Noncyclic Notch activity in the presomitic mesoderm demonstrates uncoupling of somite compartmentalization and boundary formation

Abstract

To test the significance of cyclic Notch activity for somite formation in mice, we analyzed embryos expressing activated Notch (NICD) throughout the presomitic mesoderm (PSM). Embryos expressing NICD formed up to 18 somites. Expression in the PSM of Hes7, Lfng, and Spry2 was no longer cyclic, whereas Axin2 was expressed dynamically. NICD expression led to caudalization of somites, and loss of Notch activity to their rostralization. Thus, segmentation and anterior-posterior somite patterning can be uncoupled, differential Notch signaling is not required to form segment borders, and Notch is unlikely to be the pacemaker of the segmentation clock.

Figure 1.

Segmentation in embryos with constitutive Notch activity. (A–F) In situ hybridization of embryos showing expression of the Nicd-Gfp fusion transcript throughout the paraxial mesoderm of T(s)∷Cre; R-NICD embryos (A,B) and up-regulation of Hey1 in the PSM and the somites (C,D) compared with wild-type embryos (E,F). (G,J,L) Scanning electron microscopic pictures illustrating somites in T(s)∷Cre; R-NICD (G,J) and wild-type (L) embryos. (H,I,M,N) Hemalaun-stained plastic sections of somites in T(s)∷Cre; R-NICD (H,I) and wild-type (M,N) embryos. (K) Segmented paraxial mesoderm in T(s)∷Cre; R-NICD embryos visualized by Myf5 expression. Developmental stages are indicated on top. Bars: H,I,M,N, 500 μm.

Figure 2.

Endogenous Notch1 activity in wild-type and mutant embryos. (A–H) Immunohistochemical detection of activated endogenous Notch1 in the PSM. In contrast to wild-type embryos (A–C), endogenous Notch1 activity is down-regulated in the posterior but is found in one or two stripes in the anterior PSM (arrowheads in D,E) of T(s)∷Cre; R-NICD embryos. (F) Specificity of antibody is demonstrated by the lack of staining in T(s)∷Cre; N1^loxp/loxP embryos. Loss of Dll1 (G) or Pofut1 (H) function completely abolishes Notch1 activity. (I,N,O) Segmented paraxial mesoderm in T(s)∷Cre; R-NICD; N1^loxp/loxP (I), T(s)∷Cre; R-NICD; Dll1^loxp/loxP (N), and Pofut^−/− (O) embryos visualized by Myf5 expression. (J–M) Overlapping expression of Notch1 (J,L) and Notch2 (K,M) in the anterior PSM of wild-type (J,K) and T(s)∷Cre; R-NICD (L,M) embryos. (P,Q) Sections of plastic-embedded Pofut^−/− embryos. Sections are 50 μm apart from the same serially sectioned embryo. Bar: Q, 500 μm.

Figure 3.

Patterning of the PSM in T(s)∷Cre; R-NICD embryos. (A) In situ hybridization of wild-type (panels a,c,e,g,i,k,m) and T(s)∷Cre; R-NICD (panels b,d,f,h,j,l,n) embryos with the probes indicated on top demonstrates grossly normal subdivision of the PSM of T(s)∷Cre; R-NICD embryos. (B) Cyclic expression of Lfng (panels a–c,f–h) and Hes7 (panels k–m,p–r) in wild-type and static expression throughout the PSM in T(s)∷Cre; R-NICD embryos (panels d,e,i,j,n,o,s,t). (Panels e,j) Extended color development revealed low levels of Lfng transcription throughout the posterior PSM. Cyclic expression of Axin2 both in wild-type (panels u–w) and T(s)∷Cre; R-NICD (panels x–z) embryos. Cyclic expression of Spry2 in wild-type (panels za–zc) is disrupted in T(s)∷Cre; R-NICD (panels zd,ze) embryos. (C) Tail halves of T(s)∷Cre; R-NICD hybridized with Mesp2 (panels a′,b′,c′) and Lfng (panel a), Hes7 (panel b), and Spry2 (panel c), indicating that in the anterior PSM the Mesp2 expression domain overlaps with the high Lfng and Spry2 expression and with the decreasing Hes7 expression domain. The dotted lines depict the overlapping domains.

Figure 4.

Disrupted somite polarity in transgenic embryos. Uncx4.1 (A,C,E,G,I,K) and Tbx18 (B,D,F,H,J,L) expression in wild-type (A,B), T(s)∷Cre; R-NICD (C,D), Dll1^−/− (E,F), Pofut1^−/− (G,H), T(s)∷Cre; R-NICD; Dll^lox/lox (I,J), and T(s)∷Cre; N1^loxp/loxP (K,L) embryos, indicating that constitutive Notch activity leads to caudalization of somites (C,D,I,J) and loss of all Notch activity to rostralization (E–H), whereas loss of Notch1 function alone has no significant impact on segment polarity (K,L).