How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells

Imadeldin Yahya,Gabriela Morosan-Puopolo,Beate Brand-Saberi,Marion Böing

doi:10.1007/s00418-020-01920-7

Imadeldin Yahya, Gabriela Morosan-Puopolo + Show 2 more

Open Access

https://doi.org/10.1007/s00418-020-01920-7

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Abstract

Cell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18–HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

Highlights

The neural crest is a migratory embryonic cell population that gives rise to numerous structures in vertebrates (Bronner 2012)
Sox10 is detected in migrating neural crest cells (NCCs) shortly after Slug, but is diminished as cells undergo neuronal differentiation in ganglia of the peripheral nervous system (Cheng et al 2000)
Double whole-mount in situ hybridization (ISH) is used to study the pattern of gene expression in mesodermal and neural crest cells

Summary

Introduction

The neural crest is a migratory embryonic cell population that gives rise to numerous structures in vertebrates (Bronner 2012). In the trunk, these cells migrate to form amongst others sensory and sympathetic ganglia and adrenal medulla (Aoki et al 2003; Bronner 2012; Cheng et al 2000). The transcription factor activating protein (Ap2α) has been identified as the regulatory factor for face and limb development (Shen et al 1997). Chicken Ap2α is expressed in the distal limb bud mesenchyme and its expression is associated with limb bud outgrowth (Shen et al 1997)

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