Sethi AJ , Angerer RC , Angerer LM .

R01 GM025553 NIGMS NIH HHS , GM25553 NIGMS NIH HHS , Intramural NIH HHS

	Figure 1. Gene Regulatory Networks Underlying Endomesoderm Induction(A) The micromere determinant Pmar1 (circled in red) activates the PMC-GRN in micromere progeny and is sufficient for micromere-derived endomesoderm-inducing signals. The E-EM/En-GRNs (up to 17 h postfertilization) integrate the regulatory functions of maternal and zygotic core factors that drive the earliest steps of endomesoderm progenitor specification in sea urchin embryos. The zygotically expressed core factors Z13, Eve, Wnt8, Blimp1, FoxA, and Brachyury (Bra) (circled in black) accumulate in presumptive endomesoderm during early developmental stages and could potentially respond to early inductive inputs from micromere descendants.(B) Schematic depicting an experiment that reveals micromere-derived endomesoderm inductive signals, which are sufficient to induce ectopic endo16 expression and complete archenteron formation in animal blastomeres, and are also necessary for normal vegetal endo16 expression and timely gastrulation in the sea urchin embryo. The regulatory interactions among these signals and the overall EM-GRN are unknown. GRN diagram is adapted from [8].
	Figure 2. EM-GRN Factors Induced by the Pmar1-Dependent Endomesoderm Induction(A) In all cases shown in (B–D), two-color FISH was used to detect ectopic induction of test transcripts (green) adjacent to gfp (red)-mRNA–expressing blastomeres in embryos injected with 0.02 μg/μl SpHE-gfp and 0.015 μg/μl SpHE-pmar1 (e–h in each case) compared to embryos injected with 0.02 μg/μl SpHE-gfp alone (a–d in each case).Test transcripts: (B) z13 expression at 16–18 h p.f. (C) foxA expression at 20 h p.f.(D) eve expression at 14 h p.f. Embryos shown are representative of over 80% of at least 50 embryos in each of three separate experiments in which gfp mRNA was detected in nonvegetal regions.Black scale bar in (B) d represents approximately 20 μm. DIC images in (B) d and h, (C) d and h, and (D) d and h illustrate the absence of detectable developmental defects in injected embryos. gfp-expressing cells containing Pmar1 in (C) g and h have adopted a mesenchyme phenotype and ingressed into the blastocoel at 20 h p.f.
	Figure 3. EM-GRN Factors That Are Not Induced by Ectopic Pmar1 Expression(A) In all cases shown in (B–D), two-color FISH showed the absence of ectopic induction of test transcripts (green) adjacent to gfp-mRNA–expressing (red) blastomeres in embryos injected with 0.02 μg/μl SpHE-gfp and 0.015 μg/μl SpHE-pmar1 (e–h in each case) compared to embryos injected with 0.02 μg/μl SpHE-gfp (a–d in each case).Test transcripts:(B) blimp1 expression at 14–16 h p.f.(C) wnt8 expression at 14–16 h p.f.(D) brachyury expression at 20 h p.f.Embryos shown are representative of 100% of at least 50 embryos in each of three separate experiments in which gfp mRNA was detected in nonvegetal regions. DIC images in (B ) d and h, (C) d and h, and (D) d illustrate the absence of detectable developmental defects in injected embryos. gfp-expressing cells containing Pmar1 in (D) panels g and h have adopted a mesenchyme phenotype and ingressed into the blastocoel at 20 h p.f.
	Figure 4. ActivinB-ALK4/5/7 Function Is Required for Timely Gastrulation and Pregastrular endo16 mRNA Expression(A) Embryos were injected with buffer (Control; a–c) or 1.2 mM ActivinB MO1 (ActBMO1; d–f) or 0.6 mM ActivinB MO2 (ActBMO2; g–i) and photographed at 36 (a, d, and g) and 72 (b, c, e, f, h, and i) h p.f.(B) Embryos were treated with DMSO (a and b) or 5 μM SB-431542 (c and d) for the first day of development and photographed at 36 (a and c) and 72 (b and d) h p.f. Compared to buffer-injected controls ([A] a), gut formation was delayed in embryos injected with ActivinB MO1 ([A] d) and ActivinB MO2 ([A] g). Similarly, gastrulation was delayed in SB-treated embryos ([B] c) compared to DMSO-treated controls ([B] a).(C) Reduced endo16 mRNA expression was observed at 26 h p.f. in 1.2 mM ActBMO1-injected (c) and 5 μM SB-treated (d) embryos compared to buffer-injected (a) or DMSO-treated (b) controls respectively. Images are representative of the phenotypes observed in over 80% of at least 100 embryos in three separate experiments.Black scale bar in (A) b represents approximately 40 μm.
	Figure 5. ActivinB Is Required for Pmar1-Mediated Endomesoderm InductionEmbryos were injected with either 0.02 μg/μl SpHE-gfp or 0.02 μg/μl SpHE-gfp and 0.015 μg/μl SpHE-pmar1, as indicated to the left of the images. Embryos were analyzed by two-color FISH for gfp (red) and either (A) endo16 (green) at 26 h p.f. or (B) gcm (green) transcripts at 18–20 h p.f. The presence of 1.4 mM ActivinBMO1 strongly reduces both ectopic and endogenous endo16 mRNA levels ([A], i–l vs. e–h, compare arrowheads in [A] j and k vs. f and g for residual endogenous endo16 expression in the presence of ActivinMO1), but not those of the pigment cell marker, gcm ([B] i–l vs. e–h). Embryos shown are representative of more than 80% of a minimum of 50 embryos in each of three separate experiments in which gfp was detected in nonvegetal regions of the embryo. DIC images in (A) d and (B) d, h, and l illustrate the absence of detectable developmental defects in injected embryos. gfp-expressing cells containing Pmar1 in (A) h and l have adopted a mesenchyme phenotype and ingressed into the blastocoel at 26 h p.f.
	Figure 6. ActivinB Is Required in Cells That Emit and Receive Pmar1-Dependent Induction SignalsIndividual blastomeres of embryos were injected as diagrammed to the left and analyzed by two-color FISH for gfp (red) and endo16 (green) transcripts. The concentrations of gfp mRNA, pmar1 mRNA, and ActivinB-MO1 used were 0.5 μg/μl, 0.2 μg/μl, and 1.4 mM, respectively. The presence of ActivinB MO1 in Pmar1-expressing cells (f–j) strongly inhibits ectopic and not endogenous (arrowheads in i and j) endo16 mRNA expression in the uninjected half of the embryo. When ActivinB MO1 is injected exclusively into blastomeres that respond to Pmar1-dependent induction (k–o), both ectopic and endogenous endo16 mRNA expression are strongly reduced. At the mesenchyme blastula stage shown, Pmar1 (gfp)-expressing cells in c, h, m and b, g, l acquire mesenchymal character and ingress into the blastocoel. Images represent the phenotypes observed in 16/19 (a–e), 43/47 (f–j), and 6/6 (k–o) injected embryos.
	Figure 7. activinB mRNA Distribution during Early Sea Urchin Development at the Stages IndicatedDuring early blastula and cleavage stages of development (a–c), low levels of activinB transcripts accumulate throughout the embryo. At the mesenchyme blastula stage (d), activinB mRNA is expressed in most of the embryo except one side of the ectoderm. At the late gastrula stage (e), activinB mRNA expression is detected in the gut and oral ectoderm and in the gut in prism (f) stage embryos.
	Figure 8. ActivinB-ALK4/5/7 Signaling Activates the Pmar1-Responsive E-EM/En-GRN during Normal Endomesoderm Specification(A) Embryos were treated with either (a–c) DMSO (Control) or (g–i) SB-431542 (SB; 5 μM) and analyzed by WMISH to detect (a and g) z13 at 8 h , (b and h) eve at 8 h, (c and i) foxA at 18 h. SB treatment strongly inhibits the accumulation of each of these transcripts in veg2 blastomeres at the stages assayed (g–i) compared to DMSO-treated controls (a–c). The same mRNAs were detected in embryos injected either with buffer (d–f) or ActivinB MO1 (ActBMO1; 1. 2 mM) (j–l) at 14 h (d and j), 10 h (e and k), or 20 h (f and l), respectively. z13 and foxA mRNA expression in veg2 blastomeres was strongly reduced in ActivinB MO1-injected embryos (j and l) compared to buffer-injected controls (d and f) assayed at corresponding stages. At the late cleavage stage, eve mRNA expression in the veg2 tier was unaffected by the presence of ActivinB MO1 (k vs. e).(B) Embryos were treated with either (a–c) DMSO or (g–i) SB (5 μM) and analyzed by WMISH to detect (a and g) brachyury at 18 h, (b and h) blimp1 at 14 h, and (c and i) wnt8 at 14 h. The same mRNAs were detected in embryos injected either with buffer (d–f) or ActBMO1 (1.2 mM) (j–l) at 20 h (d and j), 14 h (e and k), and 14 h (f and l), respectively. The accumulation of each of these transcripts in veg2 blastomeres was independent of ALK4/5/7 (g–i vs. a–c) and ActivinB (j–l vs. d–f) function at the stages assayed.Images in (A) and (B) represent the molecular phenotypes seen in over 80% of at least 50 embryos in three separate experiments.(C) Fate map of blastula stage embryo.
	Figure 9. Secondary Mesoderm Specification Is Independent of ActivinB-ALK4/5/7 Function(A) Embryos were treated with either (a and b) DMSO (Control) or (e and f) SB-431542 (5 μM) and analyzed by WMISH to detect (a and e) gcm at 18 h and (b and f) gataE at 18 h. The same mRNAs were detected in embryos injected either with (c and d) buffer or (g and h) ActivinB MO1 (1.2 mM) at 18 h (c, g, d, and h). The accumulation of each of these transcripts in veg2 secondary mesoderm precursors was independent of ALK4/5/7 (e and f vs. a and b) and ActivinB (g and h vs. c and d) function at the stages assayed. All images are representative of the molecular phenotypes observed in more than 80% of a minimum of 50 embryos in each of three separate experiments.(B) At 34 h p.f., (a–c) buffer-injected controls contain 29 gcm-mRNA expressing (n = 5 embryos examined) cells (green in b and c) compared to 40 (n = 5 embryos examined) such cells (green in e and f) in embryos injected with (d–f) 1.2 mM ActivinB MO1. Nuclei are stained with DAPI (blue).
	Figure 10. The Late Endoderm-GRN Is Independent of ActivinB-ALK4/5/7 Signaling(A) Embryos were treated with either (a and b) DMSO (Control) or (e and f) SB-431542 (5 μM) and analyzed by WMISH to detect (a and e) z13 at 26 h and (b and f) foxA at 26 h . The same mRNAs were detected in embryos injected either with (c and d) buffer or (g and h) ActBMO1 (1.2 mM) at 26 h (c, g, d, and h). The late accumulation of each of these transcripts in vegetal blastomeres was independent of ALK4/5/7 (e and f vs. a and b) and ActivinB (g and h vs. c and d) function at the stages assayed. Images are representative of the molecular phenotypes observed in more than 80% of a minimum of 50 embryos in each of three separate experiments.(B) Fate map of blastula-mesenchyme blastula stage embryo.
	Figure 11. Pmar1-Responsive Endomesoderm Specification Is Independent of the Micromere Signal That Clears Vegetal SoxB1(A) SoxB1 protein (green), gcm mRNA (red), and DAPI nuclear staining (blue) at 18 h p.f.: (a–c) buffer-injected controls; (d–f) 1.2 mM ActivinB MO1–injected embryos; and (g–i) 0.15 mM Delta MO-injected embryos. Images represent the phenotype observed in over 80% of at least 50 embryos in each of three separate experiments. SoxB1 protein clears normally from veg2 secondary mesoderm precursors in ActivinB (d–f vs. a–c) and Delta (g–i vs. a–c) morphant embryos.(B) (a–c) SoxB1 protein (green), z13 mRNA (red), and DAPI nuclear staining (blue) at 8 h p.f. in normal embryos showing high levels of SoxB1 protein in nuclei of z13-expressing cells at the cleavage stage of development. The optical section is slightly oblique with respect to the animal-vegetal axis, and z13-expressing cells are at the bottom.
	Figure 12. Pregastrular EM-GRN Subnetworks Mediating Micromere-Mediated Endomesoderm InductionThe micromere determinant Pmar1 activates the PMC-GRN and transmits endomesoderm-inducing signals to macromere descendants. The Pmar1-responsive genes z13, foxA, eve (and the cardinal endomesoderm marker, endo16) define a responding EM-GRN (red arrows leading to factors circled with solid red lines) that is activated in animal blastomeres. The Pmar1-unresponsive genes wnt8, blimp1, and brachyury (Bra) (circled with hatched black lines) are dispensable for ectopic Pmar1-mediated endomesoderm induction, which probably occurs without inducing ectopic nuclearization of β-catenin in animal blastomeres. In both animal and vegetal contexts, ActivinB is necessary for Pmar1-activated endomesoderm induction and eventual endo16 expression (shown at bottom right of diagram) and is an essential component of the PMC-GRN and E-EM/En-GRNs. ES refers to an early signal, thought to depend on Pmar1, that is sent from micromeres starting after fourth cleavage stage, which, like ActivinB, is required for endo16 expression and timely gastrulation. ActivinB-independent Pmar1-derived Delta signaling (blue arrow) specifies pigment and blastocoelar cell fate within secondary mesoderm precursors and regulates gcm expression in veg2 descendants (the SMC-GRN core factor, Gcm, shown in the diagram is not an element of the E-EM/En-GRNs). A third, unknown Pmar1-mediated signal clears SoxB1 (black line leading to factor circled with solid black line) from endomesoderm precursors and, contrary to previous models of pregastrular endomesoderm development, is independent of micromere-mediated specification of these blastomeres. GRN diagram is adapted from [8].

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