Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation

Abstract

The dynamic embryonic expression of germ cell nuclear factor (GCNF), an orphan nuclear receptor, suggests that it may play an important role during early development. To determine the physiological role of GCNF, we have generated a targeted mutation of the GCNF gene in mice. Germ line mutation of the GCNF gene proves that the orphan nuclear receptor is essential for embryonic survival and normal development. GCNF(-/-) embryos cannot survive beyond 10.5 days postcoitum (dpc), probably due to cardiovascular failure. Prior to death, GCNF(-/-) embryos suffer significant defects in posterior development. Unlike GCNF(+/+) embryos, GCNF(-/-) embryos do not turn and remain in a lordotic position, the majority of the neural tube remains open, and the hindgut fails to close. GCNF(-/-) embryos also suffer serious defects in trunk development, specifically in somitogenesis, which terminates by 8.75 dpc. The maximum number of somites in GCNF(-/-) embryos is 13 instead of 25 as in the GCNF(+/+) embryos. Interestingly, the tailbud of GCNF(-/-) embryos develops ectopically outside the yolk sac. Indeed, alterations in expression of multiple marker genes were identified in the posterior of GCNF(-/-) embryos, including the primitive streak, the node, and the presomitic mesoderm. These results suggest that GCNF is required for maintenance of somitogenesis and posterior development and is essential for embryonic survival. These results suggest that GCNF regulates a novel and critical developmental pathway involved in normal anteroposterior development.

FIG. 1

GCNF expression during postgastrulation and neurulation stages. (A to C) Whole mount in situ hybridization of GCNF at 7.5 (A), 8.5 (B), 8.75 (C), and 9.5 (D) dpc.

FIG. 2

Targeted mutation of the GCNF gene. (A) Gene targeting strategy. Approximately 40 kb of the GCNF locus is shown, with exons depicted as solid boxes. The targeting vector with homologous arms, neo gene, and tk gene are shown relative to the genomic structure. The recombined locus is missing the DBD exon. (B) Southern analysis to genotype ES cell clones and embryos, using a 5′ probe upstream of the targeting vector. Digestion with Bsp106I and Acc65I yielded a 21-kb band for the wild-type allele and a 9-kb band for the mutant allele. The presence of a single integration was determined by using the neo probe on the same digests to produce a 5.5-kb band. (C) PCR strategy used for genotyping produced a 239-bp wild-type DBD exon band and a 416-bp neo gene band. MW, molecular weight markers.

FIG. 3

Analysis of GCNF expression and gross morphology of GCNF^−/− embryos. GCNF is expressed in all three germ layers (gl) and in the extraembryonic tissues of the ectoplacental cone (epc) in GCNF^+/+ embryos (A). No positive signal was detected with the same DBD cDNA probe in GCNF^−/− embryos, indicating that GCNF^−/− embryos do not express any GCNF transcript (B). (C) Gross morphology of a 10.5-dpc GCNF^+/+ embryo. (D) GCNF^−/− embryo at 10.5 dpc, showing severe trunk and posterior defects, open neural tube, failure of axis rotation, and distended pericardium.

FIG. 4

Gross morphology of GCNF^−/− embryos. (A and B) GCNF^+/+ (A) and GCNF^−/− (B) embryos within the yolk sac at 8.5 dpc. Double-headed arrows indicate the extent of the curvature of the ventral body. (C and D) GCNF^+/+ (C) and GCNF^−/− (D) embryos outside the yolk sac at 8.5 dpc. A small protrusion of tissue was first seen at the base of allantois of the GCNF^−/− embryo (B and D). The brackets (B, D, F, and J) indicate a presumptive tailbud protruding from the yolk sac of a GCNF^−/− embryo. (E and F) GCNF^+/+ (E) and GCNF^−/− (F) embryos within the yolk sac at 8.75 dpc. (G and H) GCNF^+/+ (G) and GCNF^−/− (H) embryos outside the yolk sac at 8.75 dpc. Unlike GCNF^+/+ embryos, this GCNF^−/− embryo does not turn and remains in a lordotic position. The tailbud lies perpendicular to the anteroposterior body axis (H). (I and J) GCNF^+/+ (I) and GCNF^−/− (J) embryos within the yolk sac at 9.5 dpc. (K and L) GCNF^+/+ (K) and GCNF^−/− (L) embryos outside the yolk sac at 9.5 dpc. The GCNF^−/− embryo remains in a lordotic position with an opened neural tube.

FIG. 5

Posterior histologic defects in GCNF^−/− embryos. (A and B) Sagittal sections of GCNF^+/+ (A) and GCNF^−/− (B) embryos at 8.5 dpc. Arrows indicate positions of yolk sac attachment to the embryos. Note that in the GCNF^−/− embryo, the position of attachment is at the base of the allantois (al). Arrowheads indicate the large invagination formed by the posterior neural epithelium (ne) in the GCNF^−/− embryo. Brackets (B and D) indicate the region of the tailbud (tb). (C and D) Cross sections of GCNF^+/+ (C) and GCNF^−/− (D) embryos at 8.5 dpc. Both embryos contain open neural tubes at the anterior and posterior ends. The arrowhead indicates the large invagination formed by posterior neural epithelium (ne) in the GCNF^−/− embryo. (E and F) Dorsal views of GCNF^+/+ (E) and GCNF^−/− (F) tailbuds at 9.5 dpc. Arrows indicate the presence in the GCNF^+/+ tailbud (tb) but absence in the GCNF^−/− tailbud of a posterior neuropore. (G and H) Cross sections of tailbuds of GCNF^+/+ (G) and GCNF^−/− (H) embryos. The GCNF^+/+ embryo has an open neural tube (ne) on the dorsal side of the tailbud and a hindgut (hg) located centrally, whereas the GCNF^−/− embryo has a closed neural tube (ne) in the middle of the tailbud. No hindgut is seen, but the notochord (nt) lies ventrally to the neural tube.

FIG. 6

Expression of anteroposterior marker genes in GCNF^+/+ and GCNF^−/− embryos. (A) Strong Otx2 expression in GCNF^+/+ (left) and GCNF^−/− (right) embryos. (B) Detection of the HNF3β probe throughout the notochord of both GCNF^+/+ (left) and GCNF^−/− (right) embryos. Expression is also seen in the foregut (fg), (liver) (li), and hindgut (hg) of the GCNF^+/+ embryo. In contrast, only foregut expression is seen in GCNF^−/− embryos. al, allantois; tb, tailbud. (C) Hoxb-13 expression in the posterior end of both GCNF^+/+ (left) and GCNF^−/− (right) embryos. (D) Wnt-3a expression in the extreme posterior end of the tailbud of both GCNF^+/+ (left) and GCNF^−/− (right) embryos. (E and F) GCNF^−/− embryo in the yolk sac before (E) and after (F) hybridization with the brachyury T probe. (F) Brachyury T expression in the tailbud (tb), which protrudes outside the yolk sac. The high level of signal in the anterior portions of the embryo is due to trapping within the yolk sac.

FIG. 7

Expression of marker genes of somitogenesis. Whole-mount in situ hybridization was performed on GCNF^+/+ (A, C, E, G, I, and K) and GCNF^−/− (B, D, F, H, J, and L) embryos. (A and B) Expression of myogenin in both GCNF^+/+ (A) and GCNF^−/− is (B) only in the first seven differentiating somites in GCNF^−/− embryos (arrowheads in panel B). Paraxis is expressed in the somites of 8.5-dpc GCNF^+/+ (C) and GCNF^−/− (D) embryos, in somites of a GCNF^+/+ embryo at 9.5 dpc (E), and ectopically at the posterior end (arrow) of the tailbud in the GCNF^−/− embryo at 9.5 dpc (F). (G) mCer-1 expression in the two newly formed somites (arrows) of a GCNF^+/+ embryo. (H) Reduction of mCer-1 expression (arrow) in a GCNF^−/− embryo at 9.5 dpc. (I) Expression of lunatic fringe in the first two somitomeres (arrows) and a broad swath of cells (arrowhead) in the PSM of GCNF^+/+ embryos. (J) In GCNF^−/− embryos at 9.5 dpc, lunatic fringe is expressed with equal intensity in the first two somitomeres (arrows) of the PSM. Only a transient, faint expression of lunatic fringe is found at the posterior end of the PSM (arrowhead). (K and L) RALDH-2 expression in cervical mesenchyme, caudal somites, and the cloacal region of a GCNF^+/+ embryo (K) and ectopic expression of RALDH-2 at the posterior end (arrow) of the tailbud in a GCNF^−/− embryo at 9.5 dpc (L).

FIG. 8

Expression of nodal and brachyury T in the node and PSM. Whole-mount in situ hybridization was performed on GCNF^+/+ (A, C, E, G, and I) and GCNF^−/− (B, D, F, H, and J) embryos. By 8.25 dpc, nodal was expressed at the node (arrow) in both GCNF^+/+ (A) and GCNF^−/− (B) embryos. Nodal expression (arrow) decreased in GCNF^+/+ embryos at 9.5 dpc (C), and persistent and disorganized expression (arrow) was found at the node of GCNF^−/− embryos at 9.5 dpc (D). By 8.5 dpc, brachyury T was expressed in the node (arrow) and the primitive streak of both GCNF^+/+ (E) and GCNF^−/− (F) embryos. tb, tailbud. By 8.75 dpc, brachyury T is restricted to the PSM in GCNF^+/+ embryos (G) but is still expressed in the node region (arrow) of GCNF^−/− embryos (H). Additional expression domain (arrowheads) is observed between the base of the allantois and the tip of the tailbud (tb). Like in embryos at 8.75 dpc, brachyury T expression is observed in the PSM and notochord at the posterior end of GCNF^+/+ embryos at 9.5 dpc (I); in addition to expression in the node (arrow), brachyury T has a V-shape expression pattern in the tailbud (tb) of the GCNF^−/− embryo at 9.5 dpc (J).

FIG. 9

Model of ectopic tailbud formation in GCNF^−/− embryos. At 8.0 dpc in both GCNF^+/+ (A) and GCNF^−/− (B) embryos, the neural plate arises when the somites are generated from the primitive streak. Arrows indicate the direction of node regression. The orange area represents brachyury T expression in the primitive streak and PSM. (C) In the 8.5-dpc GCNF^+/+ embryos, the node, which expresses nodal (yellow), moves caudally (arrow), with a resultant lengthening of the notochord, which continues to induce the neural plate to rise to form the neural fold. The edges of the neural plate begin to elevate, forming the neural groove. (D) In GCNF^−/− embryos the node does not regress posteriorly, and invagination of neural epithelium occurs within the primitive streak region by 8.5 dpc. This invagination then pushes the primitive streak away from the original direction of regression (arrow). Arrowheads (C and D) indicate positions of yolk sac attachment to the embryos. (E) By 9.5 dpc, the posterior neuropore (PNP) in GCNF^+/+ in GCNF^+/+ embryos is closed by zipping up the neural grove caudally (arrow). (F) In 9.5-dpc GCNF^−/− embryos, the neural tube elongates inside the tailbud as the tailbud pushes out of the yolk sac (arrow). Paraxis expression in somites and PSM is shown in blue. P, posterior; P∗, new posterior.