Fail-safe function and underlying gene regulatory network circuitry.

Exclusive regulatory domains established by reciprocal repression between pmar1 and hesC. (A) Diagram of a 2.59-kb fragment upstream of the pmar1a start of translation fused with the coding sequence for the GFP reporter. Small portions of the sequence are reproduced below to indicate highlighted transcription factor target sites discussed in the text. The bent arrow indicates the start of transcription. (cf. Fig. S1). (B–E) Typical results in embryos bearing pmar1a expression constructs at 16 h postfertilization: (B) wild-type pmar1 reporter activity (this construct expressed only in micromere descendants in 124 of 135 GFP⁺ embryos); (C) pmar1 construct but with HesC target site shown in A disrupted (result is gross ectopic activity in 77 of 92 embryos harboring construct). (D) Normal skeletogenic mesenchyme (SM) expression of wild-type pmar1 construct in presence of coinjected control morpholino antisense oligonucleotide (MASO). (E) Wild-type pmar1 construct in presence of coinjected HesC MASO resulted in ectopic expression in nearly all embryos (56 of 61). (F) Summary of regulatory interactions from refs. 1 and 8 and these experiments.

pmar1 morpholino antisense oligonucleotide (MASO) effects and mechanism of regulative recovery. (A) Control MASO embryo at 24 h postfertilization, showing normal ingression of skeletogenic mesenchyme (SM). (B) pmar1 MASO-injected embryo at 24 h: Ingression is totally blocked. (C) Severe decrease in levels of expression of genes downstream of the double-negative gate at 12 h after injection of pmar1 MASO or pmar1 MASO + blimp1 MASO. (D) Control MASO-injected embryo at 31 h. (E) Recovery from pmar1 MASO by 31 h: normal SM ingression, similar to control in D. (F) Regulative recovery is seen at the molecular level by 24 h but not when the blimp1 MASO is also present. (G, H) Regulative recovery depends on specific repression of hesC by Blimp1. (G) Control experiment where in addition to pmar1 MASO a wild-type hesC BAC (“wt hesC”) is coinjected as a hesC expression vector; an additional hesC GFP reporter lacking the previously characterized intronic Blimp1 target site (“ΔBlimp hesC:GFP”) is coinjected as a marker of incorporation of the expression constructs. Reporter activity is seen in ingressed SM cells as regulative recovery takes place. (H) A hesC BAC in which the single intronic Blimp1 target site is mutated (“ΔBlimp hesC”) is introduced together with the same GFP marker construct and pmar1 MASO. Clones harboring the transgenes now fail to ingress, although SM cells lacking the transgenes recover and ingress.