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doi: 10.1242/10.1242/dev.00367

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Defective somite patterning in mouse embryos with reduced levels of Tbx6

Phillip H. White, Deborah R. Farkas, Erin E. McFadden* and Deborah L. Chapman{dagger}

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA
* Present address: UC Davis School of Veterinary Medicine, Davis, CA 95616, USA



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  		Fig. 1. Tbx6 transgenic construct and expression pattern. (A) The Tbx6 genomic locus (top) showing the positions of the exons (blue boxes), start of transcription (black arrow) and positions of the XbaI (X) sites used for cloning the Tbx6 transgenic construct (bottom). The Tbx6 transgenic construct consists of the entire coding region, upstream and downstream flanking regions and a lacZ reporter gene, which has a minimal promoter and a polyadenylation sequence. (B-D) Transgenic embryos, derived from the Tg46 founder line, were dissected from E7.5-9.5 and stained for ß-galactosidase activity. (B) Side view (posterior is towards the right) of an E7.5 mouse embryo showing X-gal staining predominantly within the primitive streak, but also widely scattered throughout the embryo. (C) By E8.5, staining is found within the primitive streak and PSM, with some stained cells found in the newly formed somites (red arrowhead). (D) Similarly, at E9.5, X-gal staining is seen in the primitive streak region and PSM, but also extending into the caudal-most somites. (E) Whole-mount in situ hybridization of endogenous Tbx6 expression in an E9.5 wild-type embryo, showing the anterior limit of Tbx6 transcripts in the PSM prior to somite segmentation (red arrowhead indicates the position of the newly formed somite). (F) Whole-mount in situ hybridization of lacZ expression in a Tg46 transgenic embryo is largely limited to the primitive streak and PSM with transcripts downregulated in the newly formed somite (red arrowhead).

 


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  		Fig. 2. Partial rescue of the Tbx6 mutant phenotype with Tbx6 transgenes. Embryos were dissected at E10.5 (A-C,E), E13.5 embryos (D), and E15.5 (F-M). Embryos were derived from the following crosses: Tbx6tm1Pa/+ Tg46/+ x Tbx6tm1Pa/+ (A-I), Tbx6tm1Pa/+ Tg130/+ x Tbx6tm1Pa/+ (J,K), and Tbx6tm1Pa/+ Tg46/+ x Tbx6tm1Pa/+ Tg130/+ (L,M). Genotypes are indicated in the panels with Tbx6+/– or Tbx6–/– denoting hetero- or homozygosity for the Tbx6tm1Pa allele, respectively and Tg46/+ or Tg130/+ denoting hemizygosity for the Tg46 or Tg130 transgene, respectively. (A-C) X-Gal staining reveals predominant expression of the Tg46 transgene in the tailbud, with perdurance of ß-galactosidase in the formed somites of both control and Tg46 rescued embryos. (B,C) A Tg46 partially rescued Tbx6 mutant, in which posterior somites are clearly seen in the high magnification of the tail region (C). X-gal staining is present in the caudal-most somites and predominantly in the dorsal region of the expanded tailbud. (D) Branching morphology of an E13.5 tail from a Tg46 rescued embryo. (E) Whole-mount in situ hybridization of Tbx6 expression in the tails of Tbx6tm1Pa/+ and Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryos, showing that although Tbx6 is expressed in both the primitive streak and the PSM, transcripts are downregulated in the Tg46 rescued embryos compared with the heterozygotes. (F,G) Gross morphological analysis reveals the short stature and short tail (red arrow indicates the tip of the tail in G) of the Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ compared with the Tbx6tm1Pa/+ Tg46/+ embryo. (H,I) Alcian Blue and Alizarin Red stained skeletons of the embryos pictured in F,G reveal fused vertebrae and fused ribs along the entire AP axis of the Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ (I). Fusions of the ribs are detected in both proximal and more distal regions of the ribs. (J,K) Alcian Blue and Alizarin Red skeletal staining of embryos derived from a Tbx6tm1Pa/+ Tg130/+ x Tbx6tm1Pa/+ cross. Tbx6+/+ or Tbx6tm1Pa/+ embryos with or without the Tg130 transgene were indistinguishable (J), while Tbx6tm1Pa/Tbx6tm1Pa Tg130/+ embryos showed almost complete absence of vertebrae and ribs (K). (L,M) Side (L) and dorsal (M) views of a Tbx6tmPa1/Tbx6tmPa1 Tg46/+ Tg130/+ embryonic skeleton showing more complete rescue of the mutant phenotype than with either transgene alone (compare L,M with I,K). Fusions were predominantly found in the cervical (not visible in these panels) and thoracic regions (red arrow in L and red asterisks in M), while the lumbar or sacral regions appeared normal.

 


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  		Fig. 3. Whole-mount in situ hybridization of Tg46 rescued mutant embryos reveals abnormalities in R-C somite patterning. Embryos, derived from Tbx6tm1Pa/+ Tg46/+ x Tbx6tm1Pa/+ crosses, were dissected at E10.5 and hybridized with Uncx4.1 (A-D), Mesp2 (E), Cer1 (Cerr-1 in figure; F), neurofilament l (G-H), Tbx18 (I-L), Dll3 (M) and Dll1 (H-M) antisense riboprobes. The genotypes of the embryos are as indicated. The designation of `normal' is given to embryos that are wild type or Tbx6tm1Pa/+ with or without the transgene, as no differences were observed between these genotypes when hybridized with the specified probes. (A) Uncx4.1 is normally expressed in the caudal region of each somite along the AP axis. (B-D) By comparison, Uncx4.1 transcripts are found throughout the somites of the Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryos. (C,D) In some cases, the posterior-most somites express stripes of Uncx4.1 transcripts; however, these stripes (red arrows in D) are found on a background of low uniform levels of Uncx4.1 expression. (E) Mesp2 is expressed in the rostral region of the forming somite in both normal and Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryonic tails (dorsal view) at comparable levels. In the tail of the top Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryo, very low levels of Mesp2 transcripts are also detected in the last somite to form (red arrow). (F) Cer1 is expressed as two stripes in the anterior PSM and in the rostral region of the newly formed somite (red arrow) in normal embryos. Cer1 transcripts are downregulated in the anterior PSM and absent in the newly formed somite of Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryos. (G) Neurofilament L (NF in figure) is expressed in the spinal ganglia and neural tube of normal embryos. Expression in the spinal ganglia appears as evenly distributed blocks along the axis, reflecting their migration route through the rostral halves of the somites. (H) By contrast, Neurofilament L expression in the spinal ganglia of the Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryo is irregularly distributed along the axis, often appearing as fused blocks. (I-L) Lateral views of normal and Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryos showing Tbx18 expression. The heads of the embryos in have been removed for genotyping. Tbx18 is normally expressed in the rostral halves of somites along the AP axis, as well as in the heart and limb buds (I,K). In the Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryos, Tbx18 is either not expressed in the paraxial mesoderm (J) or is expressed in the newly formed somites, eventually being lost in more anterior regions of the embryo (L). (M) Dorsal view of Dll3 expression in the tailbuds of normal, Tbx6tm1Pa/Tbx6tm1Pa and Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryos. (N,Q) In normal embryos Dll1 is expressed in the tailbud and PSM, and is upregulated in the next-to-form somite. Low levels of Dll1 are later detected in the caudal compartment of the formed somites (Q). (O,P,R,S) The level of Dll1 transcripts in the tails of Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryos is reduced compared with Tbx6tm1Pa/+ embryos. Weak upregulation of Dll1 expression can be seen in the anterior region of the PSM prior to somite segmentation. The expanded tail region of the Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ embryos can be seen in R and S (broken blue outline in R), where Dll1 transcripts appear dorsally localized. Low levels of Dll1 transcripts are later localized to the caudal compartment of the formed somites similar to the Tbx6tm1Pa/+ embryos. (P) Dll1 expression in the flank of the Tg46 rescued embryo marks ectopic neural tissue (red arrowhead).

 


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  		Fig. 4. Genetic interaction between Tbx6 and rib-vertebrae (rv). The genotypes are as indicated, except where `normal' is given to indicate that differences were not observed for embryos that were wild type, Tbx6tm1Pa/+ or Dll1tm1Gos+ in H. (A-E) Gross morphology and skeletal preparations of E15.5 embryos dissected from Tbx6tm1Pa/+ x rv/rv matings. (A,B) Tbx6tm1Pa/rv embryos are shorter in stature than their rv/+ littermates and have short tails (arrow in B indicates the tip of the tail). (C-E) Alcian Blue and Alizarin Red skeletal staining reveals fusions of the ribs and vertebrae along the entire AP axis of the Tbx6tm1Pa/rv embryos. (E) High magnification of the skeleton in D shows that rib fusions occur both proximally and distally and that the neural arches of the lumbar region are fused along the axis. (F) Tbx6 expression in the tail regions of rv/rv and rv/+ E10.5 embryos. Dorsal (left) and side (right) views of the tails are shown. The tailbuds of the rv/rv mutant embryos are larger than the rv/+ littermates, and show diminished levels of Tbx6 transcripts compared with rv/+ tails. (G) The Tbx6 genomic locus showing the positions of the exons (blue boxes), start of transcription (black arrow), and the position and nature of the rv lesion. A red line indicates the Tbx6 genomic region that is duplicated and inverted (red arrow) in the rv mutation. The rv mutation is caused by the insertion of this duplicated and inverted region upstream of the Tbx6-coding region. (H-K) Alcian Blue and Alizarin Red skeletal preparations from E14.5 embryos dissected from Tbx6tm1Pa/+ x Dll1tm1Gos/+ crosses. (H) Heterozygous embryos for either Tbx6 or Dll1 showed normal skeletal morphology. (I) The genetic interaction between these two mutations can be seen in compound heterozygotes (labeled as Tbx6+/– Dll1+/–), which are characterized by the presence of abnormally formed vertebrae and ribs along the AP axis. (J,K) Higher magnifications of the Tbx6tm1Pa/+ Dll1tm1Gos/+ skeleton in I, the thoracic and lumbar region is shown in J, and the lumbar and sacral regions are shown in K. (J) A rib missing its proximal portion is indicated by a red arrowhead. Abnormally formed vertebrae are seen in thoracic vertebrae in (J, red arrow) and in the lumbar vertebrae in (K, red asterisks). Limbs have been removed from all skeletons.

 


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  		Fig. 5. Whole-mount in situ hybridization of myogenin expression in embryos with reduced Tbx6 expression. Embryos were dissected at E10.5 from crosses of Tbx6tm1Pa/+ Tg46/+ x Tbx6tm1Pa/+ (A,B) and Tbx6tm1Pa/+ x rv/rv (C,D) mice, and hybridized with a myogenin antisense riboprobe. The designation `normal' is given to embryos that were either wild type or Tbx6tm1Pa/+ with or without the Tg46 transgene (A), or rv/+ (C), which showed no differences in myogenin expression compared with wild-type embryos. (A,C) Myogenin is normally expressed in the myotome compartment of the differentiating somite and thus appears as evenly spaced stripes along the AP axis. Expression of myogenin in either the Tbx6tm1Pa/Tbx6tm1Pa Tg46/+ (B) or Tbx6tm1Pa/rv (D) embryo is disorganized compared with `normal' littermates, showing areas in which the myotomes are fused or form as small isolated islands in the intersegmental regions.

 

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