Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

Summary

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel’s cartilage.

Abstract

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel's cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel’s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel’s cartilage.

Multispectral cell labeling enables scientists to demonstrate extension of the Meckel’s chondrocytes. During extension phase of the Meckel’s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Introduction

Craniofacial development requires complex molecular, cellular and tissue interactions to drive cell proliferation, migration and differentiation^1,2,3. This tightly regulated and complex process is subject to genetic and environmental perturbations, such that craniofacial deformities are amongst the most common birth malformations^1-9. While surgical interventions remain the mainstay of treatment for craniofacial anomalies, understanding the development basis is essential to innovate future therapies. Therefore, studying the morphogenesis and the mechanisms in the convergence and extension and cell integration provides novel i....

Protocol

Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) approved all procedures under protocol number #2010N000106. This is in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines.

1. Reagents and Materials Preparation

1. Prepare 1 L of 50X E3 embryo medium (See Table 1) and prepare 1 L of 1X E3 embryo medium (See Table 2).
2. Prepare E3 embryo medium with N-Phenylthiourea (PTU) by adding 30 mg of PTU to 1 L of 1X E3. Stir O/N to ensure that the PTU has completely dissolved. Store at RT.
....

Results

Traditional cartilage visualization by whole mount Alcian blue stains has been invaluable in observing the developing Meckel’s cartilage and commonly used to visualize final cellular organization¹² (Figure 1A). To further analyze the developing chondrocytes overtime, lineage tracing using sox10:Kaede transgenic lines has enabled us to study cell migration, convergence and extension in live embryos^2,12(Figure 1B). However, the organization of chondrocytes in.......

Discussion

Alcian blue and photoconvertible transgenic lines as described above complements each other to define the intricate process of cartilage and bone development. However, live cellular migration and organization during organogenesis has long been hypothesized and indirectly demonstrated but never visualized. Zebrabow-M transgenic line coupled with a cartilage specific Cre permits simultaneous live observation of all these distinct events involved in bone and cartilage formation. This technique allows the study of lower jaw .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pronase	Roche Life Sciences	10165921001	Prepare 500 μl stock aliquots at 50 mg/ml
Methylcellulose	Sigma-Aldrich	M0262
PTU (N-Phenylthiourea)	Sigma-Aldrich	P7619
Tricaine	Sigma-Aldrich	E10521
4-HydoxyTamoxifen	Sigma-Aldrich	T176
24 x 60 coverslips	Fisher Scientific	12-548-5P
18 x 18 coverslips	Fisher Scientific	12-540A
25 x 25 coverslips	Fisher Scientific	12-540C
pENTR5'-TOPO TA Cloning Kit	Life technologies	K591-20
pENTR/D-TOPO Cloning Kit	Life Technologies	K2400-20
pENTR3'-pA	Tol2Kit	302
pDEST			Gift from Geoffrey Burns labs
Bright field microscope
Fluorescent microscope
Confocal microscope
Image processing software