Bae CJ , Jeong J , Saint-Jeannet JP .

R01-DE014212 NIDCR NIH HHS, R01 DE014212 NIDCR NIH HHS

             brain development

	Figure 1: egr4 is a downstream target of Pax3 and Zic1. (a) Whole-mount in situ hybridization for egr4, pax3 and zic1. The DIG-labeled probes were used alone or in combination (pax3/egr4 and zic1/egr4). Dorsal views, anterior to top. Scale bar, 300 μm. (b) Injection in one blastomere at the 2-cell stage of MOs to block Pax3 (pax3MO; 40 ng) or Zic1 (zic1MO; 40 ng) function resulted in a reduction of egr4 expression. The injected side is to the right as indicated by the presence of the lineage tracer (Red-Gal). Dorsal views, anterior to top. Scale bar, 300 μm. (c) The graph indicates the percentage of embryos with normal (white) or reduced/lost (red) egr4 expression. The number of embryos analyzed is indicated on top of each bar. (d) mRNA encoding pax3GR and zic1GR (100 pg each), alone or in combination were injected into both blastomeres in the animal pole at the 2-cell stage. At the blastula stage (stage 9), animal cap explants were dissected and cultured for 8 hours in the presence of dexamethasone (DEX). In some samples, cyclohexamide (CHX) was used to block protein synthesis. (e) egr4 expression in pax3GR and zic1GR injected animal cap explants analyzed by qPCR.
	Figure 2: egr4 is specifically and transiently expressed in r5 and r6. (a) Analysis of the spatiotemporal expression of egr4 as compared to genes expressed in similar region of the ectoderm, including en2 (midbrain-hindbrain boundary), krox20/egr2 (hindbrain r3/r5), mafb/kreisler (hindbrain r5/r6) and hnf1b/vHnf1b (hindbrain r5/r6/r7). The probes were used alone or in combination as indicated. Scale bar, 300 μm. (b) Temporal dynamics of the expression of egr4, krox20, mafb, and hnf1b analyzed by qPCR in whole embryos at the indicated stages. The values were normalized to odc1. (c) Two-color in situ hybridization for egr4/snail2, egr4/fgf3, and egr4/mafb at the neurula stage. The expression of egr4 at the neural plate border overlaps with the neural crest marker, snail2 (arrows). egr4 is detected immediately adjacent and posterior to fgf3 expression domain in r4. egr4 and mafb have overlapping expression domains in r5/r6. Scale bar, 300 μm.
	Figure 3: Egr4 is required for caudal hindbrain development. (a) Increasing amounts of egr4MO1 10 ng (+), 100 ng (++), and 1000 ng (+++) blocks translation directed by egr4 mRNA in an in vitro coupled transcription/translation reaction. Full-length blot is presented in Supplementary Figure 1. (b) Schematic representation of the egr4 locus. The PCR primers used for the analysis of spliced transcripts are indicated (blue arrows). The position of the splice (egr4MO2) blocking MO is indicated. (c) In egr4MO2-injected embryos a larger egr4 transcript is detected (~2.0 kb) due to intron retention. For all samples, the RT-PCR was performed under the same experimental conditions. (d–f) Unilateral injection of egr4MO1 at the 2-cell stage caused a reduction of expression of krox20 in r5 (d), mafb in r5/r6 (e) and hoxa3 (f). Note the residual krox20 in r5 (arrowheads). Injection of Xenopus egr4GR (e) or mouse Egr4GR (g) mRNAs (100 pg) can efficiently rescue mafb or krox20 expression in egr4-depleted embryos. (h) Unilateral injection of egr4MO2 results in a specific reduction/loss of expression of krox20 in r5 and mafb in r5/r6. Note the residual krox20 in r5 (arrowhead; left panel). (i–j) Unilateral injection of egr4MO1 (50 ng) at the 2-cell stage caused a posterior expansion of hoxb1 and fgf3 (i), while hnf1b and meis3 were largely unaffected (j). (k) Unilateral injection of meis3MO (40 ng) at the 2-cell stage caused a reduction of egr4 and mafb expression. (l) Egr4 knockdown (egr4MO1; 40 ng) has no effect on snail2 and sox9 expression at the neural plate border. In all panels (d–l), the graphs indicate the percentage of embryos with normal (white), reduced/lost (red) or expanded/ectopic (blue) gene expression. The number of embryos analyzed is indicated on top of each bar. Dorsal views, anterior to top. (m) At stage 25 egr4MO1-injected embryos (40 ng) show a specific loss of anterior branchial neural crest stream that normally travels to the third branchial arch (red arrows). Lateral views, anterior to left (uninjected side; black arrows) or to right (injected side; red arrows), dorsal to top. Panels (d–m) scale bars, 300 μm. (n) Transverse section through an embryo injected with 40ng of egr4MO1. On the injected side (red arrow) the otic vesicle failed to form. The otic vesicle on the control side is indicated (black arrow). hb: hindbrain; ov: otic vesicle.
	Figure 4: Cross-regulation of Hnf1b and Egr4. (a) Injection of 100 pg hnf1bGR mRNA expands egr4 and mafb expression anteriorly, while repressing hoxb1 and fgf3 expression. (a) Injection of 100 pg egr4GR mRNA expands krox20 and mafb expression and represses hnf1b, hoxb1 and fgf3 expression. The graphs indicate the percentage of embryos with normal (white), reduced/lost (red) or expanded/ectopic (blue) gene expression. The number of embryos analyzed is indicated on top of each bar. (c) Similar results were obtained by injection of 100 pg mouse Egr4 (mEgr4GR) mRNA. (d) Embryos injected with 100 pg of mouse Krox20 (mKrox20GR) or Xenopus mafb (mafbGR) mRNAs exhibit a dramatic reduction of hnf1b and egr4 expression. (e) Two-color in situ hybriidization for egr4/hnf1b and krox20/hnf1b. Overtime, the anterior limit of hnf1b retracts posteriorly and segregates from the r5 egr4 and krox20 expression domains. Dorsal views, anterior to top. In all panels scale bars, 300 μm.
	Figure 5: Hnf1b directly activates egr4 expression. (a) In Xenopus Hnf1b induces mafb and krox20 indirectly through the activation of egr4. (b) In the mouse Hnf1b directly activates mafb and krox20 expression. (c) The expansion of mafb in embryos injected with hnf1bGR mRNA (10 pg) is completely blocked by co-injection of egr4MO2, but not by a control morpholino (ctrMO). Scale bar, 300 μm. The graph indicates the percentage of embryos with normal (white), reduced/lost (red) or expanded (blue) mafb expression. The number of embryos analyzed is indicated on top of each bar. (d) A conserved region upstream of the Mafb gene containing and Hnf1 binding site has been characterized as an r5/r6-specific enhancer (S5). (f) A putative S5 enhancer (pS5) downstream of Xenopus tropicalis egr4 containing two putative Hnf1 binding sites was identified using rVISTA 2.0. (e, g) Schematic representation of the S5 and pS5 enhancer around Mafb (mouse) and egr4 (Xenopus) genes, respectively. (h) Experimental procedure to test the activity of the pS5 in Xenopus embryo. (i) Fold EGFP induction analyzed by qPCR. Values are normalized to ef1α. Graph represents mean ± S.E. of 3 independent experiments. *, P<0.05; **, P<0.01; versus embryos injected with each vector alone. Student's t-test.
	Figure 6: Model for the temporal regulation of r5 identity by Hnf1b, Egr4, Mafb and Krox20 in Xenopus posterior hindbrain.
	Supplementary Figure 1: Increasing amounts of egr4MO1, 10 ng (+), 100 ng (++), and 1000 ng (+++) blocks translation directed by egr4 mRNA in an in vitro coupled transcription/translation reaction. Full blott of the cropped version presented in Figure 3a.
	The coinjection of low doses of translation (egr4MO1) and splice (egr4MO2) blocking antisense shows an additive effect on the repression of mafb expression at the neurula stage. The graph indicates the percentage of embryos with normal (white), reduced/lost (red) or expanded/ectopic (blue) gene expression. The number of embryos analyzed is indicated on top of each bar.
	egr4 (early growth response 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior up.
	Figure 1. egr4 is a downstream target of Pax3 and Zic1.(a) Whole-mount in situ hybridization for egr4, pax3 and zic1. The DIG-labeled probes were used alone or in combination (pax3/egr4 and zic1/egr4). Dorsal views, anterior to top. Scale bar, 300 μm. (b) Injection in one blastomere at the 2-cell stage of MOs to block Pax3 (pax3MO; 40 ng) or Zic1 (zic1MO; 40 ng) function resulted in a reduction of egr4 expression. The injected side is to the right as indicated by the presence of the lineage tracer (Red-Gal). Dorsal views, anterior to top. Scale bar, 300 μm. (c) The graph indicates the percentage of embryos with normal (white) or reduced/lost (red) egr4 expression. The number of embryos analyzed is indicated on top of each bar. (d) mRNA encoding pax3GR and zic1GR (100 pg each), alone or in combination were injected into both blastomeres in the animal pole at the 2-cell stage. At the blastula stage (stage 9), animal cap explants were dissected and cultured for 8 hours in the presence of dexamethasone (DEX). In some samples, cyclohexamide (CHX) was used to block protein synthesis. (e) egr4 expression in pax3GR and zic1GR injected animal cap explants analyzed by qPCR.
	Figure 2. egr4 is specifically and transiently expressed in r5 and r6.(a) Analysis of the spatiotemporal expression of egr4 as compared to genes expressed in similar region of the ectoderm, including en2 (midbrain-hindbrain boundary), krox20/egr2 (hindbrain r3/r5), mafb/kreisler (hindbrain r5/r6) and hnf1b/vHnf1b (hindbrain r5/r6/r7). The probes were used alone or in combination as indicated. Scale bar, 300 μm. (b) Temporal dynamics of the expression of egr4, krox20, mafb, and hnf1b analyzed by qPCR in whole embryos at the indicated stages. The values were normalized to odc1. (c) Two-color in situ hybridization for egr4/snail2, egr4/fgf3, and egr4/mafb at the neurula stage. The expression of egr4 at the neural plate border overlaps with the neural crest marker, snail2 (arrows). egr4 is detected immediately adjacent and posterior to fgf3 expression domain in r4. egr4 and mafb have overlapping expression domains in r5/r6. Scale bar, 300 μm.
	Figure 3. Egr4 is required for caudal hindbrain development.(a) Increasing amounts of egr4MO1 10 ng (+), 100 ng (++), and 1000 ng (+++) blocks translation directed by egr4 mRNA in an in vitro coupled transcription/translation reaction. Full-length blot is presented in Supplementary Figure 1. (b) Schematic representation of the egr4 locus. The PCR primers used for the analysis of spliced transcripts are indicated (blue arrows). The position of the splice (egr4MO2) blocking MO is indicated. (c) In egr4MO2-injected embryos a larger egr4 transcript is detected (~2.0 kb) due to intron retention. For all samples, the RT-PCR was performed under the same experimental conditions. (d–f) Unilateral injection of egr4MO1 at the 2-cell stage caused a reduction of expression of krox20 in r5 (d), mafb in r5/r6 (e) and hoxa3 (f). Note the residual krox20 in r5 (arrowheads). Injection of Xenopus egr4GR (e) or mouse Egr4GR (g) mRNAs (100 pg) can efficiently rescue mafb or krox20 expression in egr4-depleted embryos. (h) Unilateral injection of egr4MO2 results in a specific reduction/loss of expression of krox20 in r5 and mafb in r5/r6. Note the residual krox20 in r5 (arrowhead; left panel). (i–j) Unilateral injection of egr4MO1 (50 ng) at the 2-cell stage caused a posterior expansion of hoxb1 and fgf3 (i), while hnf1b and meis3 were largely unaffected (j). (k) Unilateral injection of meis3MO (40 ng) at the 2-cell stage caused a reduction of egr4 and mafb expression. (l) Egr4 knockdown (egr4MO1; 40 ng) has no effect on snail2 and sox9 expression at the neural plate border. In all panels (d–l), the graphs indicate the percentage of embryos with normal (white), reduced/lost (red) or expanded/ectopic (blue) gene expression. The number of embryos analyzed is indicated on top of each bar. Dorsal views, anterior to top. (m) At stage 25 egr4MO1-injected embryos (40 ng) show a specific loss of anterior branchial neural crest stream that normally travels to the third branchial arch (red arrows). Lateral views, anterior to left (uninjected side; black arrows) or to right (injected side; red arrows), dorsal to top. Panels (d–m) scale bars, 300 μm. (n) Transverse section through an embryo injected with 40ng of egr4MO1. On the injected side (red arrow) the otic vesicle failed to form. The otic vesicle on the control side is indicated (black arrow). hb: hindbrain; ov: otic vesicle.
	Figure 4. Cross-regulation of Hnf1b and Egr4.(a) Injection of 100 pg hnf1bGR mRNA expands egr4 and mafb expression anteriorly, while repressing hoxb1 and fgf3 expression. (a) Injection of 100 pg egr4GR mRNA expands krox20 and mafb expression and represses hnf1b, hoxb1 and fgf3 expression. The graphs indicate the percentage of embryos with normal (white), reduced/lost (red) or expanded/ectopic (blue) gene expression. The number of embryos analyzed is indicated on top of each bar. (c) Similar results were obtained by injection of 100 pg mouse Egr4 (mEgr4GR) mRNA. (d) Embryos injected with 100 pg of mouse Krox20 (mKrox20GR) or Xenopus mafb (mafbGR) mRNAs exhibit a dramatic reduction of hnf1b and egr4 expression. (e) Two-color in situ hybriidization for egr4/hnf1b and krox20/hnf1b. Overtime, the anterior limit of hnf1b retracts posteriorly and segregates from the r5 egr4 and krox20 expression domains. Dorsal views, anterior to top. In all panels scale bars, 300 μm.
	Figure 5. Hnf1b directly activates egr4 expression.(a) In Xenopus Hnf1b induces mafb and krox20 indirectly through the activation of egr4. (b) In the mouse Hnf1b directly activates mafb and krox20 expression. (c) The expansion of mafb in embryos injected with hnf1bGR mRNA (10 pg) is completely blocked by co-injection of egr4MO2, but not by a control morpholino (ctrMO). Scale bar, 300 μm. The graph indicates the percentage of embryos with normal (white), reduced/lost (red) or expanded (blue) mafb expression. The number of embryos analyzed is indicated on top of each bar. (d) A conserved region upstream of the Mafb gene containing and Hnf1 binding site has been characterized as an r5/r6-specific enhancer (S5). (f) A putative S5 enhancer (pS5) downstream of Xenopus tropicalis egr4 containing two putative Hnf1 binding sites was identified using rVISTA 2.0. (e, g) Schematic representation of the S5 and pS5 enhancer around Mafb (mouse) and egr4 (Xenopus) genes, respectively. (h) Experimental procedure to test the activity of the pS5 in Xenopus embryo. (i) Fold EGFP induction analyzed by qPCR. Values are normalized to ef1α. Graph represents mean ± S.E. of 3 independent experiments. *, P<0.05; **, P<0.01; versus embryos injected with each vector alone. Student's t-test.
	Figure 6. Model for the temporal regulation of r5 identity by Hnf1b, Egr4, Mafb and Krox20 in Xenopus posterior hindbrain.

Aoki, Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. 2003, Pubmed, Xenbase

Aoki, Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. 2003, Pubmed , Xenbase
Aragón, vHnf1 regulates specification of caudal rhombomere identity in the chick hindbrain. 2005, Pubmed
Bae, Identification of Pax3 and Zic1 targets in the developing neural crest. 2014, Pubmed , Xenbase
Barembaum, Identification and dissection of a key enhancer mediating cranial neural crest specific expression of transcription factor, Ets-1. 2013, Pubmed
Bradley, The structure and expression of the Xenopus Krox-20 gene: conserved and divergent patterns of expression in rhombomeres and neural crest. 1993, Pubmed , Xenbase
Brivanlou, Expression of an engrailed-related protein is induced in the anterior neural ectoderm of early Xenopus embryos. 1989, Pubmed , Xenbase
Chomette, Krox20 hindbrain cis-regulatory landscape: interplay between multiple long-range initiation and autoregulatory elements. 2006, Pubmed
Cordes, The mouse segmentation gene kr encodes a novel basic domain-leucine zipper transcription factor. 1994, Pubmed
Crosby, Neural-specific expression, genomic structure, and chromosomal localization of the gene encoding the zinc-finger transcription factor NGFI-C. 1992, Pubmed
Demartis, Cloning and developmental expression of LFB3/HNF1 beta transcription factor in Xenopus laevis. 1994, Pubmed , Xenbase
DEOL, THE ABNORMALITIES OF THE INNER EAR IN KREISLER MICE. 1964, Pubmed
Dibner, XMeis3 protein activity is required for proper hindbrain patterning in Xenopus laevis embryos. 2001, Pubmed , Xenbase
Fraguas, egr-4, a target of EGFR signaling, is required for the formation of the brain primordia and head regeneration in planarians. 2014, Pubmed
Frohman, Altered rhombomere-specific gene expression and hyoid bone differentiation in the mouse segmentation mutant, kreisler (kr). 1993, Pubmed
Fu, A genome-wide screen for spatially restricted expression patterns identifies transcription factors that regulate glial development. 2009, Pubmed
Gavalas, Retinoid signalling and hindbrain patterning. 2000, Pubmed
Ghislain, Neural crest patterning: autoregulatory and crest-specific elements co-operate for Krox20 transcriptional control. 2003, Pubmed
Gilardi, Krox-20: a candidate gene for the regulation of pattern formation in the hindbrain. 1991, Pubmed
Godsave, Expression patterns of Hoxb genes in the Xenopus embryo suggest roles in anteroposterior specification of the hindbrain and in dorsoventral patterning of the mesoderm. 1994, Pubmed , Xenbase
Gutkovich, Xenopus Meis3 protein lies at a nexus downstream to Zic1 and Pax3 proteins, regulating multiple cell-fates during early nervous system development. 2010, Pubmed , Xenbase
Harland, In situ hybridization: an improved whole-mount method for Xenopus embryos. 1991, Pubmed , Xenbase
Hernandez, vhnf1 integrates global RA patterning and local FGF signals to direct posterior hindbrain development in zebrafish. 2004, Pubmed
Hong, The activity of Pax3 and Zic1 regulates three distinct cell fates at the neural plate border. 2007, Pubmed , Xenbase
Irving, Progressive spatial restriction of Sek-1 and Krox-20 gene expression during hindbrain segmentation. 1996, Pubmed
Ishibashi, Distinct roles of maf genes during Xenopus lens development. 2001, Pubmed , Xenbase
Kim, The vHNF1 homeodomain protein establishes early rhombomere identity by direct regulation of Kreisler expression. 2005, Pubmed
Kolm, Efficient hormone-inducible protein function in Xenopus laevis. 1995, Pubmed , Xenbase
Lecaudey, The zebrafish Iroquois gene iro7 positions the r4/r5 boundary and controls neurogenesis in the rostral hindbrain. 2004, Pubmed
Lee, Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). 1996, Pubmed
Lee, Early development of the thymus in Xenopus laevis. 2013, Pubmed , Xenbase
Lombardo, Expression and functions of FGF-3 in Xenopus development. 1998, Pubmed , Xenbase
Luo, Induction of neural crest in Xenopus by transcription factor AP2alpha. 2003, Pubmed , Xenbase
Manzanares, The role of kreisler in segmentation during hindbrain development. 1999, Pubmed
Manzanares, Krox20 and kreisler co-operate in the transcriptional control of segmental expression of Hoxb3 in the developing hindbrain. 2002, Pubmed , Xenbase
Marín, Hindbrain patterning: FGFs regulate Krox20 and mafB/kr expression in the otic/preotic region. 2000, Pubmed
Maves, FGF3 and FGF8 mediate a rhombomere 4 signaling activity in the zebrafish hindbrain. 2002, Pubmed
Mayor, Induction of the prospective neural crest of Xenopus. 1995, Pubmed , Xenbase
Moens, valentino: a zebrafish gene required for normal hindbrain segmentation. 1996, Pubmed
Monsoro-Burq, Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction. 2005, Pubmed , Xenbase
Nonchev, Segmental expression of Hoxa-2 in the hindbrain is directly regulated by Krox-20. 1996, Pubmed
O'Donovan, The EGR family of transcription-regulatory factors: progress at the interface of molecular and systems neuroscience. 1999, Pubmed
Park, Xaml1/Runx1 is required for the specification of Rohon-Beard sensory neurons in Xenopus. 2012, Pubmed , Xenbase
Prince, Zebrafish hox genes: expression in the hindbrain region of wild-type and mutants of the segmentation gene, valentino. 1998, Pubmed
Ruiz i Altaba, Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos. 1991, Pubmed , Xenbase
Sadaghiani, Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy. 1987, Pubmed , Xenbase
Santagati, Cranial neural crest and the building of the vertebrate head. 2003, Pubmed
Sato, Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm. 2005, Pubmed , Xenbase
Schneider-Maunoury, Disruption of Krox-20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain. 1993, Pubmed
Schneider-Maunoury, Hindbrain signals in otic regionalization: walk on the wild side. 2007, Pubmed
Sham, The zinc finger gene Krox20 regulates HoxB2 (Hox2.8) during hindbrain segmentation. 1993, Pubmed
Slack, An interaction between dorsal and ventral regions of the marginal zone in early amphibian embryos. 1980, Pubmed , Xenbase
Spokony, The transcription factor Sox9 is required for cranial neural crest development in Xenopus. 2002, Pubmed , Xenbase
Studer, Genetic interactions between Hoxa1 and Hoxb1 reveal new roles in regulation of early hindbrain patterning. 1998, Pubmed
Sturgeon, Cdx1 refines positional identity of the vertebrate hindbrain by directly repressing Mafb expression. 2011, Pubmed
Sun, vhnf1, the MODY5 and familial GCKD-associated gene, regulates regional specification of the zebrafish gut, pronephros, and hindbrain. 2001, Pubmed
Topilko, Multiple pituitary and ovarian defects in Krox-24 (NGFI-A, Egr-1)-targeted mice. 1998, Pubmed
Topilko, Krox-20 controls myelination in the peripheral nervous system. 1994, Pubmed
Tourtellotte, Sensory ataxia and muscle spindle agenesis in mice lacking the transcription factor Egr3. 1998, Pubmed
Tourtellotte, Infertility associated with incomplete spermatogenic arrest and oligozoospermia in Egr4-deficient mice. 1999, Pubmed
Trainor, Patterning the cranial neural crest: hindbrain segmentation and Hox gene plasticity. 2000, Pubmed
Weisinger, Analysis of expression and function of FGF-MAPK signaling components in the hindbrain reveals a central role for FGF3 in the regulation of Krox20, mediated by Pea3. 2010, Pubmed
Wiellette, vhnf1 and Fgf signals synergize to specify rhombomere identity in the zebrafish hindbrain. 2003, Pubmed
Wilkinson, Segment-specific expression of a zinc-finger gene in the developing nervous system of the mouse. 1989, Pubmed