Fig. 5. The crucial determinant of medial IZ formation is the level of Him + Her5 inhibitory activity, probably achieved in vivo by Him/Her5 heterodimers but replaceable by a higher level of either factor alone. (A) Co-immunoprecipitation assays reveal possible interactions between the bHLH transcription factors important to prevent/promote neurogenesis at the MHB. Crude protein extracts were isolated from yeasts transformed with the following constructs combinations: (1) her5pGBKT/ + her5pGADT7, (2) her5pGBKT7 + ngn1pGADT7, (3) himpGBKT7 + her5pGADT7, (4) T-antigen pGBKT7 + p53pGADT7; (5) her5pGADT7 + lampGADT7. Isolated extracts were either probed with anti-cMyc antibodies (A') and anti-HA antibodies (A'') or immunoprecipitated with anti-HA antibodies and then probed with anti-cMyc antibodies (A'''). (B) Stringency of Her5 homodimerization and Her5/Him heterodimerization, based on beta-galactosidase activity of yeast cells expressing appropriate construct combinations (Lazo et al., 1978). Note that the interaction between Him and Her5 is significantly stronger than Her5 homodimerization. (C-E) A higher dose of Him alone can compensate for the loss of Her5 activity and maintain the MIZ. (C) Schematic representation of the transgene integrated to generate her5PAC::egfp embryos (Tallafuss and Bally-Cuif, 2003): the egfp cDNA (blue cylinder) is inserted into the her5 region coding for the bHLH domain, resulting in a dysfunctional protein unable to bind both DNA and other bHLH factors. However, the him gene, contained in the PAC, is intact. (D) Quantification of him and otx1 (control) mRNAs in her5PAC::egfp transgenic compared to wild-type embryos using real-time RT-PCR. We do not know the number of recombined PAC copies integrated into the genome in our transgenic lines; however, note that the amount of him mRNA is 1.5-fold higher in the her5PAC::egfp transgenic embryo than in wild-type siblings. The change in threshold-crossing cycle (1/R) is shown for each mRNA relative to that for pax6 (assumed as a housekeeping gene) (a decrease in threshold-crossing corresponds to increase in mRNA level). The increase in him expression in the transgenic line is significant (P<0.02 by Student's t- test). Standard deviations are indicated with red lines. (E) Blocking Her5 activity (by injecting her5MO^ATG) in her5PAC::egfp transgenic embryos fails to trigger ectopic expression of ngn1 across the MIZ (white asterisk) (flat-mounted embryo at three somites, anterior left, used markers color-coded).