(a) Schematic representation of a DA neuron synapse. AAAD: aromatic L-amino acid decarboxylase; bas-1: Biogenic Amine Synthesis related 1; cat: abnormal CATecholamine distribution; DA: dopamine; dat: DopAmine Transporter; GTPCH: GTP cyclohydrolase; TH: Tyrosine hydroxylase; Tyr: tyrosine; VMAT: vesicular monoamine transporter.
(b) Schematic representation of two different models for DA terminal differentiation. See text for explanations.
(c) Picture of an adult worm expressing GFP under the control of the full length dat-1/DAT promoter, labeling all C. elegans DA neurons. Similarly, cat-2/TH is also exclusively expressed in DA neurons (not shown), as C.elegans contains no adrenergic or noradrenergic neurons.
(d) dat-1/DAT promoter analysis. Schematic representation of the dat-1/DAT locus with its upstream region. Exons are represented as red blocks, the upstream gene is shown in grey. Below: representation of cloned and injected constructs, and expression pattern in the DA and serotonergic (5-HT) neurons. Thick black lines symbolize the promoter piece placed in front of GFP (green box). Red cross represents a mutated EBS. “+” indicates >10% penetrant expression in more than half of the transgenic lines examined; “+/−“ also means >10% penetrant expression, but the penetrance is lower than in the corresponding full-length construct, “-” indicates <10% penetrant expression in more than half of the transgenic lines examined, “n.d” means not determined.
(e-h) Analysis of the regulatory regions of all other dopamine pathway genes. “+*” means dimmer gfp expression than corresponding wild type construct. See Fig. S1 and S3 for all primary data and nature of the mutations.
(i) The sequence alignment of all functional EBSs defines a position weight matrix (PWM) that is represented by a sequence logo. The conserved core in all sequences constitutes the DA motif. See Fig. S5 for sequences used to define the DA motif.

(a) Schematic representation of ast-1 locus and the mutants available for this gene.
(b) Representative example of loss of DA fate marker in ast-1 mutants. See Fig. S7 and S8 for other examples and Table S1 for quantification of data.
(c) Summary of ast-1 null mutant phenotype. +: fate marker expressed; -: fate marker not expressed. Due to early larval lethality, only the embryonically generated DA head neurons, but not the postembryonically generated PDE neurons could be scored for developmental defects in ast-1 null mutants. Markers that were expressed in both DA and 5-HT neurons were assayed with an rfp reporter in a transgenic background in which 5-HT neurons were labeled with gfp (Is[tph-1::gfp) so as to allow for loss of expression specifically in the DA neurons. See Fig. S7 for primary data.
(d) Expression of an ast-1::yfp reporter gene 4 in DA neurons. DA neurons are labeled with dat-1::mCherry. Scale bar: 10 μm.
(e) Rescue of the ast-1 mutant phenotype. We used a hypomorphic allele, hd-1, in which dat-1 expression is unaffected (Table S1), to drive ast-1 or a mouse homolog, etv-1, under control of the DA-specific dat-1 promoter and assayed expression of cat-2::gfp (otIs199).
(f) Developmentally staged ast-1(rh300) animals, containing the heat shock-inducible ast-1 array otIs198 and the DA fate marker cat-2::gfp (otIs199), were grown under non-inducible condition to the first larval stage (resulting in an absence of cat-2::gfp expression in 100% of animals); ast-1 was then induced by heat shock at the L1 stage and animals scored 4 hours and 3 days after heat shock. Of the 40 animals found to turn on expression of cat-2::gfp 4 hours after heat shock, all lost expression after 3 days. Data with a temperature-sensitive allele of ast-1 corroborate sustained ast-1 activity (Supp.Fig.10).

(a) Representative picture of a control embryo after the 3-fold stage. dat-1::gfp expression starts at late three fold stage and can be detected in the six embryonically generated DA neurons.
(b,c) Representative picture of an embryo heat shocked four hours after the two cells stage and analyzed ten hours after the heat shock. dat-1::gfp is ectopically expressed in many cells of the embryo. Scale bar: 20 μm. In the presence of a pan-neuronal marker (rgef-1::rfp)(panel c), the ectopic DA-fate expressing cells can be identified as neurons.
(d,e) Ectopic expression of ast-1 under the control of the ectodermal promoter unc-119 and the DA /5HT-neuron specific promoter bas-1 leads to ectopic expression of dat-1::gfp in additional neurons compared to wild-type worms (red arrowheads). Similar effects were observed in multiple lines (2/2 lines for bas-1 driver; 2/3 lines for unc-119 driver). Scale bar: 100 μm.

(a,b) Coronal section TH immunostaining of a wild type (A) and Etv1 mutant (B) newborn pup (P0) olfactory bulb. Scale bar: 150 μm.
(c) Quantification of TH positive cells in wild type and Etv1 mutants at P0. Etv1 mutants show a significant reduction of the TH positive cells already at this stage (n=3, p-value=0.00009).
(d, e) Coronal section tuj1 immunostaining and DAPI staining of a wild type (d, d’) and Etv1 mutant (e, e’) P0 glomerular layer to label neurons and cell nuclei. Scale bar: 40 μm.
(f) Quantification of DAPI nuclei in wild type and Etv1 mutants at P0. Glomerular layer cell density is similar between wild type and Etv1 mutants (n=3, p-value=0.93).
(g) Overexpression of Etv1 can induce DA differentiation. Dissociated P0 olfactory bulbs were transfected with GFP and PCDNA3.1 (control) or GFP and Etv1 cloned into the PCDNA expression vector, plated and cultured for 4 days. Etv1 overexpression leads to increased number of tyrosine hydroxylase positive cells.
(h) Analysis of the activation of TH promoter by Etv1 in COS cells. The dotted line indicates the level of luciferase activation of the empty luciferase vector observed upon Etv1 transfection. “DA motif” = phylogenetically conserved match to the a VGGAWRNV consensus. n=3 independent experiments for each construct.