Fatty acid synthesis is essential in embryonic development: fatty acid synthase null mutants and most of the heterozygotes die in utero

Abstract

In animals, including humans, the source of long-chain saturated fatty acids is de novo synthesis, which is mediated by fatty acid synthase (FAS), ingested food, or both. To understand the importance of de novo fatty acid synthesis, we generated FAS knockout mice. The heterozygous FAS mutants (Fasn+/-) are ostensibly normal. In Fasn+/- mice the levels of FAS mRNA and the FAS activity are approximately 50% and 35% lower, respectively, than those of WT mice; hence, FAS levels are affected by gene dosage. When the Fasn+/- mutant mice were interbred, Fasn-/- mice were not produced; thus, FAS is essential during embryonic development. Furthermore, the number of Fasn+/- progeny obtained was 70% less than predicted by Mendelian inheritance, indicating partial haploid insufficiency. Even when one of the parents was WT, the estimated loss of heterozygous progeny was 60%. This loss of Fasn+/- pups appeared to be strain-specific and became more pronounced as the heterozygous females produced more litters. Most of the Fasn-/- mutant embryos died before implantation and the Fasn+/- embryos died at various stages of their development. Feeding the breeders a diet rich in saturated fatty acids did not prevent the loss of homoor heterozygotes. These observations are very important in considering teratogenic consequences of drugs aimed at inhibiting FAS activity, to reduce either obesity or the growth of cancerous tissues.

Fig. 1.

Targeted mutation of the Fasn locus and analysis of Fasn knockout mice. (A) Restriction map of the 5′ half of the Fasn gene. The restriction sites shown are: A, Asp718; B, BamHI; EV, EcoRV. The BamHI fragments used in the generation of the disruption construct (line 2) are indicated in line 1 as dark lines. The arrow in the HPRT gene box indicates its direction of transcription. The 5′ and 3′ probes used and the sizes of the restriction fragments they hybridize to identify WT and targeted alleles by Southern blot analysis are indicated. (B) Southern blot analysis of genomic DNAs digested with Asp718 and probed with the ³²P-labeled 3′ probe described in A. (C) A typical PCR analysis of the genotypes. Two sets of primers were used to amplify 360-bp WT and 300-bp knockout allele products as described in Materials and Methods. (D) A typical Northern blot analysis of RNA isolated from livers of mice that were either starved for 48 h and refed the fat-free diet for 48 h (lanes 1 and 3) or fed the normal diet (lanes 2 and 4). Total liver RNA from WT (lanes 1 and 2) and heterozygous (lanes 3 and 4) mice were analyzed on agarose gel and the blots were probed with a ³²P-labeled mouse FAS cDNA probe. Actin mRNA levels were used to normalize FAS mRNA levels.

Fig. 2.

Analysis of the breeding behavior of F₁ Fasn heterozygotes. F₁ heterozygotes were either interbred or bred with WT C57/129 hybrid mice, and the number of litters produced and their genotypes were determined. (A) Analysis of litters produced when heterozygous males and females were interbred. (B) Heterozygous F₁ males were bred with WT C57/129 females. (C) WT males were bred with F₁ hybrid heterozygous females. Only the positive values of the SD of the mean are indicated to reduce crowding of the plot. Genotyping was performed as described in Fig. 1 and Table 1.

Fig. 3.

Photographs of E9.5 embryos. (A) Embryos shown are in their yolk sacs. A WT embryo is on the left and a defective hemorrhagic Fasn^+/– embryo is on the right. (B) The embryos dissected from their yolk sacs. Note that the Fasn^+/– defective embryo on the right was growth retarded and did not finish turning. (Magnification: × 100.)

Fig. 4.

Immunological determination of FAS expression in developing embryos with anti-FAS antibodies. Timed matings were performed and the uteri were resected at E6.5 (A and B) and E5.5 (C–E) postcoitus. The uteri were fixed in Bouin's solution and sectioned through the decidua-containing regions. The sections were treated with anti-FAS goat antibodies, and the immune complexes were detected immunohistochemically with peroxidase-conjugated secondary antibodies (dark brown regions). (A) Small, growth-retarded E6.5 embryo. (B) Normal-looking E6.5 embryo. (C and D) Sagittal and transverse sections of normal-looking E5.5 embryos. (E) Growth-retarded E5.5 embryo. (Magnification: ×400.) Arrows indicate some of the regions expressing high FAS levels. epc, ectoplacental cone; vc, visceral endoderm; en, endoderm; d, decidual cells; e, ectoderm.

Fig. 5.

Analysis of FAS mRNA expression in developing embryos. WT embryos of E8.5, E9.5, and E10.5 were probed with antisense FAS mRNA probes. The rightmost E9.5 embryo was hybridized with labeled sense transcripts showing only background labeling. (Magnification: ×100.) Arrows indicate some of the regions expressing high levels of FAS mRNA. p, proencephalon; tb, tail bud; np, nasal pit; b, branchial arches; lb, limb bud; fm, frontonasal mass.