Development of the Viscerocranial Skeleton During Embryogenesis of the Sea Lamprey

This post is based off a paper, written by W. Martin, L. Bumm, and D. McCauley. Most information are obtained and cited from “Development of the Viscerocranial Skeleton During Embryogenesis of the Sea Lamprey, Petromyzon marinus.” The purpose of this paper is not to take credit from the authors (Martin, Bumm, and McCauley) but to use their paper to highlight and share important findings from developmental biology research projects.

By: Alexander Francis Lydon (1836-1917) Wikicommons http://commons.wikimedia.org/wiki/File:Lampreys.jpg

Terminology

Neurocranial: “back part of the skull that houses the brain” (http://en.wikipedia.org/wiki/Neurocranium)

Viscerocranial: “facial skeleton”
(http://en.wikipedia.org/wiki/Viscerocranium)

Branchial basket: “cartilaginous structure supporting the gills in lower vertebrates” (http://www.merriam-webster.com/dictionary/branchial%20basket)

Intercalate: “to insert between layers”
(http://www.merriam-webster.com/dictionary/intercalate)

Trabecular cartilage: “cartilage that acts as a support” (http://www.thefreedictionary.com/Trabecular)

Parachordal cartilage: “cartilage alongside the anterior portion of the notochord in the embryo”
(http://medical-dictionary.thefreedictionary.com/parachordal)

Subchordal cartilage: “Cartilage situated below the notochord”
(http://www.merriam-webster.com/dictionary/subchordal)

Dorsal: “relating to or situated near or on the back”
(http://www.merriam-webster.com/dictionary/dorsal)

Ventral: “of or relating to the belly”
(http://www.merriam-webster.com/dictionary/ventral)

Rostral: “situated toward the oral or nasal region”
(http://www.merriam-webster.com/dictionary/rostral)

Caudal: “of, relating to, or being a tail”
(http://www.merriam-webster.com/dictionary/caudal)

By: Original image is listed as public domain, from US NOAA Wikicommons (http://commons.wikimedia.org/wiki/File:Anatomical_Directions_and_Axes.JPG)

Introduction

Vertebrates are a group of animals that have a supporting backbone or spinal cord and are derived from invertebrates. The evolution of internal support was a crucial turning point for early vertebrates that led to the diversification of many different types of animals such as mammals, birds, amphibians, fish, and more. However, the initiation of skeletogenesis and evolution of vertebrates is currently not fully understood. Using sea lampreys as a model organism because of its close relation to early vertebrates and easy access to embryos, this research seeks to understand skeletogenesis and development of viscerocranial skeleton during embryogenesis of sea lampreys in hopes of gaining more knowledge about the origin of vertebrate skeleton.

Sea lamprey’s are descendants of jawless vertebrates known as agnathans and closely resemble the early vertebrates that had cartilaginous internal structures. Lamprey’s do not have a mineralized skeletal system but instead have cartilaginous skeletons that are responsible for supporting a number of different internal parts such as viscerocranial, neurocranial, and branchial regions. The brain, pharynx, notochord, and gills are examples of parts that are stabilized by cartilage.

The main focus of this research was to understand the development of branchial basket in sea lampreys. The vertebrate skeletal system is derived from the mesoderm and neural crest during embryogenesis, but the exact structure of where the branchial basket is derived from is uncertain. Part of the viscerocranial skeleton is the branchial basket, a structure that stabilizes the gills and pharynx in sea lampreys. Using several experimental procedures, Martin et al. provides information on morphological and physical changes occurring during the development of the branchial basket and trabecular cartilage during embryogenesis and chondrogenesis, shows that the branchial basket develops from the neural crest, and details important physiological structures of the branchial basket, all of which are important information for understanding the development of the skeletal system and origin of vertebrate animals.

Figure 1. Diagram of the lamprey branchial basket and trabecula. The figure above depicts a pictorial illustration of an adult lamprey’s branchial basket and trabecula. The anatomy of the lamprey branchial basket was important because it served as a reference image when observing the embryonic development of lampreys. Within the pictures are abbreviations that are important for understanding the anatomy and development of the sea lamprey. Below is the exact words for those abbreviations.

Abbreviations

EP – epitrematic process

HBB – hypobranchial bar

HP – hypotrematic process

NT – notochord

PA3-9– pharyngeal arch cartilages 3-9

PC – parachordal

SC – subchordal

T – trabecula

More information on Sea Lamprey: http://www.dec.ny.gov/animals/6998.html

Video: Invading Species Awareness PSA – Sea Lamprey

Methods Summary

Alcian Blue Skeletal Staining: technique that is used to stain cartilage.  Alcian blue staining was used to identify and observe differentiation and growth of cartilage and chondrocytes within the branchial system.
(http://devoasu.blogspot.com/2013/02/skeletal-staining-alcian-blue-and.html)

Muscle Staining: FITC-phalloidin provides fluorescence and labels actin, a component in muscles. Using FITC-phalloidin, muscles surrounding the branchial basket was imaged.  (http://products.invitrogen.com/ivgn/product/F432)

DiI Labeling: technique used to stain cells. DiI labeling provides fluorescence of neuronal or other cells that are easily detectable through microscopy and was used to identify neural crest movement and behavior during embryogenesis and chondrogenesis. (http://products.invitrogen.com/ivgn/product/V22885)

Results

Cartilage Differentiation in the Developing Lamprey

Figure 2. and 3. Process of chondrogenesis of branchial basket in sea lamprey. Images are oriented anterior facing left. With cartilage’s distinctive fluorescence properties, Alcian blue staining was used to observe the growth and rearrangements of differentiated chondrocytes within lamprey embryos. For several days until the branchial basket was completely formed, embryos were carefully monitored to outline the morphological changes and growth of the cartilage elements within the branchial basket. Important details about physiological changes corresponding to the day after fertilization are detailed in the Table below.

Starting on day 15 after fertilization, chondrogenic cells begin concentrating near the branchial arches to give rise to cartilaginous skeleton, and branchial basket formation was complete 30 days after fertilization.

Table 1. Description of morphological changes in sea lamprey embryos. The table below describes key anatomical growths and changes during the 30 day branchial basket development period.

Days after fertilization Key Descriptions
15 Concentration of chondrocytes within pharyngeal arches (PA)
17 PA forms and stained chondrocytes are visible on PA3
18 Cartilage cells are present in the PA3-7 of the organism with no significant development within the ventral and dorsal area.
19 Initiation of Epitrematic (EP) and hypotrematic (HP) processes from PA3 occur
20 Epitrematic and hypotrematic processes are seen in PA4-5, and EP and HP extend out from PA3
22 EP and HP extend in PA4-5 while EP and HP from PA3 are connected to form a loop. The hypobranchial bar bridge develops between PA3 and PA4
24 EP and HP are present in all PA except PA9
26 EP and HP detectable in all PA and subchordal bars (SC) are integrated to PA7-9
28 Parachordal (PC) and trabecula extends outs rostrally
30 Complete formation of branchial basket

Dorsoventral Separation of Branchial Basket Skeletal Rods

After understanding differential growth patterning of the branchial basket in sea lampreys, Martin et al.  studied  dorsoventral separation of branchial basket and skeletal rod. Based on the results, several different embryos displayed very different dorsoventral separation. For example, the most common dorsoventral separation in embryos occurred on PA9, while other’s showed variation on where the separation occurred (Table 2.).

While studying dorsoventral separation other results relevant to dorsal and ventral cartilage development were noted.

-The hypobranchial bar (hbb) is not a single linear cartilage of the branchial basket that occupies the ventral portion of the sea lamprey. Instead the hypobranchial bar grows inwards from both ends. For example, hbb of PA3 grows ventral caudal direction to PA4 while hbb of PA44 grows ventral rostral direction to PA3. All other PA’s grow in this manner.

-Two different types of cells exist within the branchial basket: the discoidal shaped cells that create the skeleton rods and the polygonal shaped cells that create the trabecular cartilage. The two types do not associate together, and there is a noticeable change in cell type composition when going from the parachordal and subchordal region to the branchial basket region. The two types of cells form different structures and functions.

-Muscle is positioned adjacent to cartilage which has the ability to bend the skeletal rod during muscle contraction. When relaxed, the muscle releases allowing the skeletal rods of the branchial basket to shape back into its original shape (Figure 7.)

Table 2. Diagram of variation of dorsoventral separation of branchial basket. This table is referenced as Table 2. in this blog, but it is Table 1. in the actual paper. In this Table, different dorsoventral separation pattern are listed. The first row explains the number of larvae showing dorsal and ventral cartilage separation within the designated pharyngeal arch position. The second row breaks down the number of larvae that showed multiple arches with separate dorsal and ventral cartilage within the pharyngeal arches. Lastly, the bottom most row depicts the number of larvae with no dorsoventral separation.

Figure 4. Diagram of branchial basket of thirty day old larva and dorsoventral separation. Thirty days after fertilization, sea lamprey larva show complete development of the branchial basket. Figure 4a. shows differing growth lengths of epitrematic and hypotrematic processes, and Figure 4h. shows a magnification of dorsoventral axis separation within PA8 and PA9 of the branchial basket.

Figure 5. Morphological image of branchial basket of thirty days and older larva. Figure 5a. and 5b. shows an image of branchial basket several days after the complete formation of the branchial basket (30 days after fertilization). The sea lamprey larva displayed increased dorsoventral axis separation compared to day 30 post-fertilization. In Figure 5c, the arrowheads indicate the point of discontinuity between the dorsal and ventral regions of the branchial basket on PA3.

Figure 6. Difference in cell composition within chordal and trabecular structures. Images of a sixty three day year old sea lamprey larva shows surprising facts about the types of cells that form different structures. The subchordal chondrocytes are polygonal shaped while the skeletal bar chondrocytes are discoidal. Figure 6e. and Figure 6f. show the different cell shapes, green is the discoidal and red is the polygonal.

Neural Crest Origin of Branchial Basket

Previous research suggested that the branchial basket originates from the neural crest (McCauley and Bronner-Fraser). However, there hasn’t been concrete data supporting the claim. By using cell labeling technique (lipophilic dye DiI), Martin et al. was able show that DiI labeled neural crest cells affected the cartilage growth within the branchial baskets, and the chondrocytes migrated along the skeletal rod forming groups of layers surrounding the skeletal rods. Based on the results, neural crests functions to promote growth of skeletal rod cartilages; however, it is unknown whether the neural crests have any effects on other cartilage elements such as parachordal or subchordal because labeled neural crest cells were not present within those cartilage structures. Lastly, further DiI labeling experiment indicated that DiI labeled cells were inserted either together in groups or separately next to unlabeled cells suggesting that there is some mechanism of cell migration from the neural crest.

Figure 7. Images of DiI labeling of neural crest cells migrating to cartilage bars and muscles associated with the branchial bars. In Figure 7a-b, muscles are attached to the branchial basket having an effect in bending and contraction of the skeletal rod. Figure 7c depicts a region where the hypotrematic process and skeletal rod connects. In Figure 7d-f, DiI labeled neural crest cells (in red) show migrating patterns and division to form aggregates. Neural crest cells divide to form groups of neural crest cells within the cartilage bars while other neural crest cells move to other regions with non-neural crest cells to divide to form more groups.

Conclusion

This study described and showed morphological growth of sea lamprey during its post-fertilization period. From this research, Martin et al. stated that branchial basket growth occurs during days 17 through 30 after fertilization, and cartilage differentiation occurs within the branchial basket skeleton rod causing a dorsoventral separation  after 30 days post-fertilization. Also, neural crest cells are inserted along the skeletal rod either in groups or next to non-neural crest cells. Results suggest that neural crest cells migrate to branchial bars and definitely influence branchial bar development. However, there are other possibilities that other regions may also control skeletal rod growth. Furthermore, dorsoventral separation can occur in any pattern, and data displayed high variation in where the dorsoventral separation occurred and in which PA it developed in. Lastly, the cartilage of the dorsal and ventral regions are morphologically different suggesting that there are two embryonic origins for the dorsal and ventral regions of the branchial basket.

In conclusion, vertebrate development is still largely a mystery and there are countless numbers of scientific communities out there trying to understand cartilage development and how it factored into evolution. Just from this paper alone, several studies have been performed indicating high levels of interest. Other ongoing research includes studies on understanding the mechanism of chondrogenesis, regulation of cartilage growth, dorsoventral separation in other animals, and differences in morphological structures of chondrocytes throughout the branchial basket.  With further studies, researchers hope to uncover the mystery of the evolution of early vertebrates.

Strengths/Weaknesses

Strengths includes very supportive research with sufficient amounts of data, experimental results are mostly images which helps readers understand, study is on a very interesting topic, the explanations are very thorough, and several previous reports are cited and explained to help readers learn more about the study.

Weaknesses include difficult and long read, most of the results become suggestions instead of supportive ideas, conclusions are not concrete, statistical tests are not performed since majority of data are from images, data is qualitative, and the article does not transition very well from different topics.

References

Martin W, Bumm L, McCauley D. Development of the Viscerocranial Skeleton During Embryogenesis of the Sea Lamprey, Petromyzon marinus. Developmental Dynamics serial online?. n.d.;238(12):3126-3138. Available from: Science Citation Index, Ipswich, MA. Accessed March 8, 2013. (http://onlinelibrary.wiley.com/doi/10.1002/dvdy.22164/abstract;jsessionid=
EC2558C9DA23048ABA75EC760CE3E6DC.d04t04
)

McCauley, DW, and M Bronner-Fraser. “Neural Crest Contributions To The Lamprey Head.” Development 130.11 (n.d.): 2317-2327. Science Citation Index. Web. 28 Apr. 2013.
(http://dev.biologists.org/content/130/11/2317)

Yao, T, K Ohtani, and H Wada. “Whole-Mount Observation Of Pharyngeal And Trabecular Cartilage Development In Lampreys.” Zoological Science 25.10 (n.d.): 976-981. Science Citation Index. Web. 28 Apr. 2013.
(http://www.tulips.tsukuba.ac.jp/dspace/bitstream/2241/113547/1/ZS_25-10.pdf)

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