This work was supported by the USDA&#160;National Institute of Food and Agriculture, NRS Project No. FLA-TRC-006176 and the University of Florida Institute of Food and Agricultural Sciences.

1. Planting and pollination
NOTE: It is important to identify compatible genotypes whose hybridization would result in fruit set&#160;and the production of viable embryos.
<ol>
	<li>Planting conditions and maintenance
	<ol>
		<li>Obtain seeds of squash genotypes (cultivars/accessions) for hybridization (Table 1).</li>
		<li>Fill 50 cell starting flats (25 cm width x 50 cm length) with potting medium amended with complete NPK fertilizer containing 1.38 g/kg N, 1.38 g/kg P, and 1.38 g/kg K.</li>
		<li>Sow seeds to a depth equal to their length and cover with potting medium. Water the flats without creating standing water. Afterward, keep the media moist by watering it by hand once per day.</li>
		<li>At the second true-leaf stage, transplant the seedlings into 25 cm to 30 cm diameter pots amended with a complete NPK fertilizer at 3 tablespoons/pot. Fertilize the plants once a week with 500 mL/pot of liquid fertilizer containing NPK 20:20:20 added to a concentration of 1 g per gallon of water.</li>
		<li>Maintain plants in the greenhouse at temperatures between 22-28 &#176;C under natural light regime. For vining genotypes, provide a supporting trellis in the greenhouse (Figure 1).</li>
	</ol>
	</li>
	<li>Performing pollinations
	<ol>
		<li>Typically, flowering in squash starts at 6 to 8 weeks from seeding depending upon the cultivar (Figure 2A,B). Begin making controlled hybridization (crosses) as soon as the plants start flowering. Under greenhouse conditions, pollination can be done year-round.</li>
		<li>Identify male and female flowers of C. pepo and C. moschata cultivars that will be ready for pollination the following day. To identify such flowers, check for flowers whose petals have a yellow hue but are not open. Gently tape the flowers closed at the top using masking tape to prevent accidental insect pollination (Figure 2C,D).</li>
		<li>The morning of the following day, the flowers are ready to pollinate. Carry out pollination before 10:00 a.m. to improve the rate of hybridization success27.</li>
		<li>Open the female and male flowers by gently removing the taped top portion of the petals. Remove the petals from the male flower and transfer the pollen by gently rubbing the anther on the stigma of the female flower (Figure 3A).</li>
		<li>After pollination, immediately close the pollinated female flower with masking tape. Use a tag to record the date of pollination and to indicate the paternal and maternal parents used in the cross (Figure 3B).</li>
		<li>A successful cross is indicated by an expanded ovary which quickly forms a small fruit within 1 week (Figure 4A). The plant is then ready for harvest 45-55 days after pollination28 (Figure 4B).</li>
	</ol>
	</li>
</ol>
2. Embryo rescue technique
<ol>
	<li>Media preparation
	<ol>
		<li>Prepare antibiotic stocks: For cefotaxime (sodium salt), dissolve the antibiotic in 4 mL of deionized or distilled water, filter through a sterile 0.22 &#181;m syringe filter, and make 0.5 mL aliquots. Store&#160;at -20&#160;&#176;C. The resulting stock solution will have a concentration of 250 mg/mL.</li>
		<li>For ampicillin (sodium salt), dissolve the powder in 10 mL of water, filter through a sterile 0.22 &#181;m syringe filter, and aliquot in 1 mL stock with a final concentration of 100 mg/mL. Store&#160;at -20&#160;&#176;C.</li>
		<li>Make Murashige and Skoog (MS) medium by dissolving 2.45 g of medium (4.91&#160;g/L concentration) in 500 mL of distilled water in a 1 L bottle. Add 1.5 g of gellan gum&#160;and autoclave at 121 &#176;C for 20 min. The gellan gum completely dissolves during autoclaving.</li>
		<li>After autoclaving, cool the medium by placing the bottle in a water bath at 50 &#176;C. Remove the antibiotic stocks from the freezer and thaw them in the laminar flow cabinet.</li>
		<li>Transfer the medium bottle to the laminar flow hood. Add 0.6 mL of cefotaxime stock solution (250 mg/mL)&#160;and 0.25 mL of ampicillin stock (100 mg/mL) to the medium bottle and mix well. Pour about 7 mL of the medium into a sterile Petri dish (60 mm x 15 mm).</li>
		<li>Allow the medium to solidify in the Petri dishes for approximately 15-20 min. Close the Petri dishes with sealing wrap and place them into a storage box. Store at room temperature.</li>
	</ol>
	</li>
	<li>Embryo rescue
	<ol>
		<li>Before starting, clean and sterilize the laminar-air-flow cabinet with 70% ethyl alcohol.</li>
		<li>Harvest the squash fruit by breaking/cutting it off the main vine and disinfect the fruit surface by washing with liquid detergent (e.g., 0.3% chloroxylenol) in the lab sink until all the loose dirt is removed (Figure 5A).</li>
		<li>Rinse with ample tap water. Dry the fruit with clean paper towels. Move the fruit to the laminar-air-flow cabinet (Figure 5B).</li>
		<li>Surface sterilize the fruit by spraying 70% ethanol on the fruit in the sterile laminar-air-flow cabinet. Bisect the fruit with a sterile knife (Figure 5C) and extract the seeds.</li>
		<li>Use sterile forceps to aseptically open the seed coat and expose the immature embryos (Figure 6). Carefully place the immature embryos in a Petri dish containing MS medium supplemented with cefotaxime and ampicillin (Figure 7A). Close the Petri dish and seal with wrapping film. 
		NOTE: Depending on the size of the embryo, it is possible to fit five or six embryos in a Petri dish.</li>
		<li>Place the sealed Petri dishes with embryos in a growth chamber under 16 h photoperiod at 25 &#176;C and 70% relative humidity. If contamination occurs, promptly subculture the uncontaminated embryos into a new plate with the same medium.</li>
		<li>The cotyledons will start expanding after 4 days and will turn green in 10 days (Figure 7B). At this point, if needed, perform subculturing into new plates containing the same medium to allow tissue expansion. The roots will start to appear at 14 days (Figure 7C), and at 21 days the plantlets will have extended roots and cotyledons (Figure 7D). 
		NOTE: The culture medium used in the protocol is appropriate for differentiation into shoots and roots without supplementary growth regulators.</li>
		<li>At this stage, remove the plantlets from the&#160;Petri dishes and gently wash off the media from the roots with tap water (Figure 8). Place the plantlets in a plastic container (14 cm x 9 cm x 4 cm) and cover the roots with wet paper towels (Figure 9A). Cover the container and remoisten the paper towels as necessary.</li>
		<li>Keep the containers at room temperature (25-28 &#176;C) and with a 16 h photoperiod. This step will acclimatize the plantlets. After acclimatization for 7-10 days in the container, the seedlings will be approximately 3-4 in long. During this period, remoisten the paper towels as needed.</li>
		<li>Transfer the seedlings into 50 cell starting flats (25 cm width x 50 cm length) amended with fertilizer as previously described and move them to the greenhouse (Figure 9B). Do not over-water seedlings to avoid rotting; add approximately 10-20 mL per cell as needed.</li>
		<li>At the second to third true-leaf stage, transplant the seedlings into 30 cm diameter pots filled with potting medium amended with fertilizer as previously described (Figure 10A). Provide trellis support for vining plants and perform controlled hybridization when the plants start flowering, as previously described (Figure 10B).</li>
		<li>Maintain the plants in the greenhouse at temperatures between 22-28 &#176;C under natural light regime. Evaluate the plants for fruit and seed characteristics.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

There are two main bottlenecks for successful interspecific hybridization between C. moschata and C. pepo: cross-compatibility barrier, which is determined by genotype responsiveness to produce hybrid embryos, and post-fertilization barriers, which hinder the development of hybrid embryos to normal seeds. As previously reported for squash, the cross-compatibility test in the current study revealed that most of the fruit developed parthenocarpically, with most of the seeds unviable16</s...

Cucurbita (2n = 40) is a highly diverse genus in the Cucurbitaceae family that contains 27 different species, of which five are domesticated1. Among these, Cucurbita moschata, C. pepo, and C. maxima are the most economically important worldwide. In the U.S., C. moschata and C. pepo are the two most important species in agricultural production. C. pepo consists of four subspecies (ovifera, pepo, fraternal, and gumala) that contain both summer and winter squash cultivar groups of crookneck, straightneck, acorn, scallop, cocozelle, vegetable marrow, zucchini, and pumpkin2,3,4,5. C. moschata primarily consists of winter squash market types including butternut, Dickinson, and cheese group1. The two species are morphologically and phenotypically diverse, with C. pepo regarded for its yield, earliness, bush growth habit, and diverse fruit traits including fruit shape, fruit size, flesh color, and rind pattern. On the other hand, C. moschata is prized for its adaptation to heat and humidity, as well as disease and pest resistance6,7. Interspecific hybridization between C. moschata and C. pepo is not only an important strategy for introgression of desirable characteristics between the two species, but also allows for the broadening of the genetic base in breeding programs7,8.
Early crosses between C. moschata and C. pepo were made to determine their compatibility and/or taxonomic barriers9,10,11, whereas later studies mostly focused on transferring desirable traits12,13,14. Interspecific hybridization between the two species has targeted the transfer of novel traits such as a bush or semi-bush growth habit and improved yield from C. pepo along with disease resistance, adaptability to abiotic stress, and increased vigor from C. moschata14,15,16. For example, specific crosses between C. pepo (P5) and C. moschata (MO3) have resulted in higher fruit yield13, while C. moschata accessions (Nigerian Local and Menina) have been widely used as the primary source of resistance to potyviruses in cultivated C. pepo cultivars17,18.
Previous studies showed that hybridization between C. moschata and C. pepo is possible but difficult8,15. The interspecific crosses could result in no fruit set (abortion), parthenocarpic fruits devoid of viable seeds (empty seeds), seedless fruits where the immature embryos fail to develop (stenospermocarpy), or fruits with few immature embryos that can be rescued into mature plants through embryo rescue15,16. For instance, no viable seeds were obtained by crossing C. pepo (table queen, maternal) with C. moschata (large cheese, paternal), however, the reciprocal cross yielded 57 viable seeds from 134 pollinations9. Hayase obtained viable seeds from C. moschata and C. pepo crosses only when crosses were made at 04:00 a.m. using pollen stored at 10 &#176;C overnight19. Baggett crossed eight different C. moschata varieties with C. pepo (delicata) and reported that out of 103 total pollinations, 83 fruits were obtained that appeared normal, but none of them contained viable seeds8. In a cross between C. pepo (S179) and C. moschata (NK), Zhang et al. obtained 15 fruits with 2,994 seeds, but only 12 of those seeds were viable while the remaining displayed only rudimentary development. These studies suggest that even though interspecific crossing between C. moschata and C. pepo is highly beneficial, obtaining fruits with viable seeds from the crosses is demanding16.
Embryo rescue has been suggested as an appropriate method to overcome problems arising from early aborting or poorly developed embryos and is one of the earliest and most successful in vitro culture techniques for regeneration of immature embryos16,20. Embryo rescue involves the in vitro culture of under-developed/immature embryos followed by transfer to a sterile nutrient medium to facilitate recovery of seedlings and ultimately mature plants21. Although embryo rescue is commonly used in squash breeding, a detailed description of the appropriate methodology that would allow its routine application is lacking. Using embryo rescue technique to overcome interspecific hybridization barriers in Cucurbita species was reported as early as 195422. However, the success of embryo rescue in the early studies was either unreported or very low. Metwally et al. reported a 10% success rate (regeneration into mature plants) among 100 interspecific hybrid embryos rescued from a cross between C. pepo and C. martinezii23. Sisko et al. reported a variable success rate of embryo regeneration among embryos obtained from different cross combinations: the regeneration rate of hybrids obtained by crossing C. maxima (Bos. Max)and C. pepo (Gold Rush) was 15.5%, for C. pepo (Zucchini) and C. moschata (Hokaido) was 20%, while for C. pepo (Gold Rush) and C. moschata (Dolga) it was 37.5%24. In addition to genotype, media and in vitro culture conditions are important factors for the success of the technique25,26. In the current study, various cross combinations between C. moschata and C. pepo were&#160;tested, and a simple methodology for utilizing the embryo rescue technique in squash was developed. The development of a simple and easily reproducible embryo rescue technique will facilitate interspecific hybridization and germplasm enhancement in squash breeding programs.

<table><tbody><tr><td>ampicillin</td><td>Fisher Scientific</td><td>BP1760-5</td><td></td></tr><tr><td>autoclave</td><td>Steris</td><td>AMSCO LAB 250</td><td></td></tr><tr><td>balance</td><td></td><td></td><td></td></tr><tr><td>cefotaxime</td><td>Sigma Alfrich</td><td>C 7039</td><td></td></tr><tr><td>centrifuge tubes (1.5 ml)</td><td>Sigma Alfrich</td><td>T9661</td><td></td></tr><tr><td>detergent</td><td></td><td></td><td></td></tr><tr><td>ethanol, 95%</td><td>Decon Labs</td><td>2805HC</td><td></td></tr><tr><td>forceps</td><td>VWR</td><td>82027-408</td><td></td></tr><tr><td>gellan gum</td><td>Caisson Laboratories</td><td>G024</td><td></td></tr><tr><td>growth chamber or illuminated shelf</td><td></td><td></td><td></td></tr><tr><td>laminar hood / biosafety cabinet</td><td>The Baker Company, Inc</td><td>Edgegard</td><td></td></tr><tr><td>masking tape</td><td>Uline</td><td>S-11735</td><td></td></tr><tr><td>media bottle</td><td></td><td></td><td></td></tr><tr><td>Murashige &amp;amp; Skoog Medium</td><td>Research Products International</td><td>M10200</td><td></td></tr><tr><td>NPK fertilizer (20-20-20)</td><td>BWI Companies, Inc</td><td>&amp;nbsp;PR200</td><td></td></tr><tr><td>Osmocote Plus fertilizer</td><td>BWI Companie,s Inc</td><td>OS90590</td><td></td></tr><tr><td>Parafilm M</td><td>Sigma Alfrich</td><td>P7793</td><td></td></tr><tr><td>Petri dish (60 x 15 mm)</td><td>USA Scientific, Inc</td><td>8609-0160</td><td></td></tr><tr><td>plant pots</td><td>BWI Companies, Inc</td><td>NP4000BXL</td><td></td></tr><tr><td>plastic food containers, reused</td><td>Oscar Mayer</td><td>4470003330</td><td></td></tr><tr><td>plastic hang tags</td><td>Amazon</td><td>B07QTZRY6T</td><td></td></tr><tr><td>potting mix</td><td>Jolly Gardener</td><td>Pro-Line C/B</td><td></td></tr><tr><td>seedling starter trays</td><td>BWI Companies Inc</td><td>GPPF128S4</td><td></td></tr><tr><td>syringe filter (0.22 um )</td><td>ExtraGene</td><td>B25CA022-S</td><td></td></tr><tr><td>trellis support</td><td>The Home Depot</td><td>&amp;nbsp;2A060006</td><td></td></tr><tr><td>water bath</td><td></td><td></td><td></td></tr></tbody></table>

embryo rescue protocol for interspecific hybridization in squash

Interspecific hybridization in Cucurbita crops (squash) is desirable for widening genetic variation and for the introgression of useful alleles. Immature embryos generated from these wide crosses must be regenerated using appropriate embryo rescue techniques. Although this technique is well established for many crops, a detailed description of the appropriate methodology for squash that would allow its routine application is lacking. Here, we describe an embryo rescue protocol useful for interspecific hybridization of C. pepo and C. moschata. To identify viable combinations for embryo rescue, 24 interspecific crosses were performed. Fruit set was obtained from twenty-two crosses, indicating a 92% success rate. However, most of the fruits obtained were parthenocarpic, with seeds devoid of embryos (empty seeds). Only one cross combination contained immature embryos that could be regenerated using basal plant growth media. A total of 10 embryos were rescued from the interspecific F1 fruit, and the success rate of embryo rescue was 80%. The embryo rescue protocol developed here will be useful for interspecific hybridization in squash breeding programs.

Fruit set and seed viability 
An initial test was conducted to determine fruit set and seed viability in a variety of cross-combinations. A total of 15 squash genotypes, four C. pepo and 11 C. moschata, were chosen (Table 1). Out of the 24 interspecific cross combinations attempted, a fruit set was obtained for 22 (Table 2), representing an overall &#62;92% success in the fruit set. No mature fruits were obtained by crossing O and M and E and J, wh...

Watch this Scientific Journal Video about Embryo Rescue Protocol for Interspecific Hybridization in Squash at JoVE.com

Embryo Rescue Protocol for Interspecific Hybridization in Squash

The&#160;article describes an embryo rescue protocol for the regeneration of immature embryos derived from the interspecific hybridization of Cucurbita pepo and Cucurbita moschata. The protocol can be easily replicated and will be an important resource for squash breeding programs.

Interspecific hybridization in Cucurbita crops (squash) is desirable for widening genetic variation and for the introgression of useful alleles. Immature ...

embryo-rescue-protocol-for-interspecific-hybridization-in-squash

Research

JoVE Journal

Biology

2.6K Views.  UF/IFAS Tropical Research and Education Center. The article describes an embryo rescue protocol for the regeneration of immature embryos derived from the interspecific hybridization of Cucurbita pepo and Cucurbita moschata. The protocol can be easily replicated and will be an important resource for squash breeding programs.

Video: Embryo Rescue Protocol for Interspecific Hybridization in Squash

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

Developmental Biology

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Neuroscience

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.

This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques.

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as ...

Genetics

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Embryo Rescue Protocol for Interspecific Hybridization in Squash

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Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

Double Whole Mount in situ Hybridization of Early Chick Embryos

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Generating Chimeric Zebrafish Embryos by Transplantation

Wholemount In Situ Hybridization for Astyanax Embryos

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function