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Lauren Witty
BIOL 415
2/11/21

Limb morphology and development is a widely studied area of biology and is commonly

used to determine phylogenies. In vertebrates, elements of limbs such as the stylopodium,

consisting of the upper arm and leg, the zeugopodium, which makes up the lower arm and leg,

and the autopodium, containing the hand and foot, have all been identified and are used to map

evolution. However, not much emphasis has been placed on the specific genes that influence

these different developmental patterns. In their paper, “Hoxd13 expression in the developing

limbs of the short-tailed fruit bat, Carollia perspicillata,” Chen and his colleagues attempt to

understand the role and expression patterns of Hoxd13 during limb development. Previous

studies have identified both the importance of the expression patterns of Hoxd genes in mice, and

the essential regulating function of Hoxd13 in vertebrate autopod development, piquing the

researcher’s interest in the Hox genes. One specific study found that mice that had a mutant,

unfunctional, Hoxd13 gene developed severe defects in their autopodal elements (Dollé et al.

1993). Therefore, these previous studies suggest that the biochemical activity and pattern of

Hoxd13 expression may regulate the expression of other Hox genes, and lead to different

developmental limb patterns.

The previous work conducted on evolution and limb development paved the way for

Chen and his colleagues to conduct this study. However, these studies were all conducted on

mice and other widely studied species. Chen and his colleagues realized that there was a gap in

research, as by continually studying the same organisms, researchers were failing to understand

the different mechanisms of limb development in vertebrates and the diversity of morphologies

that those cause. Therefore, they decided to conduct a study that would provide more models to
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explain limb diversity among vertebrates. Based on this goal, Chen and his colleagues

hypothesized that “Hoxd13 expression may be a regulator of vertebrate limb morphological

diversity,” (Chen et al. 2005). This hypothesis was well supported by background research, as

the role of Hoxd13 during autopodal development was established and it was known that the Hox

genes are highly conserved among vertebrates. It was also testable, as there was a wealth of

available knowledge of other model organisms that they could use as a comparison for their new,

selected species in order to examine limb diversity among vertebrates.

To test this hypothesis, Chen and his colleagues chose C. perspicillata, or short-tailed

bats, as their primary model organism due to the unique modifications of these mammals’ limbs.

These bats, of the order Chiroptera, are the only mammals capable of sustained flight due to their

unique limb modifications and the presence of their wing membranes, also known as patagia.

The modifications of their forelimbs include a distal expansion of the autopodium with continued

interdigital tissue, which makes up the wing membrane. Bats also have different body plans than

humans and mice, as their zeugopodium is about twice as long as the stylopodium of the

forelimb, even though these two elements are the same size in the other two mammals. However,

bats have a reduced set of hindlimbs that develop symmetrically. This morphological distinction

implies a difference in expression of transcription factors within the forelimbs and hindlimbs of

the bat, as well as other mammals, that were yet to be studied by the time this paper was written.

These differences among species highlight the importance of limb development, as different

morphological structures can dictate the abilities and structure of life for these animals.

Therefore, by selecting C. perspicillata, the researchers chose an organism that was easily

testable and representative of related species, allowing them to test their hypothesis and examine

the diversity of limb development among vertebrates.

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To begin examining limb development and diversity, the researchers had to catch

pregnant female C. perspicillata in Trinidad. By utilizing their knowledge of mating and

copulation patterns of these bats, the researchers were able to catch bats that had embryos of

desired age. The researchers chose embryos at specific dates, in between 42 and 50 days

post-conception. This was important, as the researchers needed to monitor the embryos’ limb

development and Hoxd13 expression patterns, and forelimb and hindlimb development occurs

shortly after 50 days post conception. Once caught, the researchers isolated the Hoxd13 gene of

these embryos by utilizing PCR with primers derived from the conserved protein sequence of

human, mice and chick HOXD13 proteins. Once PCR had amplified the C. perspicillata Hoxd13

gene, it was cloned and ran though VISTA, a system that compares genomes of different species.

Through this process, the researchers also examined mice embryos. They chose mice as their

control group as they were the main subjects of many of the previously cited background studies.

The influence of Hoxd13 on mice development was also used to justify their hypothesis, so it

was essential that they be included in this study. Therefore, mice were a good choice of a control

group, as the mechanisms behind limb development in mice were well understood and the wealth

of knowledge around mice allowed for more morphological and biochemical comparisons to be

made between the two species.

From this comparative genome analysis, the researchers discovered that bats had

HOXD13 proteins that were characteristically mammalian. The bat HOXD13 shared 95.2% of its

sequence with humans and 94.59% with mice. All of these animals shared the same

homeodomain, polyalanine and polyserine stretches within the protein, most likely due to the

conserved nature of the HOXD13 sequence (Figure 2). Since the proteins all had similar
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sequences, the researchers then posed the question of why the bat’s limb development is so

different from the other two mammals if their protein composition is similar.

To answer this question, Chen and his colleagues decided to compare the development of

bat and mouse embryos. In early stages of development, both embryos had similar

anterior-posterior length to proximal-distal length ratios. Both species continued to grow their

forelimbs so that the proximal-distal length was longer than the anterior-posterior length.

However, as the embryos continued to develop, the bats had greater posterior growth length, due

to it’s different Hoxd13 gene expression. This small difference allowed for the growth of the

plagiopatagium, also known as the wing membrane. However, bats have a more symmetric

distribution of Hoxd13 in the hindlimbs, which starkly contrasts the asymmetric hindlimb

development of mice. The mouse showed Hoxd13 expression that was biased to the posterior,

leading to another difference between these two mammals’ body plans.

After observing these embryos, the researchers came to the conclusion that there are both

spatial and temporal differences in Hoxd13 expression, which leads to the unique development of

the C. perspicillata. While all of the observed mammals showed a similar spatial arrangement of

Hoxd13 during early development of the forelimbs, the bat quickly shifts expression patterns

towards the posterior, allowing the wing membrane to grow and the autopodium to expand. The

temporal difference among the species is represented by the delayed expression of Hoxd13 in the

hindlimbs of the bat. This temporal difference allows for the hindlimbs to grow more

symmetrically, which generates the C. perspicillata body plan that is capable of flight. From

these results, the researchers concluded that the expression, both spatially and temporally, of

Hoxd13 can generate a variety of unique vertebrate morphologies. This was a valid conclusion,

as their results directly showed differences in Hoxd13 expression within the C. perspicillata, and
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their well studied control species all showed different Hoxd13 expression patterns that led to

vastly different limb morphologies.

From these results, the researchers began to generate new models to explain the great

diversity that exists within the limb development of vertebrates. When comparing bats and mice,

the researchers suggest that the initiation of Hoxd13 is begun by a similar signaling center that

regulates other signaling molecules within the two species. To explain the difference between

hindlimb and forelimb development in bats, the researchers thought that the signaling center in

the hindlimbs either is not established correctly during the beginning of development, or is

inhibited by other factors, preventing the signaling molecules from being activated for a period

of time. This model is mostly governed by four important, previously studied, genes that would

participate in such signaling centers: SHH, FGF4, BMP and GLI3. While the paper does not go

into detail about the functions of the genes, they do propose that C. perspicillata has a unique

antagonist system between these 4 genes that leads to the posterior expression of Hoxd13 in the

forelimbs. This new model was important to the field of biology, as it provided a new organism

to utilize when mapping out limb development and provided suggestions about conserved genes

that lead to different morphological patterns. Therefore, by studying bats, this paper gave other

researchers the opportunity to look at species besides mice, allowing them to study the Hox

genes and their role in evolution from a new perspective.

Not only did this paper establish a new, influential model for examining limb

development, but it also provided a good example of how to clearly communicate in a research

article. This paper did an excellent job of explaining the background research that inspired this

study. They clearly articulated basic concepts, giving novice readers the opportunity to

understand their work. Additionally, they provided eight figures that clearly mapped out the
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development of limbs, Hoxd13 expression, as well as the sequences of the genes and proteins

being studied. This variety of figures allowed the readers to have a comprehensive visual

representation of their work, allowing them to gain a thorough understanding of the study, it’s

methods, and results.

However, not every paper is perfect. The hypothesis could have been more explicitly

stated, as it was only briefly mentioned in the “Methods” section. Additionally, SHH, FGF4,

BMP and GLI3 were all mentioned within the “Discussion” section, yet not introduced in the

“Introduction.” This could have left readers with little knowledge of limb development confused.

Briefly explaining these genes’ roles in development would have given their model more

credibility and allowed more people to understand their proposition. Finally, throughout the

paper, there were many species mentioned, besides bats and mice. At different points in their

research, Chen and his colleagues compared bats to humans, chicks, horn sharks and zebrafish.

These comparisons were slightly confusing, as no background was given as to why they chose

these species for comparison. Mentioning their motives behind species selection would have

clarified their goals for this study and strengthened their paper. Overall, the main critique for this

paper is that they needed to expand on their reasoning behind choosing specific genes for their

model or comparison species. By expanding on these topics, the researchers would have

provided a clear and logical path of reasoning for their study, and increased the credibility of

their new model of limb development.

Looking forward, this paper opened a new avenue for limb development diversity

research. In their discussion, Chen and his colleagues provided a few suggestions for the unique

hindlimb development of C. perspicillata. They were unsure if the late-development of the

hindlimbs was due to a late establishment of a signaling center or the influence of inhibitory
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factors which prevented the expression of Hoxd13 in the hindlimbs. A future experiment, aiming

to determine the genes responsible for this temporal difference in the development of the

hindlimbs, could be to knock out one of the following growth related genes in the forelimbs of

bats: SHH, FGF4, BMP and GLI3. They could then compare the forelimb development to the

hindlimb development, and see if the loss of one of these genes leads to symmetrical

development in all four limbs. If knocking out one of these genes led to similar development in

the forelimbs and hindlimbs, researchers would be able to understand the importance of the

temporal difference in development and identify the genes that are required for that difference.

Then, by monitoring the limb development and gene expression patterns of other mammals, they

could then determine if the same growth related genes are conserved among mammals, or if

these genes participate in different developmental pathways. This could further develop the C.

perspicillata model for limb development diversity, and continue to add to our breadth of

evolutionary biology knowledge.

“Hoxd13 expression in the developing limbs of the short-tailed fruit bat, C. perspicillata”

is a paper that was greatly influential in the field of evolution and development. By beginning

research on relatively understudied organisms, Chen and his colleagues inspired others to

research the signaling pathways that determine mammal limb development. In 2012, a paper

titled “The Evolution and Development of Mammalian Flight,” built upon Chen’s research by

citing his work and researching these signaling pathways that lead to limb development in

short-tailed bats (Cooper et al. 2012). Other papers have also utilized Chen’s results, expanding

his work to different species of bats in order to map bat evolution. Therefore, this paper made a

lasting impact on the field of biology, and will continue to be cited as more research on limb

development emerges.
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WORKS CITED

Chen, Chih-Hsin et al. “Hoxd13 expression in the developing limbs of the short-tailed fruit bat,
Carollia perspicillata.” Evolution & development vol. 7,2 (2005): 130-41.
doi:10.1111/j.1525-142X.2005.05015.x

Cooper, L.N., Cretekos, C.J. and Sears, K.E. “The evolution and development of mammalian
flight. WIREs Dev Biol, 1 (2012): 773-779. https://doi.org/10.1002/wdev.50

Dollé, P et al. “Disruption of the Hoxd-13 gene induces localized heterochrony leading to mice
with neotenic limbs.” Cell vol. 75,3 (1993): 431-41. doi:10.1016/0092-8674(93)90378-4