Creating Avian Forebrain Chimeras to Assess Facial Development

Podsumowanie

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain during craniofacial development.

Streszczenie

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

Wprowadzenie

Much contemporary research in developmental biology focuses on the role of genes in shaping embryos. There are good tools to examine developmental mechanisms from a genetic perspective. However, embryos are assembled and undergo morphogenesis in response to tissue interactions. The avian system is a classic tool used to assess the variety of tissue interactions that regulate development for the following reasons: the embryology is well-understood, the embryos are easily accessible, tools for analysis of avian systems are well-developed, and the embryos are inexpensive.

The avian transplantation system has been widely employed for lineage tracing and to assess tissue interactions during development for almost a century^1,2,3,4. This system was used to investigate a signaling center, the Frontonasal Ectodermal Zone (FEZ), that regulates morphogenesis of the upper jaw⁵, and a video was published describing that technique previously⁶. In addition to quail-chick, other species have also been used to produce chimeras for analysis of tissue interactions. For example, the mouse FEZ was transplanted from wild type⁷ and mutant mice⁸, and others have used a duck, quail and chick systems to assess the role of neural crest in patterning the facial skeleton^9,10,11,12.

In this work, the role of the forebrain in regulating the pattern of gene expression in the FEZ by transplanting the ventral forebrain reciprocally among quail, duck, and chick embryos was assessed, because a signal from the forebrain is required to induce Sonic hedgehog expression in the FEZ. Forebrain transplants are not unique in the field. These transplants were used to assess development of motility in quail and duck embryos¹³, although in these experiments tissues that contributed to non-neural derivatives were also transplanted. In other work, auditory circuits in birds have been assessed by forebrain transplantation¹⁴, but these transplants contained presumptive neural crest cells, which contribute to facial shape^9,10 and participate in regulating SHH expression in the FEZ¹⁵. Hence, a system to transplant just the ventral forebrain from one species of bird to another prior to closure of the neural tube was devised to assess the role of the brain in facial shape¹⁶ (Figure 1A,B). This method was devoid of neural crest contamination of the graft. In this article, the method is illustrated and the expected results are described, and the challenges faced are discussed.

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Protokół

White Pekin duck (Anas platyrhynchos), white Leghorn chicken (Gallus gallus) and Japanese quail (Cortunix coturnix japonica) are incubated at 37 °C in a humidified chamber until stage-matched at HH7/8¹⁷.

1. Preparing the donor tissue

NOTE: Preparation of reagents and tools and how to open eggs for experimental manipulation has been described⁶.

1. Prepare DMEM media with neutral red, a glass transfer pipette, and sharpened tungsten needles.
2. Expose embryos (as shown in⁶).
3. Harvest tissue grafts from the left side of basal forebrain of stage 7/8 embryos. Use curved sharpened tungsten needles⁶ to gently incise a piece of the forebrain measuring ~0.3 mm in length by 0.2 mm in width, making sure to not include the underlying endoderm by sliding the needle beneath the forebrain so that the needle is parallel to the axis of the neural tube.
4. Using the glass transfer pipette, pick the graft up from the donor embryo.
5. Transfer the graft into DMEM containing Neutral Red (0.01% in PBS, 23 °C) for 2 minutes to stain it, and then place the stained graft into DMEM that does not contain Neutral Red until ready for engraftment.

2. Preparing the host

1. Incubate fertilized eggs from white Leghorn chicken (Gallus gallus) at 37 °C in a humidified chamber until HH7/8 ¹⁷.
2. Expose embryos (as shown in⁶).
3. Using sharpened tungsten needles, prepare the graft site by gently cutting, then removing a 0.3 mm by 0.2 mm piece of basal forebrain from the left side to accommodate the graft as was done to isolate the donor tissue.
4. Take care to avoid excessive disruption of the underlying endoderm, which will be evident as yolk granules will begin to leak through any tear that is made. This takes practice and not all attempts will be successful.
5. After transferring to the host⁶, position the graft to replace the extirpated basal forebrain of the host.
6. Place tape tightly over the hole and return the embryo to the 37 °C incubator for the appropriate length of time for analysis.

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Wyniki

Assessment of Chimerism and Transplant Contamination
In order to assess the chimeras, the extent of chimerism and contamination of the graft with other cell types should be addressed. Creating chimeras by transplanting quail tissues into chick embryos allows for this type of analysis. Using the QCPN antibody quail cells can be visualized and distinguished from the host tissues (Figure 1 C,D). In this case, only tissues derived from the ventral forebrai...

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Dyskusje

The method described allows examination of the tissue interactions between the basal forebrain and the adjacent ectoderm. This approach differs from previous forebrain transplant methods, because the donor tissue was restricted to the ventral forebrain. This eliminates transplantation of the neural crest cells, which have been shown to participate in patterning facial morphology^9,10. Hence, restricting the graft to the basal forebrain was essential to evaluate th...

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Materiały

Name	Company	Catalog Number	Comments
1x PBS	TEK	TEKZR114
35x10 mm Petri dish	Falcon	1008
DMEM	Thermofisher	11965084
Needle holder	Fine Science Tools	26016-12
Neutral Red	Sigma	553-24-2
No. 5 Dumont forceps	Fine Science Tools	11252-20
Pasteur Pipets	Thermofisher	13-678-6B
QCPN antibody	Developmental Studies Hybridoma bank, Iowa University, Iowa, USA
Scissors	Fine Science Tools	14058-11
Tungsten Needle	Fine Science Tools	26000