A method for inducing neural tube defects in zebrafish using all-trans retinoic acid (ATRA)

Abstract

The present invention relates to the field of applied basic medical research, specifically to a method for inducing a zebrafish neural tube defect model using all-trans retinoic acid (ATRA). This invention establishes, for the first time, a zebrafish model of neural tube defects induced by all-trans retinoic acid (ATRA), providing a new zebrafish model for studying the mechanisms and treatments of neural tube defects caused by early pregnancy ATRA. The technical problem addressed by this invention is to establish a convenient, time-saving method that allows for direct in vitro observation of the embryonic neural tube development process. This novel method for inducing a zebrafish neural tube defect model has a high embryonic malformation rate and a low mortality rate.

Description

Technical Field

[0001] The present invention relates to the field of applied basic medical research, and in particular to a method for inducing a neural tube defect zebrafish model using all-trans retinoic acid (ATRA). Background Art

[0002] Neural tube defects (NTDs) are the most common and severe congenital malformations of the central nervous system. Their incidence varies across different regions, with the highest incidence in northern China. With recent adjustments to China's fertility policy, the number of older, high-risk pregnancies has increased significantly, leading to a steadily increasing incidence of NTDs. The etiology of NTDs is complex, resulting from the interplay of genetic and environmental factors. NTDs, caused by abnormal neural tube closure, lead to malformations from the fetal head to the spinal column, including anencephaly, encephalocele, craniospina bifida, and spina bifida. The neural tube is the foundation of central nervous system development, developing into the brain and spinal cord in the late embryonic stage. Neural tube closure is a highly complex and dynamic process, involving numerous cellular events precisely controlled by the microenvironment and genetic factors. During this process, adverse factors, both genetic and environmental, can lead to impaired NTD closure. Therefore, in-depth research into the pathogenesis of embryonic NTDs and the development of appropriate preventive and treatment strategies are urgent and crucial for improving population quality.

[0003] Human NTDs are limited in number, making the development of animal models crucial for understanding the mechanisms and prevention of neural tube defects. Currently, model organisms used for NTD research include rats, mice, rabbits, and sheep. However, mammalian gestation is long, and direct visualization of the embryonic nervous system development in the uterus is not possible. Early pregnancy is a critical period for neural development, coinciding with the gradual transformation of the neural tube from a single layer of neuroepithelial cells into a neural embryo. The neural tube develops in four orderly stages: neural plate formation, neural plate shaping, neural plate folding, and neural fold fusion. Disruption of any of these stages can lead to the development of NTDs. Mammalian embryonic development is a complex, spatiotemporally dynamic process, making direct observation of the dynamics of embryonic development in vivo difficult with current techniques. Researchers have attempted to study mammalian embryonic development using various methods, but each currently suffers from various limitations. For example, tissue staining of fixed embryos cannot capture the dynamics of development; imaging methods such as ultrasound or magnetic resonance imaging have limited resolution and cannot monitor embryonic development in real time for extended periods of time. In recent years, through research on the in vivo developmental environment, complete culture media for in vitro embryo culture have been developed. Mouse embryos removed from the uterus and cultured in in vitro culture media for 24 to 48 hours can be imaged from pre-implantation to early organogenesis using optical or fluorescence microscopy. However, despite efforts to mimic the internal environment of the uterus, as the placenta forms, the umbilical cord provides nutrients through the blood circulation to support embryonic development. Currently, there are no research methods to simulate the umbilical cord's provision of nutrients. Because it is impossible to reconstruct fetal-placental nutrient exchange, embryos cannot be cultured in vitro for extended periods of time. Therefore, there is an urgent need to establish an animal model that can directly visualize the process of embryonic neural development during early pregnancy, which will provide a new platform for studying the mechanisms and treatments of neural tube defects.

[0004] NTDs are caused by a failure of neural tube closure due to multiple factors, including genetics and environmental factors. While folic acid has been shown to be effective in preventing and treating NTDs in recent years, approximately 30% of NTDs remain unpreventable. Therefore, other factors have also attracted attention. Currently, zebrafish are mostly genetically modified to study the mechanisms of neural tube defects, but a stable and universal zebrafish model is lacking. This present invention, for the first time, utilizes all-trans retinoic acid (ATRA) to induce an animal model of neural tube defects in zebrafish embryos. This model allows for direct in vitro observation of embryonic development, with a short experimental cycle, high efficiency, low cost, simple method, ease of use, and excellent reproducibility. This provides a new visual animal model for studying the mechanisms and treatments of neural tube defects. Summary of the Invention

[0005] To address the challenges of existing technologies, this present invention establishes for the first time a zebrafish model of neural tube defects induced by all-trans retinoic acid (ATRA), providing a novel zebrafish model for studying the mechanisms and treatments of neural tube defects caused by early pregnancy ATRA. This method, described in this invention, provides a novel zebrafish model for inducing neural tube defects, resulting in a high embryonic malformation rate and a low mortality rate. It is easy to operate, saves time, and allows for direct in vitro observation of the embryonic neural tube development process.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0007] The present invention provides a zebrafish model of neural tube defects, which is characterized in that all-trans retinoic acid (ATRA) is used to induce zebrafish embryos to construct the neural tube defect animal model.

[0008] Furthermore, the model is used in the study of direct observation of neural tube development in vitro.

[0009] The present invention also provides a method for constructing the above-mentioned zebrafish neural tube defect model as follows:

[0010] S1: Select zebrafish of about 4 months old and place male and female adult zebrafish in a breeding tank according to the ratio. Separate them with a partition. Remove the partition on the next day to allow the male and female fish to mate.

[0011] S2: Use a filter to collect the fertilized eggs at the bottom of the external breeding tank and wash the eggs 2 to 3 times with breeding water;

[0012] S3: After the fertilized eggs were sorted and washed, the eggs collected in S2 were placed in a culture dish using a pipette, and all-trans retinoic acid (ATRA) was added. After culturing for 24 hours, the culture water was changed to normal culture to obtain a zebrafish model of neural tube defects.

[0013] Furthermore, it is characterized in that the zebrafish strain in S1 is a wild-type or transgenic zebrafish such as the Tuebingen (Tu) strain or the AB strain.

[0014] Furthermore, the ratio of female to male adult zebrafish described in S1 is: female:male is 1:1 or 2:1.

[0015] Furthermore, the collection of fertilized eggs in S2 should be performed within 20-1h min.

[0016] Furthermore, the fertilized eggs within 20 minutes to 1 hour are for the same tank of broodstock, and the time interval between two collections of fertilized eggs shall not exceed 1 hour to ensure the consistency of the developmental stage.

[0017] Furthermore, the all-trans retinoic acid described in S3 was dissolved in dimethyl sulfoxide (DMSO), and the same concentration of DMSO was used as a control group. The dosage of all-trans retinoic acid was 1-20 nM.

[0018] Furthermore, the neural tube defect model prepared by the above-mentioned method for constructing a zebrafish neural tube defect model is used in the study of directly observing the neural tube development process in vitro.

[0019] Furthermore, the neural tube defect model prepared by the above-mentioned method for constructing a zebrafish neural tube defect model is used in studying the mechanism and treatment of neural tube defects.

[0020] Compared with the prior art, the present invention has the following beneficial effects.

[0021] 1. This invention uses all-trans retinoic acid (ATRA) as a teratogenic factor. During the early stages of fertilized eggs developing into the early stages of neural tube formation, this is the first time that all-trans retinoic acid (ATRA) is used to intervene and induce a zebrafish model of neural tube defects.

[0022] 2. The present invention uses all-trans retinoic acid (ATRA) to interfere with zebrafish fertilized eggs, which is easy to operate and can directly observe the neural tube development process in vitro, thereby realizing the direct visualization of the neural development process in early pregnancy.

[0023] 3. The method adopted by the present invention is simple, has good repeatability, high deformity rate and low mortality rate for fertilized eggs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Morphological observations of zebrafish larvae. A: Images of normal zebrafish larvae and zebrafish larvae with NTDs induced by all-trans retinoic acid (ATRA). B: HE staining of tissue sections from normal zebrafish larvae and zebrafish larvae with NTDs induced by all-trans retinoic acid (ATRA).

[0025] Figure 2 Images of normal zebrafish larvae and zebrafish larvae with NTDs induced by all-trans retinoic acid (ATRA) at different time points. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0027] Implementation example.

[0028] 1. Experimental animals and materials.

[0029] Zebrafish (Tuebingen (Tu) strain, male and female, approximately 4 months old) were obtained from the Zebrafish Platform of the Laboratory of Pediatric Congenital Malformations, National Health Commission, Shengjing Hospital, China Medical University. All-trans retinoic acid (ATRA) was purchased from Sigma at a stock concentration of 0.01 mol / L, and dimethyl sulfoxide (DMSO) was purchased from Lablead.

[0030] 2. Experimental instruments.

[0031] Olympus SZX10 stereo microscope, SHP-250 zebrafish fertilized egg incubator, ESEN-AW-DU5SS zebrafish breeding system, Satorius BS124S electronic balance, ULUPURE UPR-II-15TNZ ultrapure water instrument, SW-CJ-2FD clean bench, refrigerator, Thermo Scientific ultra-low temperature refrigerator, Thermo Scientific centrifuge, Thermo Scientific paraffin embedding machine, Leica RM2255 fully automatic paraffin sectioner, YT-7FB biological tissue baking machine, fully automatic immunohistochemistry staining machine, Nikon ECLIPSE Ci microscope, Nikon ECLIPSE 80i fluorescence microscope, NIS-Elements image analysis system.

[0032] 3. Model preparation.

[0033] Tu strain zebrafish, approximately four months old, were selected as broodstock and mated to obtain zebrafish eggs. The eggs were maintained at room temperature (28–29°C), a pH of 7.2–7.6, and a 14:10 light-dark cycle. The broodstock were replaced and discontinued until the experiment was completed or the broodstock were too old. The afternoon before modeling, after feeding the zebrafish, sexually mature zebrafish were selected and placed in a breeding tank at a female:male ratio of 1:1 or 2:1, with males separated by a partition. On the morning of modeling, within 10 minutes of turning on the lights, the partition was removed, and natural mating was initiated. Fertilized eggs were collected within 20 minutes to one hour (i.e., for broodstock from the same tank, the interval between collections should not exceed one hour to ensure developmental stage consistency). After the fertilized eggs were sorted and washed, they were collected and placed in a culture dish using a pipette. Different concentrations of all-trans retinoic acid (ATRA) were added, and the same concentration of DMSO was used as a control group.

[0034] Two to three fish were mated at a time, and approximately 200 eggs were laid in a single tank of the same parental generation (female:female ratio 2:1). The eggs were randomly divided into nine groups: ① blank control; ② 1% DMSO (volume ratio); ③ 1 nM ATRA; ④ 2.5 nM ATRA; ⑤ 5 nM ATRA; ⑥ 10 nM ATRA; ⑦ 20 nM ATRA; ⑧ 100 nM ATRA; and ⑨ 1 μM ATRA. The grouping and number of fertilized eggs are shown in Table 1. Zebrafish were cultured in a 28.5°C incubator. Embryonic development was observed under a stereomicroscope every 24 hours. Surviving and dead fertilized eggs were photographed and recorded, as were neural tube malformations at different time points (24 dpf, 48 dpf, 72 dpf, and 96 dpf). Malformation rates were calculated. Normal zebrafish embryos and deformed zebrafish embryos were fixed in 4% paraformaldehyde, and then the zebrafish embryos were embedded in paraffin for tissue sectioning.

[0035] Paraffin embedding and tissue sectioning of zebrafish embryos: (1) Sampling and fixation: Zebrafish embryos were fixed in 4% paraformaldehyde (PFA). (2) Dehydration: The zebrafish embryos were dehydrated in a gradient of alcohol. The basic process is as follows: 50% ethanol, 10 min → 70% ethanol, 10 min → 95% ethanol 1, 10 min → 95% ethanol 2, 10 min → anhydrous ethanol, 10 min → anhydrous ethanol, 10 min. (3) Transparency: The dehydrated zebrafish embryos were placed in xylene for tissue transparency. The basic process is as follows: 50% anhydrous ethanol + 50% xylene, 5 min → xylene 1, 5 min → xylene 2, 5 min → xylene 3, 5 min. (4) Wax immersion: The paraffin was melted and the temperature was maintained at about 55°C. The wax immersion time was 1h-2h. (5) Embedding: The wax-soaked zebrafish embryos were placed in a mold containing wax liquid and embedded on an embedding machine. (6) Sectioning: Place the trimmed wax block on a paraffin slicer and slice. Adjust the angle and position of the wax block. (7) Spreading: Use a brush to carefully transfer the sliced ​​fragments to clean water on a 38-40°C slide table. After a few minutes, the slices are completely wrinkle-free. Select slices with intact tissue shape and no knife marks for slicing. (8) Baking: Place the slices in a 60°C oven and bake them. Then perform HE staining.

[0036] HE staining: (1) Dewaxing: Dewaxing the baked embryo sections, xylene I, 5 min-10 min → xylene II, 5 min-10 min → xylene III, 5 min-10 min. (2) Over-watering: 100% ethanol I, 5 min-10 min → 100% ethanol II, 5 min-10 min → 95% ethanol I, 5 min-10 min → 95% ethanol II, 5 min-10 min → 75% ethanol, 5 min-10 min, rinse with running water for 10 min. (3) Hematoxylin staining for 1 min, rinse with running water. (4) Acid differentiation for 30 s, rinse with running water. (5) Eosin staining for 1 min, rinse with running water. (6) Dehydration: 95% ethanol II, 5 min → 95% ethanol I, 5 min → 100% ethanol II, 5 min → 100% ethanol I, 5 min. (7) Xylene I, 5 min → Xylene II, 5 min → Xylene III, 5 min. (8) Apply neutral gum and seal the slide. Use a microscope to take photos and observe the development of the zebrafish embryonic neural tube.

[0037] 4. Experimental results.

[0038] As attached Figure 1 The images show morphological observations of zebrafish larvae. The images show that zebrafish embryos treated with all-trans retinoic acid are smaller and exhibit overall developmental delay. The embryonic zebrafish brain is malformed, with smaller ventricles, thinner brain parenchyma, and disorganized nerve cell arrangement. The embryonic zebrafish spine is curved, the spinal cord is malformed, the notochord is narrowed, and the nerve cells within the spinal cord are disorganized.

[0039] Attachment Figure 2 Images of normal zebrafish larvae and zebrafish larvae with NTDs induced by all-trans retinoic acid (ATRA) at different time points. The development of zebrafish embryos was observed using a stereomicroscope at 24, 48, 72, and 96 dpf. The images show abnormal neural tube development in ATRA-induced zebrafish embryos, including reduced size, developmental delay, brain malformations, spinal curvature, and spinal cord abnormalities.

[0040] Table 1 shows the effects of various concentrations of all-trans retinoic acid (ATRA) on neural tube defects in zebrafish embryos. As shown in Table 1, high ATRA concentrations of 1 μM, 100 nM, and 20 nM resulted in high mortality and model failure. Lowering the ATRA dose range to 1–20 nM resulted in a high model success rate. At 2.5 nM, the ATRA dose produced the highest neural tube defect rate and a relatively low percentage of resorbed embryos, representing the optimal modeling dose.

[0041] Table 1 is a summary of the neural tube defects induced by different concentrations of all-trans retinoic acid (ATRA) in zebrafish embryos

[0042] .

[0043] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for constructing a zebrafish neural tube defect model, as follows: S1: Select 4-month-old zebrafish and place male and female adult zebrafish in a breeding tank according to the ratio. Separate them with a partition. The next day, remove the partition to allow the male and female fish to mate. S2: Use a filter to collect the fertilized eggs at the bottom of the outer tank of the breeding tank and wash the eggs with breeding water 2 to 3 times; S3: After the fertilized eggs were sorted and washed, the eggs collected in S2 were placed in a culture dish using a pipette, and all-trans retinoic acid was added. After culturing for 24 hours, the culture water was changed to normal culture to obtain a zebrafish model of neural tube defects.

2. The model building method according to claim 1, characterized in that The ratio of female to male adult zebrafish described in S1 was 1:1 or 2:1 for female to male.

3. The model building method according to claim 1, characterized in that The all-trans retinoic acid described in S3 was dissolved in dimethyl sulfoxide, and the same concentration of dimethyl sulfoxide was used as a control group. The dosage of all-trans retinoic acid was 1-20 nM.

4. Use of the neural tube defect model prepared by the method for constructing a zebrafish neural tube defect model according to any one of claims 1 to 3 in direct in vitro observation of the neural tube development process.

5. Use of the neural tube defect model prepared by the method for constructing a zebrafish neural tube defect model according to any one of claims 1 to 3 in studying the mechanism and treatment of neural tube defects.

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