Retinal Ganglion Cell Development and Its Effects on Neural Systems
Neuroscience: A Scoping Review
September 10, 2021
This paper reviews Retinal Progenitor Cell (RPC) development and fate, development of Retinal Ganglion Cells (RGCs) and different parts of RGCs (axon, dendrite, and synapses), and the interactions between RGCs and other cells in the different neural circuits of the brain. In the field of RPC development, the Ciliary Marginal Zone of the retina is an RPC niche, and RPC differentiation is driven probabilistically rather than deterministically. The intrinsic and extrinsic factors that control this probability include notch signaling pathways, Fibroblast Growth Factors (FGF), Hedgehog morphogens, Igf signaling, transcription factors, and microRNA. They affect mechanisms such as the proliferation of RPCs, and more. In the field of RGC development, many features of dendrite development and remodeling have been discovered, including transcription factors controlling dendrite guidance and how action potentials are unnecessary for proper development. The growth of the axon initial segment stems from the cisternal organelle and light input during development, and the myelin sheath is a very plastic region during development that changes in length and features depending on location and other factors. RGCs form synapses with amacrine and bipolar cells in the IPL. These synapses, and gap junctions, develop and affect the receptive fields of RGCs. RGCs also interact with glial cells, amacrine cells, and bipolar cells, which play regulatory roles for RGCs. The regions of the brain that RGCs project to and affect include the circadian system, defense response system, and memory circuits. RGCs play a
large role in the proper function of each of these systems.
The visual pathway is a vital process that is in constant use. Light is converted into communicable information between the neurons in the retina and brain in a complex and fascinating process. Beginning with the cells that make up the retinal layer at the back of the eye, photoreceptor cells (rods and cones) constantly release glutamate. Glutamate is an excitatory neurotransmitter, and is released when the photoreceptor cell is in a state of depolarization or in the absence of light. The chromophores of the pigments in these photoreceptor cells absorb light, inducing a pathway that leads to the hyperpolarization of the cell and the termination of glutamate release. The termination of this release conveys information onto off-center and on-center bipolar cells by inhibiting off-center bipolar and ganglion cells (off-BC and off-GC), and exciting on-center bipolar and ganglion cells (on-BC and on-GC). This is reversed in the dark. This excitatory response happens when the respective type of bipolar cell produces a graded potential that, once reaching a certain threshold potential, induces the release of glutamate to the ganglion cells; a threshold potential is the level that a membrane needs to be depolarized to generate an action potential, which is a rise and fall in voltage of the cell. This changes the gradient across the cell membrane, changing the driving force of the ions inside and outside the cell. The excited ganglion cells, which make up the optic nerve, fire action potentials at a changing rate depending on their state (depolarized or hyperpolarized).
In this review, I will go over the latest evidence of how the development of retinal ganglion cells (RGCs) is integral to the developmental process of the connections between the different regions of the brain and other cells. Over 30 types of RGCs are incredibly important to many systems and processes in the brain; in a study by Martersteck et al., 2017, there were shown to be over 56 retinorecipient regions of the mouse brain.1 This paper will review the latest findings on the growth and development of RGCs, their connections to other cells, and how the early development of retinal ganglion cells, and their defects, heavily affect the development of brain systems such as the visual interpretation system, eye movement, circadian system, defense response, memory, and more.
The process of how individual retinal ganglion cells (RGCs) develop, called neurogenesis, is mostly well-established. During development, RGCs are the first cells to develop in the human retina, followed by amacrine, bipolar, and horizontal cells. These cells are derived from the same progenitor cell called retinal progenitor cells (RPCs). Before differentiation, RPCs develop over a number of weeks in a process called retinal neurogenesis.2
Retinal neurogenesis, or retinogenesis, begins with the production and development of RPCs. RPCs are abundant in early developmental stages. Recent evidence from Marcucci et al., 2016, has shown that the ciliary marginal zone (CMZ), an area between the neural retina and the retinal pigment epithelium, is a crucial source of RPCs throughout life. Studying the CMZ has led to many findings about regulatory and other features of RPC production and movement. Recent time lapse imaging of mouse retinas found that CMZ cells moved to the peripheral retina to become RGCs, showing how important the CMZ is for the development of a fully functional retina.3 The mechanism behind this cell migration from the CMZ in humans is not entirely understood; although this migration mechanism was discovered in zebrafish by Wan et al., 2016. In zebrafish, the cells near the periphery of the CMZ divide, causing one daughter cell to stay in this niche area and the other to be pushed more centrally. However, in humans, it is unknown whether this cell division causes the migration, or if it is due to tissue growth pushing the cell out of the CMZ.4
RPCs differentiate into all the types of neurons present in the retina, and also proliferate to produce more RPCs. There is a chronological order to the development of different types of retinal neurons and differentiation of RPCs. Early (E10-18) RPCs differentiate into RGCs or cones, and late (E18-P0) RPCs differentiate into the multiple other types of neurons in mouse retinas. RPCs begin differentiating into RGCs around E10, 9 days after fertilization, and are often found around the head of the optic nerve.5 In 1985, Ursula C. Dräger found that RGC differentiation starts at the head of the optic nerve and goes outward to the peripheral retina.6 The chronological order of RPC fate was recently found to be probabilistic rather than deterministic. The probabilistic model itself is driven by extra and intracellular signals; however, these signals do not determine RPC fate in a fixed way. The probability of development for an early RPC decreases over time while the probability for a late one increases over time. Also, RPCs start off with a higher likelihood to divide into two progenitors, then a higher likelihood to divide into one differentiated cell and one progenitor, and then a higher likelihood to divide into two differentiated cells.5
The probability for RPCs to either differentiate into, for example, an RGC or to continue to go through the cell cycle as an RPC is controlled by extracellular signals and intracellular signals. Extracellular signals include Notch signaling pathways, Fibroblast Growth Factors (FGF), Hedgehog morphogens, and Igf signaling, among others. Notch signaling inhibits RPC differentiation, hedgehog morphogens stimulate RGC differentiation, and FGF stimulates RGC differentiation.5 Each of these extrinsic factors play a crucial role in retinal development. Recently, inhibition of notch signaling has been proven to be a possible way to prevent retinal fibrosis, as it was found that inhibiting notch ligands promotes proliferation of cells, demonstrating how important Notch pathways are to the development of a functional retina.7 Igf signaling has also been found to be a very important signaling pathway in retinal neurogenesis, particularly in the CMZ. The Igf1 receptor is very important to the proliferation of RPCs in the CMZ; it was found that initiating this signaling pathway leads to an increase in proliferation of RPCs in the CMZ and in the size of the retina in general, while also keeping the retina’s structure sound.8
RGC specification is controlled by intrinsic factors, too. Recently, RNA sequencing has revealed many intrinsic regulators that contribute to the specification of RGCs. Many transcription factors, mainly the POU Domain and Atoh7, regulate the differentiation of RGCs. Other important transcription factors include SoxC and Dlx. SoxC promotes the differentiation of certain types of RGCs and also helps with axon guidance, and Dlx is important for the terminal differentiation process of RGCs.5 One study by Melo et al., in 2020 discovered that the knockout of Dlx genes leads to more RGC death and a reduced RGC layer. Atoh7 also helps express other important transcription factors and helps RPCs leave the cell cycle rather and differentiate rather than continuing to proliferate.9 The POU domain promotes RGC specification while also inhibiting the differentiation into other retinal cell types.10
Another important intrinsic regulator whose function is less clear is microRNA, or miRNA. Its exact function is hard to pinpoint, but some have theorized that it regulates timing of differentiation, and others are trying to find its function by finding the effects of the removal of its regulation, using miRNA microarrays.
More recently, a group of mice with diabetic retinopathy were found to have a decreased number of RGCs, which corresponded with 11 miRNA being upregulated and 4 being downregulated. When treated with a formula (HuoXue JieDu Formula or HXJDF) that removed these changes in miRNA expression, along with reducing the hemodynamic changes caused by diabetic retinopathy, the normal RGC number was restored. It may be possible to use the inhibition of miRNA regulation to help treat retinal disorders such as diabetic retinopathy.11
Once RPCs differentiate into RGCs, the different parts of RGCs must develop in the correct places and ways. This development process is heavily influenced by extrinsic factors such as adjacent cells and extracellular signals, but much of the development is also in response to intrinsic factors.
A. Ribosomes bind to mRNA to translate genetic information into a chain of amino acids using tRNA. When miRNA binds to mRNA, ribosome attachment is inhibited and the genetic information carried by mRNA is not translated.
B. This lack of gene expression and protein formation causes cells to differentiate differently, such as into an amacrine or horizontal cell.
RGCs need to be able to integrate inputs from photoreceptor cells, amacrine cells, horizontal cells, bipolar cells, and more. Because of this, the different types of RGCs form “parallel pathways”, in multiple layers called retinal lamination. Each layer of this system serves important functions; for example, very recently, one of the more mysterious regions called the optic nerve lamina region (ONLR) was found to be a neural progenitor cell niche, or a region that produces progenitor cells. This study found that upon the removal of ONLR progenitor cells, the mouse retina showed hypomyelination and retinal dysfunction. Also in these layers, functional segregation plays a large part in the formation of the system; recent studies suggest that certain genes are linked to the cell type, which influences the behavior of the cell and the functional segregation of the whole system.12
The dendrite development process is incredibly important, as the dendrites allow RGCs to receive and integrate signals from multiple outside cells. First, during the development of these circuits, RGC dendrites are constantly being pruned and remodeled; this dendrite remodeling has been somewhat recently confirmed to play an important role in the formation of lamina-restricted circuits and the synapses between cells. In fact, RGCs are born usually with an excess of dendrites that are mostly pruned away. Dendritic remodeling is its own process mostly separate from the remodeling of the axons. This has been proven due to the ability of cells to continue remodeling dendrites without the presence of action potentials, which have a much larger effect on axon remodeling.13 A study found that after chronic injections of tetrodotoxin, a neurotoxin that removes action potential activity from the cell by blocking sodium channels, the dendrite remodeling process continues, while injections during axonal remodeling cause large changes.13 These dendrites, which reach into the Inner Plexiform Layer (IPL) of the retina, form into layers during the development process. All these locations of the dendrite branches, axons, and more have recently been found to be decided by a few transcription factors that, when expressed, allow dendrites to form correctly.14 These transcription factors are currently under investigation.
During development, as neurites develop into axons, the parts of the axon are created: the axon hillock, the axon initial segment (AIS), the axon terminals, and axon telodendria. AIS development was not fully understood until recently; researchers studying the the roles of different parts of the AIS in its development have uncovered more important functions and interesting regulatory features of the region.15, 16
RGCs’ AIS have stacks of Endoplasmic Reticulum (ER) that form the cisternal organelle (CO), an organelle that has been shown to play an important role in homeostasis, elongation, and plasticity of the AIS.15 Using the CO marker synaptopodin, researchers found that the cluster sizes of synaptopodin increased when the mice were dark reared, and that light deprivation also made the AIS longer. As the AIS is the initial site of action potentials in the cell, this could suggest that the AIS grows in size when there is no activation because it wants to generate action potentials but cannot. The CO and synaptopodin are the mechanisms in the cell that regulate AIS length and size, that cause the AIS to grow in these light-deprived circumstances.15
Another study by Schlüter et al. 2019 also found that in mice lacking CO, the AIS shortened in the dark, further showing the developmental role of CO during visual deprivation.16 Another role of the AIS is to backpropagate action potentials to the soma, where they are remade by sodium channels. Schluter et al. 2019 again found that the AIS has to adjust its characteristics for this propagation to occur, further showing the plasticity of the region in response to its environment. This dynamic change in levels of synaptopodin when the mice are dark reared also shows that the AIS and the CO go through structural remodeling in response to different environments, suggesting that the AIS is a very important neuroplastic site in the visual circuit.16
The growth and development of the more studied regions of the axons have had multiple developments in recent years, too. For example, Pax6, a major regulatory gene in retinal axon guidance, has been found to play a much more important role in axon development than previously thought. It has been found to help with axon guidance even after the fate specification of an RPC. Upon knocking out Pax6, there was an increase in the expression of extracellular matrix molecules, decreased expression of neuron specification, and decreased expression of axonal guidance molecules. The development of the axons is also heavily influenced by action potentials in the cells, since axons must develop in a certain way to be able to disseminate action potentials. Axons are not guided correctly without action potentials.17
Another important part of axon anatomy in RGCs is the myelin sheath, whose length and structure affects the conduction of signals through the cell. The myelin sheath also goes through remodeling during development, as axons and dendrites do, but it is also much more adaptable.18 The myelin can adapt to certain needs of a circuit. This is controlled by intracellular circuits; in fact, it was recently found that, after removing individual sheaths, mature axons can still remodel the myelin sheath. This shows that the cell can control sheath length and growth patterns, with constant remodeling taking place during development, and the length of the myelin sheath as a whole is controlled by the cells themselves and interactions with oligodendrocytes around them.19
The synapses at the dendrites and axons of RGCs, have a very unique developmental process that allows for the visual circuit to be interconnected. This process, also called synaptogenesis, causes much of the dendrite and axon remodeling we see. Synaptogenesis is a highly selective process, with certain subtypes of RGCs only forming synapses with specific cells. There are around 30 subtypes of RGCs1, previously thought to be around 20, and they selectively form synapses with bipolar and amacrine cells. This process is also highly ordered and affected by the environment. These parallel pathways between cells happen in different lamina as well, in a specific order.
Amacrine cells first develop synapses, then synapses between photoreceptors and horizontal cells, and then the synapses between RGCs and bipolar cells. RGCs form synapses with amacrine and bipolar cells in the IPL.20 In fact, a large number of RGCs have a network of dendrites branching near the center of the IPL ready to synapse to bipolar cells after eye opening. The amount of synapse formation is affected by the environment and intracellular circuits. For example, it was found that the amount of synapses in the IPL increases after eye opening, possibly due to the activation of both ON and OFF-RGCs as light levels fluctuate in the environment.20 Light deprivation was also found to increase the amount of conventional synapses.
The synapses themselves, which are of 2 main types (ribbon and conventional), have been studied more in recent years. The major difference between the two types is the presence of the ribeye protein in ribbon synapses.21 The anatomy of the synapses is very specific. Neurotransmitters are released at active zones, which, in ribbon synapses, are a variation of conventional synapses’ active zones. Calcium ion influx, the first step in synaptic signal transferral, is linked to the synaptic vesicles at the presynaptic release sites. Large scaffold proteins are needed at this site. A delineated synaptic pathway is developed between two cells, with the receiving neuron usually having a type of glutamate receptor, such as mGluR4.20 The gene Ngl2 in RGCs was relatively recently found to play a very important role in the formation of this synapse system, in terms of formation, restoration, and regulating axon growth. Knockout or overexpression of this gene resulted in a proportional decrease and increase in number of synapses, respectively.22 More important regulatory mechanisms, such as genes and vital molecules, are being found that contribute to the complete formation of this system.
Synaptic inputs and dendritic placement closely relate to the receptive field (RF) of RGCs. The layout of receptive fields in the visual circuit is important to the connectivity and communication between cells. However, inputs from other RGCs through gap junctions and more can also affect receptive fields.23 One such layout of receptive fields is an offset on-off receptive field that forms between two specific subtypes of RGCs (F-mini-off & F-mini-on). A recent study found that the network of gap junctions between these types of RGCs forms this offset receptive field layout, which is important in forming a combined electrical channel between cells.23
In addition to studying the development of the different parts of the axon such as the AIS, the myelin sheath, and the synapses & gap junctions, much recent research has been conducted on axons as a whole and specifically axon regeneration, since degenerate axons can cause irreversible blindness. For example, one new development is that the decrease in levels of certain microRNAs such as microRNA-19a decreases the suppression of axon regeneration, which means the regulation of microRNAs may be a viable therapeutic treatment for axon degeneration in the future.24 Research on macrocircuits along with the microscopic parts of the circuit are important for a full understanding of what is happening during development of RGCs and how it can affect vision.
The development of RGCs is affected by other cells, but also affects the shape and development of cells around them. Usually, much of the parts of the cell grow and develop through signals from extrinsic factors such as the cells around them. For example, there are multiple important interactions between RGCs and retinal glial cells. The amount of activated retinal glial cells is directly proportional to the growth of RGC neurites.25 Retinal glial cells, such as astrocytes, produce apolipoprotein E which stimulates the growth of these branches during developmental stages. Even after RGCs have matured, retinal glia activation stimulates axon regeneration when there is damage. Some glia also play a regulatory role in cell death or apoptosis, as they were recently found to, when stimulated, activate transient receptor potential vanilloid 4 (TRPV4) for a prolonged period of time, increasing calcium ion levels in RGCs and inducing apoptosis. Apoptosis may be induced during RGC development if the cell is defective. Other molecules that glial cells produce, such as PEDF or others, serve multiple regulatory functions such as neuroprotection, or the inhibition of intrinsic cell death pathways and more.26 Recent studies have even found that it is possible to use retinal glia to regenerate RGCs and possibly be a treatment for retinal degenerative diseases.26 The multiple interactions between glia and RGCs are still slightly unclear, and glial cells and RGCs continue to be studied and analyzed using different methods such as high resolution mass spectrometry.
Amacrine and bipolar cell outputs to RGCs cause a multitude of different responses. Specific connections form between RGCs, bipolar cells, and amacrine cells during development that affect vision, the strength of signals, and the types of signals. One study that measured light-evoked postsynaptic currents in certain types of ganglion cells found that a larger conductance change in the amacrine-RGC synapses than in the bipolar-RGC synapses results in RGC responses, or activation.27 Amacrine-RGC synapses are inhibitory, and bipolar-RGC synapses are excitatory. Another recent study to do with the connections between bipolar cells and six types of RGCs in marmoset monkey retinas found that one type of OFF bipolar cell, Db3a, favors outputting signals to one type of RGC, OFF-parasol. This was found when RGCs that connected with Db3a cells were labelled, and the number of contacts between Db3a and the 6 types of RGCs that were studied were measured.28 These “preferences” usually form during development when, possibly, different factors such as location of the dendrites, the lamina, and more lead to a connection between two types of cells. In this case, around 30% of the output from Db3a cells went to OFF-parasol RGCs, and the other 70% went to multiple other RGC types.28
Martersteck et al. 2017 found 59 total retinorecipient regions of the mouse brain, 35 from the contralateral side and 24 from the ipsilateral side. They measured all axonal projections into the brain, and found projections into the hypothalamus, thalamus, midbrain, and amygdala. RGC axons protrude into the optic nerve, optic chiasm, and other parts of the visual circuit, such as the optic radiation, superior colliculus, and lateral geniculate nucleus, as well as other regions of the brain.1 These retinal projections are incredibly important for not only the visual system, but other systems as well.
Circuits or regions of the brain that are affected by the retinal cells include the defense response mechanisms, memory regions, circadian circuit, and more. There is both established and circumstantial evidence for RGC projections into these regions.
Some defense responses are controlled by certain thalamic circuits, a region confirmed to be projected into by Martersteck et al. 2017.1 Visual stimuli such as a predator, or any form of danger, cause defense responses. A common defense mechanism is freeze or flight, a mechanism studied by Lees et al. 2020. The study measured the effect of causing defects in the RGCs of a mouse on this defense response. When confronted with a looming stimulus, the fleeing response in mice with the defective retinas (knockout of Brn3b) was largely and selectively removed, while the freezing response remained.29 This confirms the role of RGCs in defense mechanism circuits, but the exact types of RGCs present in these circuits are yet to be found.
Vision is incredibly important to the circadian system, with visual stimuli being important to the sleep/wake cycles, appetite, emotions, and more. The circadian system is mainly controlled by the hypothalamus, again a region found to be projected to by Martersteck et al. 20171 The suprachiasmatic nucleus (SCN), present in the hypothalamus, is an important regulator of circadia timings in the body, and it has been established that it takes direct input from ipRGCs in the retina. The SCN directly communicates with the pineal gland, which releases hormones required for sleep.30 RGCs also help with circadian photoentrainment and appetite. It was recently found that in Parkinson’s, when the number of melanopsin-containing RGCs (mRGCs) is decreased, this leads to sleep schedule and general circadian rhythm issues.31
The effect of RGC projections on memory is slightly more unclear. However, RGCs have been found to play a large role in spatial and more visual types of memory. In fact, recent studies have found that a certain type of RGC (SMI-32-expressing ON-type retinal ganglion cells) do indirectly connect to the neurons in the nucleus reuniens (NRe), a section of the thalamus that deals with spatial memory. The activation of these RGCs activates NRe neurons and promotes spatial memory.32
Studying Retinal Ganglion Cells (RGCs) is crucial to understanding the visual system, memory, defense response, circadian rhythm, and more. Understanding RPC development and differentiation, controlled probabilistically by different extrinsic and intrinsic factors, and the features of the development of the different parts of RGCs, give us insight toward the inner workings of these systems. Using these findings can also help researchers discover new and viable treatments for retinal degenerative diseases and other devastating retinal conditions.