Sensory Development in the Chicken Inner Ear

Introduction

The study of sensory development is important to humans as a way to understand how disorders and deficiencies of sense occur.  From the video above, we can see that hearing loss is relevant to everyone, as we all lose hearing ability over time. While not all parts of ear development are conserved, vertebrates have enough conservation that different model organisms can be used together to find candidate genes and mechanisms to compare to human development.

In this study by Neves et al (2011), the development of sensory tissue in the chicken embryo ear is examined to connect gene expression to Notch signalling. Hair cells, the means by which vertebrates transfer sound signals to the brain, are differentiated through Notch signalling. The genetic patterning that leads to this differentiation starts in the otocysts during early embryo development. These are sac structures formed from the ectoderm which develop into all the ear components. The focus of the study is to manipulate these pre-ear sacs to understand their development.

The development of the chicken ear from days 3 to 16 of embryogenesis, viewed by filling the the inner ears with paint. (1) ©2000 by National Academy of Science

The development of the chicken ear from days 3 to 16 of embryogenesis, viewed by filling the the inner ears with paint. Note the small sac-like otocyst(the otic vesicle) at E3, and how it grows and develops into a vertebrate type ear. (1)
©2000 by National Academy of Science

An image of the basilar papilla of a chicken, homologous to the corti in humans, where hair cells have been stained  flourescent white.

An image of a chicken ear, where hair cells have been stained fluorescent white. Notch signalling is a part of this pattern formation, where hair cells(light) are distinct from the supporting cells(dark) around them due to the context of nearby trans-membrane ligands. If Jag 1 is what turns Notch on for these hair cells, how is Jag 1 manipulated in earlier development to induce the Notch patterning we see in this image? (6) ©2000 by National Academy of Science

Notch is a highly conserved trans-membrane protein that is commonly activated by a trans-membrane ligand on a neighboring cell. (5) This means that the cells in contact with a cell displaying Notch are important to whether or not the pathway proceeds. While there are other atypical ways to involve Notch in signalling, this is the type of pathway related to the study by Neves et al (2011). In this case, we explore how Jag 1, a ligand for Notch, is inducing Notch signalling in nearby cells. It was already known that Jag 1 and Notch are related, and that hair cell differentiation may depend on Notch signalling. What was not known was how Jag1 and Sox2, other genes expressed in early embryo otocysts, interact to pattern the potential inner ear, and how this affects the induction of Notch signalling for proper differentiation of sensory organs.

Jag1 and Sox2 Patterning

The localization of Jag1 and Sox2 to prosensory patches within the inner ear occurs before the development of sensory organs and is one main focus of the paper.  Sox2 is an HMG box domain transcription factor which is “expressed in nuerogenic and sensory progenitors, being downregulated in differentiated neurons and hair cells”. (Neves et. al. 2007) It is being expressed in an early stage of development where neurons and sensory organs will develop, but not expressed in the developed cells. This indicates that Sox2 could be a part of preparing the prosensory areas for differentiation. In the chicken embryo, Sox2 is first expressed in large patches of the inner ear and later restricted only to areas expressing Jag1. Sox2 expression fades except for areas around Jag1 expression. The restriction follows a dorsal to ventral sequence that is consistent with the dorsal to ventral order of cell differentiation in the ear, further implicating Sox2 and Jag1 in the patterning of development.

From Figure 2A of Neves et al (2011): Red indicates Sox2 and green is the green florescence (GFP) indicating hJag1. The

From Figure 2A of Neves et al (2011): Red indicates Sox2 and green is the green florescence (GFP) indicating hJag1. Electroporation (EP) is used to deliver hJag1, and those cells and their progeny are traced using the GFP. The EP treated otic vesicle shows a higher amount to Sox2 expresstion in the regions where hJag1 is induced, although it is not able to induce Sox2 anywhere Sox2 has already faded.

Jag1 as a ligand for the Notch-1 receptor is conserved between humans, chickens, zebrafish, and mice among other vertebrate model organisms. (3) This means that homologous versions of the protein can be used in these experiments, in this case human hJag1. The localization of Sox2 by Jag1 is explored by using hJag1 to attempt to induce Sox2 outside of its usual boundaries. While hJag1 is able to induce Sox2 outside of regular prosensory regions, it is not able to do so in places that had no previous Sox2 expression, or where Sox2 expression had already faded. This shows that Jag1 is probably maintaining rather than inducing Sox2. Sox2 being an indicator of prosensory patches, this may be a mechanism to control how long specific areas have to differentiate into neural or sensory organ tissue. The affect of irregualr Sox2 expression on development can be seen in figure 5 of the paper, where broader maintenance of Sox2 using hJag1 leads to enlarged sensory organs.

From Neves et al (2011) Figure 5B: The developing otic vesicle on the right has an irregular shape compared to the typical vesicle seen on the left. The parts of the ear are broad and larger in the hJag1 affected vesicle. MyoVilla is in red as a marker for hair cells, which have developed in a scattered pattern where hJag1 EP has been applied (in the green area). The maintenance of Sox2 by hJag1 has a direct affect on morphology.

From Neves et al (2011) Figure 5B: The developing otic vesicle on the right has an irregular shape compared to the typical vesicle seen on the left. The parts of the ear are broad and larger in the hJag1 affected vesicle. MyoVilla is in red as a marker for hair cells, which have developed in a scattered pattern where hJag1 EP has been applied (in the green area). The maintenance of Sox2 by hJag1 has a direct affect on morphology.

Lateral Induction of Notch

Since Jag1 is a ligand for Notch signalling, the possibility that Notch is required for the Jag1-Sox2 interaction was investigated. DAPT was used to inhibit Notch signalling while hJag1 was tested again to maintain Sox2. hJag1 was unable to interact with Sox2 while Notch was inhibited, so Notch signalling is required for the patterning of Sox2 by Jag1 in the prosensory patches.

From Neves et al (2011) Figure 4A, B: Part A shows that the mRNA levels of Jag1 increase when hJag1 is added. This occurs through lateral induction of Notch signalling. In part B, the middle and right column indicate that the extra Jag1 is accumulating where the hJag1 was added(in green). This is much higher than the control in the left column.

From Neves et al (2011) Figure 4A, B: Part A shows that the mRNA levels of Jag1 increase when hJag1 is added. This occurs through lateral induction of Notch signalling. In part B, the middle and right column indicate that the extra Jag1 is accumulating where the hJag1 was added(in green). This is much higher than the control in the left column.

The connection between Sox2-Jag1 expression and Notch signalling is lateral induction. This is where Jag1, being a ligand for Notch, induces Notch signalling in nearby cells, and Notch signalling induces further expression of Jag1. This creates a positive feedback loop where Jag1 is up-regulated by the induction of the Notch signal. Jag1 is maintained by Notch, but is also the inducing agent. Jag1 was induced by hJag1 in chick embryos as evidence of this mechanism. While hJag1 did not increase Notch transcription, Jag1 was induced in neighboring cells presumably through activation of existing Notch receptors. Other Notch ligands did not display this behavior. This maintenance of Jag1 allows Jag1 to maintain Sox2, and links Notch signalling to the cell determination that Sox2 provides.

From Neves et al (2011) Figure 3C: When DAPT is being used to block Notch signalling, Sox2 is not induced at high levels.

From Neves et al (2011) Figure 3C: When DAPT is being used to block Notch signalling, Sox2 is not induced at high levels.

The Big Picture

The conclusion of the paper is a better model for ear sensory development:

Sox 2 is a consistent marker of neurosensory/prosensory regions. After neurogenesis, Jag1 restricts Sox2 to the prosensory patches. Differentiation occurs through Notch signalling at many stages. Lateral induction is important to maintaining the potential of different patches for later differentiation,

From Neves et al (2011) Figure 7: Sox 2 is a consistent marker of neurosensory/prosensory regions. After neurogenesis, Jag1 restricts Sox2 to the prosensory patches. Lateral induction is important to maintaining the potential of different patches for later differentiation.

Some parts of the model reference work from an earlier paper. (4) The part of the model relevant to Sox2 and Jag1 patterning in the 2011 paper is the transition from Neurogenesis to prosensory development in part A. The figure shows, through the red dots, how Sox2 expression is broadly associated with regions which are competent for neuro-sensory development. Sox2 becomes more restricted in expression to the light green cells representing Jag1. The loss of Sox2 in surrounding cells gives them a non-sensory roll. This is consistent with the Sox2 patterning we have seen so far. We know that Notch is important to this patterning, shown in part B of the model, toward the bottom. The positive feedback of Jag1 is displayed, as well as the role of Sox2 in determining sensory cells. While a lot of this model is not directly from the results of the 2011 paper, it is important to understanding how the patterning we discussed is involved in neurogenisis as well as sensory organ development, and the larger scheme of factors involved that were not explored in this paper.

Strengths and Weaknesses

The paper provides a simple model that conveys the importance of temporal control in development, and is fairly easy to comprehend. Many of the figures were difficult to decipher for a non-expert, but they did have helpful overlays to point out important details and subtle differences. Also, the model in the conclusion of the paper includes much more than what was explored, including previous work. This is a good thing to understand all the research, but it would have been helpful to have an isolated model of the Sox2, Jag1, Notch system of prosensory development. While I would have liked more information in the paper about the induction of Jag1 and Sox2 to the neurogenesis area, but this may be included in the 2007 paper.

References

1. Brigande J V et al. PNAS 2000;97:11700-11706 [link]

2. Neves J, Parada C, Chamizo M, Giraldez F (2011) Jagged 1 regulates the restriction of Sox2 expression in the developing chicken inner ear: a mechanism for sensory organ specification.Development 138: 735–744. [link]

3. NCIB Gene ID: 182, Jag1 Homo Sapien, updated on 29-Mar-2014 [link]

4. Neves, J., Kamaid, A., Alsina, B. and Giraldez, F. (2007). Differential expression of Sox2 and Sox3 in neuronal and sensory progenitors of the developing inner ear of the chick. J. Comp. Neurol. 503, 487-500. [link]

5.  Emma R. Andersson, Rickard Sandberg, and Urban Lendahl. Notch signaling: simplicity in design, versatility in function. Development 2011 138:3593-3612; doi: 10. 1242/dev.063610 [link]

6. Eddison M. et al. PNAS 2000;97:11692-11699 [link]

 

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