DEV01859 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Generation of the M1-GIC BAC and comparison of Mash1 and GFP. (A) BAC RPCI 428P21 from chromosome 10 containing the Mash1 locus was modified using homologous recombination (Yang et al., 1997). This modification used the shuttle vector pSV-GIC that contains a GFP-IRES-CRE cassette flanked by 1 kb arms homologous to sequence in the Mash1 locus. This recombination precisely replaces the coding sequence of Mash1 with the GFP-IRES-CRE-coding cassette. The resulting modified BAC is called M1-GIC. Transgenic mouse lines were generated with M1-GIC. The GFP reporter is low but it reflects the Mash1 pattern throughout multiple stages and multiple regions, including the caudal neural tube (shown here), the telencephalon and diencephalon, sympathetic and enteric nervous systems (data not shown). (B-E) Immunofluorescence on transverse sections of neural tubes from E11.5 M1-GIC embryos with antibodies to GFP (green) and Mash1 (red) show GFP reflects the pattern of Mash1 with boundaries matching precisely in the dorsoventral axis. Persistence of GFP in lateral regions is probably due to the stability of GFP relative to Mash1. (B,F) Immunofluorescence using antibodies to detect GFP demonstrate expression of GFP in the Mash1-null background (F) is dramatically increased over wild type (B). These two images were taken at the identical GAIN to illustrate the difference in levels. This observation is consistent with the negative autoregulation of the Mash1 locus previously reported (Horton et al., 1999; Meredith and Johnson, 2000). (G) Because the persistence of GFP is found continuously throughout the lateral regions adjacent to dI3-dI5, but Mash1 is not required for dI4, we used Lhx1/5, a marker of dI4/6 to examine co-labeling with the GFP. Immunofluorescence using antibodies to detect GFP (green) and Lhx1/5 (red) shows very little overlap of GFP from the M1-GIC line with the dI4/6 population, consistent with conclusions drawn from Fig. 4. Scale bar: 175 mm in B,F; 100 mm in C-E; 50 mm in G.

• Supplemental Figure 2 -

  Fig. S2. Expanded ventricular zone and premature differentiation in the Mash1 null neural tube. Immunofluorescence of transverse sections of E11.5 wild-type (A,C) or Mash1^–/– (B,D) neural tube showing Lbx1 expression (red) and BrdU incorporation (green). (A,C) In the wild type, Lbx1 expression is in post-mitotic cells that are lateral to the ventricular zone and are almost exclusively BrdU negative (one positive in this section, arrow). (C) Higher power image of the area boxed in A. (B,D) In the Mash1-null, the ventricular zone is expanded and appears to bulge. There is no obvious increase in BrdU incorporation but Lbx1+ cells are aberrantly located in the ventricular zone suggesting cells are initiating at least some differentiation program but are unable to leave the ventricular zone. Although a few cells incorporate BrdU in lateral regions, rarely have the Lbx1+cells within the ventricular zone incorporated BrdU. Taken together, the coordinated events that normally occur during neuronal differentiation in the dorsal neural tube are disrupted in the Mash1 null. The bulge seen in the dorsal neural tube appears to reflect the inability of cells to completely initiate their program of differentiation and move out of the ventricular zone. Scale bar: 70 mm in A,B; 25 mm in C,D.