Studying In Vivo Retinal Progenitor Cell Proliferation in Xenopus laevis

Abstract

The efficient generation and maintenance of retinal progenitor cells (RPCs) are key goals needed for developing strategies for productive eye repair. Although vertebrate eye development and retinogenesis are well characterized, the mechanisms that can initiate RPC proliferation following injury-induced regrowth and repair remain unknown. This is partly because endogenous RPC proliferation typically occurs during embryogenesis while studies of retinal regeneration have largely utilized adult (or mature) models. We found that embryos of the African clawed frog, Xenopus laevis, successfully regrew functional eyes after ablation. The initiation of regrowth induced a robust RPC proliferative response with a concomitant delay of the endogenous RPC differentiation program. During eye regrowth, overall embryonic development proceeded normally. Here, we provide a protocol to study regrowth-dependent RPC proliferation in vivo. This system represents a robust and low-cost strategy to rapidly define fundamental mechanisms that regulate regrowth-initiated RPC proliferation, which will facilitate progress in identifying promising strategies for productive eye repair.

1. Introduction

Studies of neural development in the African clawed frog, Xenopus laevis, have contributed significantly to the existing knowledge on vertebrate eye formation, including the identification of the eye-field transcription factors (EFTFs) and retinogenesis [1-8]. There are several features that make Xenopus a versatile system to study the eye [9, 10]. First, it is well suited for in vivo studies as Xenopus embryos develop rapidly and externally and can be generated in large numbers. Second, many molecular and cellular tools are available for investigating gene function [10-12]. Third, Xenopus embryos have relatively low culture costs as compared to mammalian models. Lastly, the mature Xenopus eye and the human eye have comparable structures due to the close evolutionary relationship between Xenopus and humans [13]. Notably, Xenopus laevis is also an established model for retinal and lens regeneration [14-22]. The high regenerative ability of Xenopus laevis, coupled with its well-understood eye developmental processes, makes it an ideal and unique platform for devising and testing strategies to promote productive eye repair.

Retinal progenitor cells (RPCs) are of strong interest because of their potential as treatment strategies for restoring visual function in the context of injury and/or disease [23]. During eye development, the multipotent RPCs derive from cells of the optic cup and generate all retinal neuron cell types and the Muller glia [24]. It is known that a number of developmental mechanisms are used during retinal regeneration [16, 25-27]. Thus, a key objective in building strategies for productive eye repair is to identify the differences and similarities between developmental and regenerative retinal progenitor cell (RPC) proliferation. However, current retinal regeneration studies are largely focused on mature eye models, making it challenging to undertake effective comparisons with developmental events, which occur in a very different context. To facilitate such studies, a developmental model of eye repair is needed. We found that Xenopus tailbud embryos at developmental stage (st.) 27 successfully regrew eyes after surgical ablation ([28]; Fig. 1). The regrowth process was rapid, completing within 5 days after ablation (Fig. 1a-h). The regrown eye was age-appropriate; contained the expected structures including the retina, lens, and pigmented epithelium; connected to the optic nerve; and showed visual function (Fig. 1i). Our studies also showed that eye regrowth is age-dependent, with st. 32 embryos losing this ability [29].

Fig. 1.

Eye regrowth following surgical ablation. Images show normal eye development (a–d) and eye regrowth progression (e–h) at 0, 1, 2, and 5 days post surgery. Closed arrowheads indicate surgery site; open arrowheads indicate age-matched unoperated eye. Regrown eyes have the same eye structures compared to an unoperated sibling control. (i) Hematoxylin- and eosin-stained section of an unoperated sibling control (left panel) and a regrown eye at 5 days post surgery (right panel). (a–h) Up = dorsal, down = ventral, left = anterior, right = posterior. (i) Up = dorsal, down = ventral. Scale bars: (a–h) = 200 μm, (i) = 50 μm (reprinted from Experimental Eye Research, 169/April, 2018, Kha, C.X., Son, P.H., Lauper, J., and Tseng K.A.-S., A model for investigating developmental eye repair in Xenopus laevis, 38–47. Copyright 2018, with permission from Elsevier)

This new developmental model for eye repair now enables a detailed examination of how regenerative RPC proliferation can drive multi-tissue eye regrowth. Within the first 24 h after surgical ablation, there was a significant increase in proliferation in the regrowing eye but not in sham-operated eyes [28]. This result showed that productive regrowth requires multipotent RPC proliferation after injury. Moreover, regenerative RPC proliferation was specific for regrowth and not due solely to injury. Another interesting finding is that the eye regrowth observed after st. 27 ablation is not due to the retinal stem cells in the ciliary margin zone (CMZ) as these cells are only present much later at st. 35 [30]. Together, the results indicated it is most likely the embryonic RPCs that regulate eye regrowth.

Here, we provided detailed methods to study in vivo RPC proliferation in the context of Xenopus embryonic eye regrowth, including embryo culture, ablation surgery, and functional approaches to define cellular and molecular mechanisms that regulate this process. We have successfully used this model to identify apoptosis (programmed cell death) as a regeneration-specific mechanism that is required for eye regrowth [28].

In summary, the Xenopus developmental eye repair model described here represents a new and robust platform to interrogate in vivo retinal progenitor cell proliferation in a model vertebrate. It will enable rapid progress in distinguishing between developmental and regenerative eye mechanisms, facilitate new approaches toward stimulating RPC proliferation in vivo, and provide opportunities for translating these findings toward identifying suitable populations of stem cells for eye repair and promoting mammalian RPC proliferation in vitro and in vivo.

2. Materials

2.1. Instruments and Dissecting Tools

1. A dissecting stereo microscope

2. Two pairs of surgical forceps, No. 5 (Dumont)

3. Two pairs of AA-style forceps

4. Transfer pipets, disposable, 7.5 mL

5. Plastic Petri dishes, 60 mm × 15 mm

6. Plastic Petri dishes, 100 mm × 15 mm

7. Delicate task wipers (Kimwipes)

8. Vibratome, Leica VT1000 S or similar

2.2. General Solutions

1. 70% Ethanol in deionized water.

2. 0.1× Marc’s Modified Ringer (MMR) medium: 0.1 M NaCl, 2.0 mM KCl, 1 mM MgSO₄, 2 mM CaCl₂, 5 mM HEPES, and pH 7.8.

3. 1% Agarose (Sigma-Aldrich) solution dissolved in 0.1× MMR. Heat to dissolve.

4. MEMFA fixative medium: 100 mM MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSO₄, and 3.7% (v/v) formaldehyde [31].

5. Dejellying solution: 3% cysteine solution in deionized water and pH 7.8.

6. 4% Low-melt agarose (VWR) solution dissolved in 0.1× MMR. Heat to dissolve.

2.3. Solutions for Eye Tissue Removal Surgery

1. 5% Tricaine methanesulfonate (MS222, Sigma-Aldrich): dissolved in deionized water and stored at 4 °C.

3. Methods

3.1. Embryo Culture

1. For general Xenopus laevis care, induction, and fertilization of embryos, follow published protocols.

2. A protective layer of jelly surrounds the eggs. The jelly is removed from the embryos after fertilization using a 3% cysteine dejellying solution [32]. After dejellying is completed, wash the embryos several times with 0.1× MMR to completely remove the cysteine solution. Fill a 100 mm × 15 mm Petri dish with 0.1× MMR (30–35 mL). Use a transfer pipet to transfer 60–80 embryos into the dish (see Note 1).

3. Culture embryos to the desired stage. Embryos are staged using the Nieuwkoop and Faber developmental staging series [33] and can be grown in temperatures ranging from 14 to 25 °C. The rate of development is dependent on the culture temperature and embryo density. Here are general guidelines for developmental timeframe: one-cell embryos develop into st. 27 embryos in ~1.5 days at 22–25 °C, ~2 days at 18 °C, and ~3 days at 14 °C (see Note 2).

4. Monitor the growth of embryos daily. Use AA-style forceps to move and examine embryos under a stereo microscope. It is critical to maintain clean and healthy cultures. Use a transfer pipette to remove any dead embryos. Medium should remain clear. Replace cloudy medium with fresh 0.1× MMR as needed.

3.2. Preparations for Surgery

1. Set up a clean work area for surgery by wiping all surfaces with 70% ethanol, including the dissection microscope stage and surgical forceps. Spray 70% ethanol onto Kimwipes, and use the wipes to gently clean forceps tips.

2. An agarose-lined dish can be used as an aid to hold embryos in place for surgery (see Note 3). First, dissolve 1% agarose in 0.1× MMR using a microwave. Allow the solution to slightly cool before pouring the solution into a 60 mm × 15 mm Petri dish until the bottom is fully covered (~10 mL). After the agarose has solidified and cooled to room temperature, create an indentation using the tip of a 200 μL pipette tip to create a well in the agarose wide enough to hold the embryos in place. Next, fill the dish with 10–15 mL of 0.1× MMR. Add in one to two drops of 5% MS222 with a disposable transfer pipet, and then gently swirl the plate to mix to reach a final concentration of 0.02% (see Note 4).

3. Set aside two additional 60 mm × 15 mm Petri dishes for washing the animals out of the anesthetics used. Fill each dish with 10–15 mL of 0.1× MMR.

4. Fill one 100 mm × 15 mm Petri dish with 30–35 mL of 0.1× MMR to use as a culture plate for animals after surgery.

3.3. Eye Ablation Surgery

1. We have carefully studied the eye regrowth process at st. 27 tailbud embryo and observed robust retinal progenitor proliferation ([28]; see Note 5). Tailbud embryos at st. 27 can be identified by examining their external morphology as described by Nieuwkoop and Faber [33]. Our studies showed that eye regrowth ability is lost after st. 32 [29].

2. To anesthetize the embryos, use a transfer pipette to gently transfer five to ten tailbud embryos to the 1% agarose dish containing 0.02% tricaine in 0.1× MMR. The embryos should become unresponsive within a few minutes.

3. Place the dish containing embryos underneath a stereo microscope to visualize the embryos and to perform surgical procedures.

4. A transparent vitelline membrane surrounds the tailbud embryo. This membrane needs to be removed prior to surgery to allow direct access to the eyes [34]. To remove the vitelline membrane, first use a pair of No. 5 forceps to pinch the membrane in the middle posterior region of the embryo while holding the embryo in place. With a second pair of No. 5 surgical forceps, pinch the membrane at a location adjacent to the first pair. While holding the membrane with both pairs of forceps, pull the forceps in opposite directions to gently break apart the membrane and release the tailbud embryo (Fig. 2a). Allow the embryos 10–15 min to gradually straighten (Fig. 2b) out prior to beginning the next steps.

5. Place the embryos into the indentation(s) made in the agarose plate. Using a pair of AA-style forceps, orient the embryos laterally with the same side (either right or left) facing upward (see Note 6).

6. In st. 27 tailbud embryos, the eye is easily identified at the head region as it protrudes out. At this stage, the embryonic eye contains the differentiating lens placode and an eye cup, with retinogenesis having started at st. 24. Use a pair of sharp No. 5 surgical forceps to make an initial surgical incision into the eye. This can be done by angling the forceps tips to make a small incision at the protruding edge of the eye (Fig. 2c). At the same time, a second pair of forceps can be used to brace the body of the animal during surgery (see Note 7).

7. After the initial cut, some eye tissues will bulge out slightly (Fig. 2c, d). Using the sharp tips of the forceps, continue cutting around the outline of the eye until the protruding tissues are completely excised and removed from the embryo (Fig. 2e-g; see Note 8).

8. After surgery, allow the embryo to recover in 0.1× MMR for 3–5 min. Remove the embryo from the tricaine solution by gently transferring the operated embryo to a Petri dish containing 0.1× MMR using a transfer pipet (see Note 9).

9. Perform a second wash by transferring the operated tailbud embryos to a second Petri dish containing 0.1× MMR. Maintain animals in 0.1× MMR at all times. It is important to minimize the amount of solution transferred between dishes to avoid transferring residual tricaine during the wash steps.

10. After the second wash, transfer the operated tailbud embryo to the culture plate. Observe embryos for normal wound closing at the surgery site (2–3 h). Culture the embryos in a 22 °C incubator for 1–5 days as needed.

11. For individual experiments, generally, 20–30 embryos are needed. Set aside a similar number of age-matched unoperated embryos to serve as controls.

Fig. 2.

Key steps in the eye ablation protocol. (a) The vitelline membrane (indicated by an arrow) is translucent and encases the tailbud embryo. (b) Removal of the vitelline membrane enables the embryo to straighten and allows access to the eye. (c–g) Images showing eye ablation surgery. (c) An initial cut is made using sharp forceps. (d) The cut is continued around the outline of the eye. (e–g) The eye tissues protrude during the surgery, and the tissues can be removed as one intact embryonic eye. ov optic vesicle, cg cement gland. Up = dorsal, down = ventral, left = anterior, right = posterior. Scale bars: (a–g) = 500 μm

3.4. Assessment of Eye Tissue Removal

1. Assessment of eye surgery can be performed using a combination of tissue sectioning and immunofluorescence microscopy. To quantify the amount of tissue removed by surgery, first fix operated embryos after surgery in MEMFA for 1–3 h at room temperature or overnight at 4 °C. Embed fixed embryos in 4% low-melt agarose, and generate sections through the eye region using a vibratome as described in [35].

2. For each tailbud embryo, generate three to four transverse sections of 50 μm thickness through the surgery site. Immunostain sections with primary antibodies to identify eye tissues (Figs. 3a and 4a, b; Table 1). The pan-neural marker, Xen1, identifies neural tissues, including the eye cup [28]. The basement membrane surrounding the eye can be visualized using an anti-laminin antibody [28]. To assess the extent of the surgical ablation, obtain digital images of eye sections ([28]; see Note 10). Select the section containing the largest amount of remnant eye tissue (as labeled by the Xen1 antibody). Measure the area of the remnant eye tissue and the contralateral unoperated control individually to calculate the percentage of eye tissue ablated (Fig. 3a).

3. If the eye surgery is performed correctly, the embryonic eye tissues are removed without damage to the surrounding neural and mesodermal tissues (Fig. 3a). We consistently remove ~83% of the embryonic eye tissue and observe full eye regrowth by 5 days post surgery ([28]; see Note 11).

Fig. 3.

Assessment of eye ablation and eye regrowth. (a) Shown are representative images after surgery to quantify the extent of tissue removal. Images are immunostained, transverse sections through the eye of a st. 27 tailbud embryo after surgery. Closed arrowheads indicate surgery site; open arrowheads indicate unoperated eye. Blue color indicates nuclear staining (DAPI). Green color indicates the basal lamina (anti-lamina), and outlines the optic vesicle. Red color indicates neural tissues (Xen1). (b) Representative images of a regrown eye following 5 days post surgery. Each regrown eye was scored based on four phenotype categories. Full = eye of appropriate size with lens. Partial = eye with minor abnormalities and comparably smaller. Weak = eye tissues with abnormal and/or absence of most eye structures. None = no visible eye tissues. (a) Up = dorsal, down = ventral. (b) Up = dorsal, down = ventral, left = anterior, right = posterior. Scale bar: (a) = 25 μm, (b) = 300 μm (reprinted from Experimental Eye Research, 169/April, 2018, Kha, C.X., Son, P.H., Lauper, J., and Tseng K.A.-S., A model for investigating developmental eye repair in Xenopus laevis, 38–47. Copyright 2018, with permission from Elsevier)

Fig. 4.

Methods to study eye regrowth and RPC proliferation. (a) Whole-mount immunostain of a st. 46 tadpole. Green color indicates neural tissues (Xen1). Magenta color indicates nuclear signal (TO-PRO-3). (b) Vibratome-generated transverse eye section showing a st. 40–41 eye immunostained with anti-Pax6 and Xen1 antibodies. Green color indicates retinal cells in the ganglion and inner nuclear layers (anti-Pax6). Red color indicates neural tissues (Xen1). (c) Targeted microinjection of GFP mRNA into the dorsal blastomere of four-cell embryos resulted in high expression of GFP in the eye region by st. 22. (d, e) Chemical inhibitor treatment of embryos with a (d) DMSO-vehicle control and (e) MG132, a cell-permeable proteasome inhibitor. (a) Up = anterior, down = posterior. (b) Up = dorsal, down = ventral. (c–e) Up = dorsal, down = ventral, left = anterior, right = posterior. Scale bars: (a and c) = 500 μm and (b) = 50 μm

Table 1.

Published antibodies for Xenopus eye

Target	Antigen	Source	Suggested dilution (IF)	References
Ganglion cells and inner nuclear layer cells	Anti-Islet-1, clone 39.4D5	Developmental Studies Hybridoma Bank, AB_2314683	1:200	[28, 36, 37]
Horizontal cells	Anti-PROX1	AB_37128	1:400	[38]
Müller glia, intermediate filament	Vimentin (14h7)	Developmental Studies Hybridoma Bank, AB_528507	1:50	[39, 40]
Müller glia, intermediate filament	Anti-Glutamine Synthetase	MilliporeSigma, G2781, AB_259853	1:500	[28, 36, 41]
Cone photoreceptor cells	Anti-Calbindin D-28K (EG-20)	Sigma-Aldrich, C2724, AB_258818	1:500	[28, 36, 42]
Rod photoreceptor cells	Anti-Rhodopsin, clone 4D2	EMD Millipore, MABN15, AB_10807045	1:200	[28, 43]
Rod photoreceptor cells	Anti-XAP-2, clone 5B9	Developmental Studies Hybridoma Bank, AB_528087	1:25	[40, 44]
Pax6	Anti-Pax6	BioLegend, AB_2749901	1:300	[45]
Neurofilament associated	3A10	Developmental Studies Hybridoma Bank, AB_531874	1:100	[46, 47]
Pan-neural, neural specific	Xen1, clone 3B1	Developmental Studies Hybridoma Bank, AB_531871	1:100	[28, 48]
Retinal pigmented epithelium (RPE)	Anti-Retinal Pigment Epithelium 65 (RPE-65)	MilliporeSigma, MAB5428, AB_571111	1:250	[17]
Basement membrane	Anti-Laminin	Sigma-Aldrich, L9393, AB_477163	1:300	[28, 49]
Cleaved caspase-3	Anti-Cleaved Caspase-3 (Asp175)	Cell Signaling Technology, 9661, AB_2341188	1:300	[28]
Mitosis marker	Anti-Histone H3, phospho (Ser10)	EMD Millipore, 06-570, AB_310177	1:500	[28]

3.5. Quantification of Eye Regrowth Quality

1. 1. To enable the comparison of the quality of eye regrowth between different groups of embryos, a Regrowth Index (RI) was established [28] (Fig. 3b). The RI is based on four phenotype categories: (1) Full, a fully regrown eye with lens that is comparable in size and external morphology to an unoperated age-matched sibling; (2) Partial, a regrown eye with minor abnormalities and a visible reduction in eye size; (3) Weak, a regrown eye with no lens and severely reduced in size or a malformed regrown eye with most normal structures missing; (4) and None, no visible tissue regrowth of the ablated eye.

2. The phenotypic scoring for each regrown eye is normally performed at 5 days post surgery (dps), when embryos have reached the tadpole stage (the Xenopus eye is considered to be mature by st. 42 as it contains all the structures found in an adult eye). Anesthetize tadpoles in 0.1× MMR containing 0.02% tricaine. Examine each regrown eye, and assign the appropriate phenotype category.

3. To calculate the RI for a group of tadpoles, the following formula is used:

   $$\begin{gathered} 100\times \{[3\times (\text{number of full regrown eyes})]+[2\times (\text{number of partial regrown eyes})] \\ +[1\times (\text{number of weak regrown eyes})]\}∕(\text{total number of tadpoles}) \end{gathered}$$

   The RI is a value ranging from 0 to 300. A value of 0 denotes no eye tissue regrowth in any individual, and a value of 300 denotes full regrowth of eye tissues in 100% of individuals in a group. Following this protocol, eye regrowth in st. 27 tailbud embryos consistently generates RI values between 280 and 290.

3.6. Molecular and Cellular Approaches to Understand RPC Proliferation

1. Retinal progenitor proliferation is required for functional eye regrowth [28]. The RI can be used to assess regrowth out-comes from loss- or gain-of-function molecular studies. We utilized this method to discover a required role for apoptosis during embryonic eye regrowth [28].

2. Operated embryos can be treated with specific chemical inhibitors dissolved in 0.1× MMR to assess the effects of inhibition on eye regrowth (Fig. 4: compare 4d (DMSO control) to 4e (treatment with MG132)) [50, 51]. Molecular inhibition using gene-specific morpholinos can be achieved with targeted injections during early embryonic stages to restrict expression of the morpholino to the eye (Fig. 4c shows GFP mRNA injection as an example) [52, 53]. When used in combination, chemical and molecular inhibition approaches represent a robust platform to identify and define mechanisms that are required for retinal progenitor proliferation in vivo during productive eye repair.

3. Gain-of-function molecular studies can be performed by microinjections of target mRNAs into embryonic cells fated to become eye tissues to induce gene overexpression (Fig. 4c).

4. To assess gene expression during eye regrowth, follow published protocols using either RNA in situ hybridization of immunohistochemistry ([54]; see Note 12).

4. Notes

1. It is common to culture Xenopus laevis embryos in the antibiotic gentamicin [55]. However, we have found that embryos can develop healthily without gentamicin. In this case, the embryos are monitored daily, and the culture medium is changed as needed.

2. Xenopus laevis developmental stage series is available online at Xenbase (http://www.xenbase.org/anatomy/alldev.do) [56]. The developmental time periods listed are approximate. Embryo density also affects developmental timing. Embryo crowding (>100 embryos per 100 × 15 mm dish) tends to delay development.

3. It is recommended to perform eye surgeries using a Petri dish lined with 1% agarose, especially for beginners. The indentations created in the agarose help to hold embryos in place and restrict movement during the procedures. Please note that the tips of No. 5 surgical forceps are sharp, delicate, and easily dented/broken, especially if they make contact with the plastic bottom of Petri dishes. The agarose plate also acts as a soft surface that protects the fine tips of surgical forceps. In general, specific care should be taken to prevent damage to the tips of the forceps. Damaged tips can be re-sharpened using sharpening tools.

4. To anesthetize embryos, use 0.01–0.03% tricaine (final concentration) in 0.1× MMR. A drop of liquid using a transfer pipet is ~50 μL. Avoid incubating embryos in tricaine for >10 min.

5. Within an individual culture plate, natural differences in growth rates will result in embryos that are in a range of developmental stages. Embryos at st. 27 can be identified by the formation of a translucent fin along the dorsal and posterior of the embryo [33]. For surgery, make sure to only select healthy tailbud embryos without developmental defects.

6. Our results indicate that there are no observable differences in the eye regrowth process between the right and left eyes (unpublished data). However, eye surgery should be performed on the same side for all embryos in an experiment to maintain experimental consistency. Anesthetized tailbud embryos are mostly stationary. However, tailbud embryos are capable of lateral movement due to the presence of epidermal cilia on the body of the embryo. Tricaine does not inhibit ciliary movement; therefore, occasional embryonic lateral movement may occur.

7. All surgical procedures should be performed with clean surgical forceps. Remaining tissues on the forceps should be cleaned off in-between surgeries to avoid contamination.

8. The initial incision will result in a protrusion of the eye tissues. Do not dig deep into the wound site when continuing the cut around the outline of the eye cup. This may result in damage to the optic stalk and underlying brain structures.

9. Always keep the embryos submerged in the MMR solution. If the open wound of an embryo is exposed to the air-water interface, it will break open the embryo.

10. Quantitative analyses of our surgeries showed that on average, ~83% of eye tissues were consistently removed [28]. About 40% of operated embryos have less than 10% of eye tissues remaining in the embryo.

11. If embryo sections show that surrounding tissues (especially the neural tissues) are damaged during surgery, then adjust surgical excision technique by decreasing the depth of the forceps incision in the eye.

12. Xenbase (xenbase.org) contains information on commercial antibodies that have been successfully used in Xenopus laevis.