functionally. The results of the
study implicated plating as being important in the force transmission in vertebrates and in the evolution of
axial bending abilities in Syngnathidae. In order to determine the evolution of muscle complexion in
seahorses and how is it different from other teleost fishes, the experimentation of the proliferation of
muscle cells and its regulator must be examined such as the expression of cdc25a that acts as regulator
and an important expression for determining muscle fates in the posterior region in the embryo such as
the expression of myoD and myf5 that are involved as regulator factors in muscle development. What
remains to be fully understandable and answered is what is the further examination should be done
concerning the evolution in the mechanics of posterior muscles along with its relationship with the
vertebrae posteriorly. What are the leading candidate genes and related fates that might have happened
evolutionarily that led to such changes in the tail functions. How can the evolution be responsible in such
changes from pipefishes that swim horizontally and sea horses that
swim vertically and can move their tails ventrally? Furthermore,
experimentation of the tail bud organizers that are the main
regulators of the tail formation in the posterior region of zebrafish
and this such as the morphogenetic protein (BMP), Nodal and Wnt8
signaling pathways are required. This information can help identify
the starting point of where to start researching and indicated the hox
genes regulation that can be responsible for such organization and at
what point are these signaling pathways activated in order to initiate
the tail bud formation in sea horses (Agathon, 2003).
Mail brooding structures
Sea horses species is also known for its unique evolutionary novelty, the male brooding structure
and its role reproduction. However, there are always exceptions for any standard rule such as male and
female role in reproduction, and especially in the world of biodiversity; the role of females and males in
the Hippocampus and Syngnathids family species are totally different in terms of reproduction and mating
ecology. Somehow there is a different and an exclusive understanding of mating and reproduction in both
Syngnathids and Hippocampus species (Sommer et al. 2012). What is ideally unique to the pouch of
males, how it adapted throughout parallel evolution? There are some pouch adaptations throughout
organisms of the subfamily of pipefish and seahorses; the pouch changes are between the abdominal
pouch and tail pouch. This evolutionary adaptation is defined as the parallel evolution that led to the
(Agathon,2003)

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remarkable male brooding organisms today (Wilson et al. 2003). In my proposal it is important to mark
the significant evolutionary novelty that is considered to be unique throughout the family of these species
and what are the reasons behind the appearance of such novelty? This structure raises many questions
about what happened evolutionary that have applied such change, there are many explanations such as the
changes in regulatory cis-elements that can change there
binding sites during formation along with hox genes
regulation, or mutations that might have naturally selected
and adapted new functions and therefore led to the rise of
male brooding structures, however, the disappointing part of
sea horses is the few fossil records that could have somehow
helped with the determination of the genes evolution and
examining the extant species of sea horses to detect the
genes that adapted new function and present it as a new morphological feature. This can experiment and
compared to the female uterus development and formation in order to build a basis on which we can track
the formation of the brood structure in sea horses. The eggs can be tracked by antibody staining’s of the
regulation sites during the embryonic development of male sea horses.
Conclusion
As a conclusion, sea horses have been an interesting aquatic species that can be attractive in many
ways, the way they camouflage with their surroundings, their skin colors that are tremendously attractive
to divers and people that watch them. They seem to be friendly and interestingly to know more about
them. However, there are broader interesting views about them from the evolutionary and development
perspective; their body posture, tail behavior, vertical locomotion unlike all fishes and the male brooding
pouches that resembles them from other fish species. Therefore, more future researches and experiments
are essential and crucial for the model system of Hippocampus species in order to answer the adaptive
evolutionary questions about such species.
http://www.seahorse.org/librar
y/articles/anatomy_files/seahor
se_male_female.gif
Figure3: This figure shows and indicates the difference between the
pouch presence in male and the absence in females in Hippocampus
species.

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