Fig. 4

FurinA Levels Control the Expansion of the Spaw Expression Domain in the LPM

(A and B) For the purpose of mathematical modeling, we have considered the left LPM of the developing zebrafish embryo as a linear domain (displayed as a rectangle here) with a source of Spaw at the posterior end (x = 0); (B) behavior of Spaw in the competent LPM described in (A). Synthetized intracellular Spaw (S_i), mature intracellular Spaw (S_i^m), and extracellular Spaw (S_e).

(C) Partial-differential equation model of the system described in (B). Parameter definitions, analysis of the model, and details on the numerical simulation and boundary conditions are available in Supplemental Experimental Procedures.

(D) Snapshots of Movie S1 showing a simulation of the model defined in (C). The speed of progression of S_e, and consequently of the domain of spaw expression in the LPM, increases with the level of FurinA.

(E) The model predicted that increasing FurinA levels, resulting in enhanced maturation of Spaw, results in increased length of the spaw expression domain at a given time (180 min here).

(F) Quantification of the length of the spaw expression domain (anterior-posterior) in embryos with no (MZaoh; n = 12), low (Zaoh mutants; n = 7), normal (WT; n = 15), or high (WT injected respectively with 25 pg; n = 25 and 50 pg; n = 17 furina mRNA) FurinA levels. Histograms display average value ± SEM; p < 0.05, p < 0.01, and p < 0.005 in Student’s t test.

(G) Cartoon illustrating the effect of FurinA on the signaling range of Spaw in the LPM. In a WT situation, Spaw is cleaved prior to secretion by cells at the posterior end of the LPM (10 somite stage, 13 hpf). Spaw induces its own expression in a paracrine fashion, and the spaw expression domain expands toward the anterior end of the developing LPM, reaching the heart field at the 23-somite stage (20 hpf). Spaw also induces expression of Lft1 at the midline, which prevents it from reaching the right LPM. Spaw expression is consequently limited to the left LPM and establishes LR patterning. In the MZaoh mutants, the absence of FurinA processing of Spaw results in failure to induce Spaw expression in the LPM and of Lft1 in the midline. As a consequence, LR patterning is affected. Overexpression of FurinA results in increased presence of mature Spaw in the extracellular space. The activation of Spaw in the LPM progresses faster toward the anterior left LPM. LR patterning is affected, likely as a result of an excess of Spaw protein overcoming the Lft1 midline barrier, Kupffer’s Vesicle (KV) and midline (M).