Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Summary

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

Abstract

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

Introduction

Preimplantation embryonic development can be divided into several distinct stages, from the one-cell stage to the morula and the blastocyst. Much evidence suggests that polarity and positional cues play a role in early cell fate determination into the trophectoderm (TE) and the inner cell mass (ICM). However, the nature of the cues, how they are transduced into cellular signals, and the physiological contexts in which such signals are capable of initiating cell lineage differentiation are not known. These differentiated cells then undergo specialization, beginning to take on characteristic structures and functions needed for embryo proper formation and growth of the p....

Protocol

NOTE: All procedures for animal breeding and cares were conducted according to the IACUC regulations of Sahmyook University, Seoul, S. Korea.

1. Superovulation, Breeding and Oviduct Isolation

1. Maintain inbred C57BL/6 mice under the following conditions: 22 ± 3 °C, ~60% humidity, 12 hr light/dark cycle, and water and food ad libitum.
2. Induce superovulation of 3-5 week-old female mice by an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG) at 0.1 ml/head followed 48 hr later by an intraperitoneal injection of 5 IU human chorionic gonadotropin (hCG) at 0.1 ml/head.
3. Immediately following hCG injec....

Results

We discovered that neogenin is transiently expressed during the early developmental stages of preimplantation mouse embryos, appearing as early as at the 2-cell stage and lasting until the early morula but becoming deficient at the late morula and blastocyst stages (Figure 2A). In addition, the spatial distribution of neogenin was restricted mainly to outside cells. The results of RT-PCR analysis were consistent with the early and transient nature of neogenin expression, .......

Discussion

In the present protocol, we demonstrate novel methods of microinjection of genetic materials, either cDNA or shRNA, into the 2-PN mouse embryos to explore the role of neogenin in early cell differentiation during mouse embryogenesis. Genetic modification of preimplantation embryos is a powerful technique in uncovering key information about underlying molecular mechanisms for, for example, the first cell lineage determination. Genetic modification is one of the most commonly used methods for ascertaining gene function. Wi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
M16 medium,	Gibco BRL (Grand Island, NY)	M7292 LOT# 11A832
Goat serum,	Dako (Glostrup, Denmark)	X0907
PMSG	Folligon, Intervet, Holland	G4877-1000IU LOT#SLBD0719V
Mineral oil	Sigma Aldrich	CG5-1VL LOT# SLBC6783V
human chorionic gonadotropin and pregnant mare serum gonadotropin	(Intervet, Holland)	(invitrogen , 15596-026)
SuperScript® III Reverse Transcriptase	lifetechnologies	18080-044
PCR pre mixture	(Bioneer, Daejon, S. Korea)	GenDEPOT , A0224-050
Agarose (molecular grade)	BioRad	161-3101
Paraformaldehyde Solution, 4% in PBS	Affymetrix USA	19943
polyclonal rabbit anti-neogenin antibody	Santa Cruz Biotechnology, US	SC-15337
Alexa fluor 488 labeled anti-rabbit antibody	Molecular Probes (Invitrogen, USA)	A-11094
Alexa fluor 555 labeled anti-rabbit antibody	Molecular Probes (Invitrogen, USA)	A-21428
Alexa Fluor® 555 Phalloidin	Molecular Probes (Invitrogen, USA)	A34055
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Invitrogen, USA	D1306
Recombinant Human RGM-C/Hemojuvelin	R&D systems	3720-RG
all plasmid constructs	Prof. Wen Cheng Xiong at Georgia Health Sciences University (Agusta, GA).		for the present studies were kindly provided by
Neon®  Transfection System	lifetech	MPK10096
Stereo Microscrope	Nikon	SMZ1000
Micromainpulation system with Nikon	Nikon	Narishige ONM-1
Micro Injector	Nikon Narishige	GASTIGHT #1750
Holder	Nikon Narishige	Narishige IM 16
Microfuge	Nikon Narishige	Narishige MF-900
grinder	Narishige	EG400
Puller	Sutter Instrumnet company	P-97
CO2 incubator	Thermo Scientific Forma	EW-39320-16
Veriti® 384-Well Thermal Cycler	lifetechnologies	4388444