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First published online 16 May 2007
doi: 10.1242/dev.004929

Development 134, 2251-2260 (2007)
Published by The Company of Biologists 2007
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Distinct functions of the major Fgf8 spliceform, Fgf8b, before and during mouse gastrulation

Qiuxia Guo and James Y. H. Li*

Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.


Figure 1
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  		Fig. 1. Generation of splice site mutation to abolish Fgf8b expression in mouse. (A) Schematic representation of mutations in the Fgf8 locus. The change of sequences (bottom; mutations are shown in red) at the junction of intron (lowercase letters) and exon (capital letters) mutates the 5' alternative splice acceptor and creates an NdeI site (underlined). (B) Southern blot analysis to identify targeted embryonic stem (ES) cell clones. Asterisks indicate non-specific signals. (C) PCR and restriction digestion analysis to verify the point mutation. (D) Schematic representation of Fgf8 (top), Fgf8{Delta}b-neo (middle, left), Fgf8{Delta}b (middle, right) and Fgf8neo (bottom) loci, and alterations in RNA splicing due to the point mutation and neo insertion. The Fgf8-neo hybrid transcript results from a cryptic splice donor and acceptor in the neo gene and in the intronic region 360 bp downstream to the neo gene, respectively. (E) Reverse transcriptase (RT)-PCR analysis of different Fgf8 splice variants in E7.5 embryos of indicated genotypes using primers 1 and 2 (shown in E). Fgf8 splice variants (a-h and unknown) are marked to the left. Notice that Fgf8b (asterisk) is missing in Fgf8{Delta}b-neo/{Delta}b-neo and Fgf8{Delta}b/{Delta}b embryos, whereas the neo insertion has no effect on the alternative splicing of the first four exons. (F) RT-PCR assay using primers 3 and 4 (indicated in D) reveals that the insertion of neo results in the production of Fgf8-neo hybrid transcripts. B, BamHI; E, EcoRI; X, XbaI.

 

Figure 2
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  		Fig. 2. Fgf8{Delta}b-neo/{Delta}b-neo embryos have more severe defects than Fgf8{Delta}b/{Delta}b embryos. (A-D) Morphology and histology of murine wild-type (A,B) and Fgf8{Delta}b-neo/{Delta}b-neo (C,D) embryos at E7.5. (B,D) Hematoxylin and Eosin (H&E) analysis of sagittal sections of the embryos shown in A and B, respectively. Notice the mesodermal cells (arrowheads) accumulated at the primitive streak (brackets) in the Fgf8{Delta}b-neo/{Delta}b-neo embryo. (E,F) Lateral view of a wild-type embryo (E) and dorsal view of an Fgf8{Delta}b-neo/{Delta}b-neo embryo (F) at E8.5. (G-J) Morphology and histology of wild-type and Fgf8{Delta}b/{Delta}b embryos at E8.5. Arrowheads indicate the rough appearance of the amniotic membrane of Fgf8{Delta}b/{Delta}b embryos, compared with the control. (I,J) H&E analysis of sagittal sections of the embryos in G and H, respectively. PSM, presomitic mesoderm.

 

Figure 3
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  		Fig. 3. Pattern formation of the neural plate of murine Fgf8{Delta}b/{Delta}b embryos is largely normal. (A-J) Expression of region-specific markers, as indicated to the left, in the anterior neural plate of wild-type and Fgf8{Delta}b/{Delta}b embryos: Otx2, telencephalon and mesencephalon; Gbx2, rhombomere 1 (r1); Fgf8, r1; En1, mesencephalon and r1; Shh, head process and axial mesoderm. Black arrows mark the mid-hindbrain junction. Arrowheads in A,B,I and J demarcate Krox20 expression in r3 and r5 (A,B) and Hoxb1 expression in r4 (I,J). Notice that the head process (red arrow; I,J) appears abnormal in the mutants.

 

Figure 4
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  		Fig. 4. Fgf4 and Wnt3a are expressed in the primitive streak of murine Fgf8{Delta}b/{Delta}b, but not in Fgf8{Delta}b-neo/{Delta}b-neo, embryos at E7.5. (A) Expression of Fgf4 in embryos of indicated genotypes. (B-D) Expression of Wnt3a in wild-type (B), Fgf8{Delta}b/{Delta}b (C) and Fgf8{Delta}b-neo/{Delta}b-neo (D) embryos. Notice that the morphologically distinct node (arrowhead) is absent in Fgf8{Delta}b/{Delta}b and Fgf8{Delta}b-neo/{Delta}b-neo embryos. Arrows mark the mass of mesodermal cells, which is variable and tends to be smaller than that found in Fgf8{Delta}b-neo/{Delta}b-neo embryos, at the primitive streak of Fgf8{Delta}b/{Delta}b embryos at E7.5.

 

Figure 5
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  		Fig. 5. The regionalization of the primitive streak and the specification of the mesoderm are partially maintained in murine Fgf8{Delta}b-neo/{Delta}b-neo embryos. (A-L) In situ hybridization analysis of markers (indicated to the left) characteristic for Fgf8 signaling and mesoderm in wild-type and Fgf8{Delta}b-neo/{Delta}b-neo embryos at E7.5. (A-D,I,J) Adjacent sagittal sections of wild-type (A,C,I) and Fgf8{Delta}b-neo/{Delta}b-neo (B,D,J) embryos. Inset in B shows whole-mount analysis of Fgf8 expression. Notice that axial mesoderm marked by T and Foxa2 expression is absent anterior to the streak of Fgf8{Delta}b-neo/{Delta}b-neo embryos, indicating a lack of mesoderm migration. (E,F) Asterisk indicates Evx1 expression at the proximal end of the primitive streak.

 

Figure 6
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  		Fig. 6. Fgf8b is required for the normal induction of Lefty2 and T, and for proper alignment between the AP axis and the shape of the embryo. (A-C) In situ hybridization with an RNA probe detecting both Lefty1 and Lefty2 in E6.5 murine embryos of the indicated genotypes. Expression of Lefty1 in the anterior visceral endoderm (AVE; marked by arrowhead) is unaffected in Fgf8{Delta}b/{Delta}b (B) or Fgf8-/- (C) embryos, whereas Lefty2 expression in the emerging primitive streak (arrow) is missing in the mutants. Insets show distal views of the embryos in A and C. Double-headed arrow in A indicates the height of the epiblast (see J). (D-F) Analysis of T expression in E6.5 embryos of the indicated genotypes. Arrowhead marks the T expression domain in the distal extraembryonic ectoderm, while the arrow marks T expression in the posterior epiblast. (G,H) Expression of Cer1 in the AVE of wild-type (G) and Fgf8{Delta}b/{Delta}b (H) embryos at E5.75. Insets show distal views of the embryo. Broken double-headed arrow marks the long axis of the embryo. (I) Schematic summary of the AP polarity with respect to the shape of wild-type and Fgf8{Delta}b/{Delta}b embryos at E5.75 and E6.5. Notice that the shift of AP axis fails to occur in the Fgf8{Delta}b/{Delta}b embryo. (J) Distribution of the ratio of AP and LR dimensions relating to the height of the epiblast (indicated by double-headed arrow in A) between E6.0 and E6.75 from intercrosses of Fgf8+/{Delta}b mutants. Each dot represents one embryo. AP, anteroposterior; LR, left-right. Scale bars: 50 µm in F for A-H, including insets in G and H; 50 µm in inset in C for insets in A-C.

 

Figure 7
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  		Fig. 7. Loss of Fgf8b expression alters the orientation of the AP axis, but not the long axis, of the embryo with respect to the longitudinal axis of the uterine horn. (A) Schematic of orientation the of the embryo within the uterus and the relative position of the sectioning plane. (B) Schematic representation of the relationships between the axes of the embryo and uterus found in two groups (I and II) of embryos at E6.5 from intercrosses of Fgf8+/{Delta}b parents. (C,D) Immunohistochemical analysis of EGFP, which marks the anterior visceral endoderm (AVE; brown) on transverse sections of E6.5 embryos within the uterus. Insets show embryos at higher magnification. (E,F) In situ hybridization of T transcripts on cross sections of wild-type and Fgf8{Delta}b/{Delta}b embryos at E7.5 within the uterus. (A-F) Red arrow represents the AP axis; black double-headed arrow marks the long uterine axis. Notice the abnormal bulge of cells (arrowhead) at the primitive steak of Fgf8{Delta}b/{Delta}b embryos in F. (G) Distribution of the angles ({alpha}) between the long axes of the embryo and the uterus at E6.5 for groups I and II. Each dot represents one embryo. The average angles (horizontal blue and red bars) and standard deviations (vertical bars) are indicated. (H) Distribution of the angles ({alpha}) between the AP axes and the uterus at E7.5 for control (red) and Fgf8{Delta}b/{Delta}b (green) embryos relating to the ratio of the AP:LR dimension. Green and red broken lines indicate the average angle for control and Fgf8{Delta}b/{Delta}b embryos, respectively. A, anterior; D, distal; L, left; P, posterior; Pr, proximal; R, right.

 




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