A vital role for glycosphingolipid synthesis during development and differentiation

^*Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; and ^†Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Gerhard-Domagk-Strasse 1, 53121, Bonn, Germany

Edited by Stuart A. Korneld, Washington University School of Medicine, St. Louis, MO, and approved June 3, 1999 (received for review March 3, 1999)

Abstract

Glycosphingolipids (GSLs) are believed to be integral for the dynamics of many cell membrane events, including cellular interactions, signaling, and trafficking. We have investigated their roles in development and differentiation by eliminating the major synthesis pathway of GSLs through targeted disruption of the Ugcg gene encoding glucosylceramide synthase. In the absence of GSL synthesis, embryogenesis proceeded well into gastrulation with differentiation into primitive germ layers and patterning of the embryo but was abruptly halted by a major apoptotic process. In vivo, embryonic stem cells deficient in GSL synthesis were again able to differentiate into endodermal, mesodermal, and ectodermal derivatives but were strikingly deficient in their ability to form well differentiated tissues. In vitro, however, hematopoietic and neuronal differentiation could be induced. The results demonstrate that the synthesis of GSL structures is essential for embryonic development and for the differentiation of some tissues and support the concept that GSLs are involved in crucial cell interactions mediating these processes.

Footnotes

↵ ‡ To whom reprint requests should be addressed at Building 10, Room 9D-20, 10 Center Drive, MSC 1810, National Institutes of Health, Bethesda, MD 20892. E-mail: proia{at}nih.gov.
This paper was submitted directly (Track II) to the Proceedings Office.
↵ § Ganglioside nomenclature used in this paper is that of Svennerholm (1).
ABBREVIATIONS:

E,

embryonic day;

ES cells,

embryonic stem cells;

GSL,

glycosphingolipid;

kb,

kilobase;

H & E,

hematoxylin and eosin;

PCNA,

proliferating cell nuclear antigen;

TUNEL,

terminal deoxynucleotidyltransferase-mediated UTP end labeling;

HPTLC,

high-performance thin-layer chromatography;

C6-NBD-ceramide,

6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine