BIOCHEMISTRY. for the Article ''Regulatory DNA Required

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BIOCHEMISTRY. For the article ‘‘Regulatory DNA required for pnas.012584599), the authors note that the color quality in Figs. vnd͞NK-2 homeobox gene expression pattern in neuroblasts,’’ by 1–3 was not adequate. Higher quality versions of the figures and Xiaoping Shao, Keita Koizumi, Neil Nosworthy, Dong-Ping Tan, their legends appear below. Ward Odenwald, and Marshall Nirenberg, which appeared in number 1, January 8, 2002, of Proc. Natl. Acad. Sci. USA (99, 113–117; First Published December 18, 2001; 10.1073͞ www.pnas.org͞cgi͞doi͞10.1073͞pnas.112186899

Fig. 2. Endogenous Vnd͞NK-2 expression. ␤-Gal expression regulated by the Ϫ5.3͞ϩ0.35-kb DNA fragment from the 5Ј-upstream region of the vnd͞NK-2 gene in neuroectoderm compared with NBs. Red, Vnd͞NK-2 protein; green, ␤-Gal protein. (A) Neuroectoderm layer of a double-labeled embryo at early stage 11. A column of neuroectodermal cells, one cell wide, that contain NK-2 protein is seen on each side of the mesectoderm. The arrowhead points to an NB that is close to the neuroectoderm layer (possibly 6–1) that has a higher level of ␤-Gal expression than that of neuroectodermal cells. (B) NB layer of double-labeled embryo; ␤-Gal expression pattern (green), directed by vnd͞ NK-2 (Ϫ5.3͞ϩ0.35)-␤-gal construct almost completely overlaps the endogenous Vnd͞NK-2 pattern; however, Vnd͞NK-2 but not ␤-Gal was expressed in NB 7-2 (arrow). The bar corresponds to 5 ␮m.

Fig. 1. Identification of Vnd͞NK-2 expressing NBs in the ventral nerve cord. To identify the NBs expressing Vnd͞NK-2, the enhancer trap NB marker Drosophila lines en-lacZ, wg-lacZ, and hkb5953 were used. In each line, ␤-Gal is expressed in identified NBs. Antibodies to Vnd͞NK-2 protein (red) and ␤-Gal protein (green) were used for double staining. (A) Double-stained en-lacZ embryo at stage 11; ␤-Gal is expressed in the engrailed pattern in posterior compartment cells. NBs 1-2, 6-1, 7-1, and 7-2 were shown to express both Vnd͞NK-2 and ␤-Gal proteins. The bar corresponds to 5 ␮m. (B) Double- stained wg-lacZ embryo at stage 11; NBs 5-1 and 5-2 are Vnd͞NK-2 and ␤-Gal positive. (C) Double-stained hkb5953 embryo at stage 11; only NB 2-1 contains both Vnd͞NK-2 and ␤-Gal proteins. (D) Schematic summary of the identity of Vnd͞NK-2 positive NBs. At stage 11, the following nine NBs (orange centers) were identified that are Vnd͞NK-2 positive per hemisegment: 1-2, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, and 7-2. MP-2 and 1-1 also express Vnd͞NK-2 earlier in development and are not shown here. Light blue, en-lacZ positive NBs; green, wg-lacZ positive NBs; purple, hkb5953 positive NBs (29).

8458 ͉ www.pnas.org Downloaded by guest on September 26, 2021 CORRECTIONS

Fig. 3. Regulatory DNA required for Vnd͞NK-2 pattern formation in NBs. Fragments of DNA from the 5Ј-upstream region of the vnd͞NK-2 gene, subcloned in the 5Ј-ﬂanking region of an enhancerless ␤-gal gene in a P- element, were used to generate transgenic lines of Drosophila. Antibodies directed against Vnd͞NK-2 protein (red) or ␤-Gal (green) were used to double- label NBs in the ventral nerve cord of stage 11 transgenic embryos. The neuroblasts are identiﬁed in the panels on the right. An NB with an orange center surrounded by a green ring expressed both Vnd͞NK-2 and ␤-Gal. A NB that expressed Vnd͞NK-2, but not ␤-Gal, is shown in red. Cells that ectopically expression of ␤-Gal in E1 and E3 cells. (D) ␤-Gal expression regulated by the express ␤-Gal are shown with white centers surrounded by green. (A) ␤-Gal Ϫ5.3͞Ϫ3.5-kb DNA fragment from the 5Ј-upstream region of the vnd͞NK-2 expression regulated by the Ϫ5.3͞Ϫ2.8-kb DNA fragment from the 5Ј- gene. ␤-Gal is expressed strongly in two NB, 1-2 and 3-1, and weakly in NB 2-1 upstream region of the vnd͞NK-2 gene in a transgenic embryo. The bar and NB 4-1. (E) ␤-Gal expression directed by the Ϫ5.3͞Ϫ4.0-kb DNA fragment corresponds to 5 ␮m. (B) ␤-Gal expression regulated by the Ϫ4.7͞Ϫ2.8-kb DNA from the 5Ј-upstream region of the vnd͞NK-2 gene in a transgenic embryo. fragment from the 5Ј-upstream region of the vnd͞NK-2 gene. The arrow and The four NBs that express both ␤-Gal and Vnd͞NK-2 shown in D also express arrowheads indicate cells (E1 and E2) that ectopically express ␤-Gal. (C) ␤-Gal both proteins in E; however, ␤-Gal expression in the embryo shown in E is expression regulated by the Ϫ4.0͞Ϫ2.8-kb DNA fragment from the 5Ј- weaker than the expression shown in D. The levels of expression of ␤-Gal in upstream region of the vnd͞NK-2 gene. Arrowheads indicate the ectopic NBs 1-2 and 3-1 are greater than the expression found in NBs 2-1 and 4-1.

PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 ͉ 8459 Downloaded by guest on September 26, 2021 Regulatory DNA required for vnd͞NK-2 homeobox gene expression pattern in neuroblasts

Xiaoping Shao*, Keita Koizumi†, Neil Nosworthy*, Dong-Ping Tan*, Ward Odenwald†, and Marshall Nirenberg*‡

*Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, and †Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-4036

Contributed by Marshall Nirenberg, November 1, 2001 Vnd͞NK-2 protein was detected in 11 neuroblasts per hemiseg- dorsal columns in the neural tube (5, 6, 12–14). Vnd͞NK-2 ment in Drosophila embryos, 9 medial and 2 intermediate neuro- homeodomain protein initiates neural development in the ven- .(blasts. Fragments of DNA from the 5؅-flanking region of the tral neuroectoderm by activating the proneural gene achaete (4 vnd͞NK-2 gene were inserted upstream of an enhancerless ␤- Vnd͞NK-2 also probably functions as part of the molecular galactosidase gene in a P-element and used to generate transgenic address of ventral neuroectodermal cells. Vnd͞NK-2 also is fly lines. Antibodies directed against Vnd͞NK-2 and ␤-galactosi- required for delamination and specification of NBs derived from dase proteins then were used in double-label experiments to the ventral column of neuroectodermal cells (15). correlate the expression of ␤-galactosidase and Vnd͞NK-2 proteins The vnd͞NK-2 gene is activated initially by Dorsal protein in identified neuroblasts. DNA region A, which corresponds to the (16). Twist is a coactivator of the vnd͞NK-2 gene (16); whereas, ؊4.0 to ؊2.8-kb fragment of DNA from the 5؅-flanking region of Snail represses vnd͞NK-2 in the mesoderm (16). Vnd͞NK-2 the vnd͞NK-2 gene was shown to contain one or more strong protein, directly or indirectly, promotes the continued expression enhancers required for expression of the vnd͞NK-2 gene in ten of the vnd͞NK-2 gene (15, 17). Single-minded represses the neuroblasts. DNA region B (؊5.3 to ؊4.0 kb) contains moderately vnd͞NK-2 gene in the mesectoderm (16), mediated by an strong enhancers for vnd͞NK-2 gene expression in four neuro- unidentified repressor (18). Vnd͞NK-2 homeodomain protein blasts. Hypothesized DNA region C, whose location was not iden- represses ind (5) and msh (15, 19). Ind also represses vnd͞NK-2.§ tified, contains one or more enhancers that activate vnd͞NK-2 Transplantation of dorsal neuroectodermal cells to the ventral gene expression only in one neuroblast. These results show that neuroectoderm converts the developmental fate of the trans- nucleotide sequences in at least three regions of DNA regulate the planted cells to that of ventral neuroectoderm (20), and the expression of the vnd͞NK-2 gene, that the vnd͞NK-2 gene can be change in developmental fate requires functional Drosophila activated in different ways in different neuroblasts, and that the EFG receptors (21). EGF receptors are activated by Spitz pattern of vnd͞NK-2 gene expression in neuroblasts of the ventral protein (22) secreted from neighboring ventral midline mesec- nerve cord is the sum of partial patterns. todermal cells (23) and͞or secreted Vein protein (24). The EGF receptor is a tyrosine protein kinase; activation of the EGF homeobox ͉ Drosophila receptor by Spitz or Vein activates the Ras–mitogen-activated protein kinase signaling pathway, which triggers the activation of ertilization of a Drosophila egg initiates 13 rounds of nuclear a protein kinase cascade and results in the entrance of activated Fdivision, resulting in an embryo that consists of a syncytium mitogen-activated protein kinase into the nucleus, which mod- of 5,000 to 6,000 nuclei. Before cellularization, two gene net- ulates gene expression by catalyzing the phosphorylation of transcription factors (for review, see ref. 25). These and other works, one functioning in the antero-posterior axis and a second ͞ functioning in the dorso-ventral axis, initiate regulatory cascades results (26–28) suggest that the vnd NK-2 gene also is activated that regulate the unique identity of each of 30 neuroblasts (NBs) by an unknown mechanism mediated by EGF receptors of in each hemisegment. By the time NBs form, segment polarity ventral neuroectodermal cells, via the Ras–mitogen-activated BIOCHEMISTRY genes define the identity of each NB in the antero-posterior axis. protein kinase signaling pathway. In contrast, ventral neuroec- Extrinsic signaling and intrinsic regulators including the tran- todermal cells transplanted to dorsal neuroectoderm continue to develop autonomously as ventral neuroblasts (20). scription factor Dorsal, the epidermal growth factor receptor ͞ (EGF) ligand Spitz, and the Bmp-4 family member Dpp, estab- We have been studying regulation of vnd NK-2 gene expression to determine how a pattern of NBs is formed in the central lish expression domains of three homeodomain proteins that in Ј turn establish unique NB identities in the dorso-ventral axis. nervous system. Previously, segments of DNA from the 5 - upstream region of the vnd͞NK-2 gene were ligated to an Neural pathways of development are initiated independently ␤ ␤ in three longitudinal columns of neuroectodermal cells along the enhancerless -galactosidase ( -gal) reporter gene in a P- dorso-ventral axis of the central nervous system. The ventral element vector and used to generate transgenic lines of flies (17). ͞ The Ϫ8.4͞ϩ0.35-kb DNA fragment from the 5Ј-flanking region nervous system defective (Vnd NK-2) homeodomain protein ͞ (1–3) initiates neural development in the ventral (i.e., medial) of the vnd NK-2 gene was shown to contain the sequences needed for expression of ␤-galactosidase (␤-Gal) in a pattern portion of neuroectoderm (4), and Intermediate neuroblast ͞ Ϫ ͞ϩ defective (Ind) homeodomain protein initiates neural develop- similar to that of Vnd NK-2; whereas a 5.3 0.35-kb DNA fragment expressed ␤-Gal in a pattern that was similar to that of ment in the intermediate column of neuroectoderm (5, 6). The ͞ mechanism of initiating neural development in the dorsal neu- Vnd NK-2 in the ventral nerve cord, but expression was lacking roectoderm column has not been identified; however, Muscle in some cephalic neuroectodermal cells or NBs (17). Smaller segment homeodomain (Msh) is expressed in the dorsal column of neuroectoderm and is required for specification of some Abbreviations: NB, neuroblast; EGF, epidermal growth factor; ␤-Gal, ␤-galactosidase. dorsal neuroblasts (7, 8). The segmentation genes, wingless (9), ‡To whom reprint requests should be addressed. E-mail: [email protected]. gooseberry distal (10), and hedgehog (11) function in the neuro- §Zhao, G. & Skeath, J.B. (2001) 41st Annual Drosophila Meeting, Washington, D.C., ectoderm to specify the identity of neuroblasts along the ante- abstr. 186. rior–posterior axis of the embryo. Vertebrate homologs of ͞ The publication costs of this article were defrayed in part by page charge payment. This vnd NK-2, ind, and msh, such as mouse NKx-2.2, Gsh, and Msx, article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. respectively, also are expressed in ventral, intermediate, and §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.012584599 PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 113–117 fragments of DNA from the 5Ј-flanking region of the vnd͞NK-2 Immunohistochemistry. The embryos from each transgenic fly line gene did not regulate ␤-Gal expression in the pattern expected were collected and fixed with 3.7% formaldehyde in PBS. The for Vnd͞NK-2 (17). Estes et al. (18) recently reported the embryos were stained first with rabbit antibody against ligation of fragments of DNA from the 5Ј-flanking region of the Vnd͞NK-2 full-length protein (1͞500 dilution) and then with ␤ vnd͞NK-2 gene to a ␤-gal reporter gene in a P-element and mouse monoclonal antibody against -Gal (Promega) (1:100 showed in transgenic flies that Single-minded (Sim) represses dilution). The embryos then were stained with CyTM5- the vnd͞NK-2 gene indirectly, mediated by an unidentified conjugated goat anti-rabbit IgG (1:100-fold dilution) and fluo- repressor that binds to one or more nucleotide sequences in rescein-conjugated goat anti-mouse IgG (1:100 fold dilution) DNA between Ϫ3.6 and Ϫ3.1 kb in the 5Ј-flanking region of the (Jackson ImmunoResearch). The embryos were washed with vnd͞NK-2 gene. In addition, they showed that three regions of PBT (PBS, 0.1% BSA, and 0.1% Tween 20) before and after incubation with each antibody (15 min per wash, four washes) at DNA in the 5Ј-flanking region of the vnd͞NK-2 gene, room temperature and then incubated in PBT plus 0.5% normal Ϫ5.3͞Ϫ4.2, Ϫ4.2͞Ϫ3.1, and Ϫ3.1͞Ϫ2.8 kb, are required for goat serum for 30 min at room temperature before application vnd͞NK-2 gene expression (18). of each antibody. Double-stained embryos were preserved in In this report, information is provided on the identification of ͞ Vectorshield anti-fading medium (Vector Laboratories), and NBs that express Vnd NK-2 homeodomain protein and on examined by using a Zeiss LSM 410 confocal microscope. regions of DNA from the 5Ј-flanking region of the vnd͞NK-2 gene involved in regulation of vnd͞NK-2 gene expression in Results and Discussion specific NBs. Preliminary reports of some of these results were Identification of Neuroblasts That Express vnd͞NK-2. ͞ ʈ The vnd NK-2 presented previously.¶ gene is expressed initially in the Drosophila embryo during the 12th nuclear doubling (stage 4) in two longitudinal stripes of Materials and Methods nuclei, each stripe about five nuclei in width (16). Each Fly Strains. All fly stocks were maintained under standard culture Vnd͞NK-2 positive stripe of nuclei identifies the ventral (i.e., conditions. Their sources were as follows: ⌬2–3 Sb͞Ser was medial) column of neuroectoderm (2, 3, 16). At late stage 8, the obtained from Akira Chiba, hkb5953 from Krishna M. Bhat, first NBs delaminate from the neuroectoderm layer. The Emory University (29), and wingless-lacZ (wg-lacZ) and en- vnd͞NK-2 gene is expressed in all medial NBs. The expression grailed-lacZ (en-lacZ) from Judith A. Kassis, National Institutes of Vnd͞NK-2 in neuroectoderm is narrowed progressively, and of Health. by completion of neuroblast formation (late stage 11), expression is restricted to the ventral column of neuroectodermal cells. Preparation of DNA Constructs. The following five DNA fragments Some NBs that express the vnd͞NK-2 gene were identified in from the 5-flanking region of the vnd͞NK-2 gene, Ϫ5.3 to Ϫ2.8 previous studies (15, 16). Antibody double-label studies on the ͞ kb, Ϫ5.3 to Ϫ3.5 kb, Ϫ5.3 to Ϫ4 kb, Ϫ4.7 to Ϫ2.8 kb, and Ϫ4.0 identity of NBs that express the vnd NK-2 gene are shown in Fig. ␤ to Ϫ2.8 kb, were subcloned into a P-element vector, 1. Transgenic fly lines that express -Gal protein in identified 5953 pCaSpeRhs43lacZ (30) containing an enhancerless lacZ re- NBs (31), such as en-lacZ, wg-lacZ, and hkb , were used with an antibody directed against ␤-Gal, whereas an antibody against porter gene, by using EcoRI and BamHI restriction sites, re- ͞ ͞ spectively. The Ϫ5.3 kb to Ϫ2.8-kb fragment was generated by full-length Vnd NK-2 protein was used to detect Vnd NK-2 protein. Vnd͞NK-2 protein was detected in all medial NBs; NBs digestion with restriction enzymes; the other four fragments 1-1, MP-2, 5-2, and 7-1 in late stage 8 and early stage 9 embryos were synthesized by PCR. The primers for PCR were as follows: (data not shown). Vnd͞NK-2 expression in NB 1-1 is weaker Ϫ5.3 to Ϫ3.5-kb construct, 5Ј-CGACAGGCATTTTGAATTC- Ј Ј than in other NBs and disappears by stage 11. In addition, NB AAGTCTCCGTTTG-3 and 5 -CGGGATCCCCGGAAAT- MP-2 disappears by stage 11. The en-lacZ embryo shown in Fig. GCATCATTACCTATG-3Ј; Ϫ5.3 to Ϫ4-kb construct, 5Ј-CGA- ␤ Ј Ј 1A expressed -Gal (green) in the cytoplasm and nuclei of cells CAGGCATTTTGAATTCAAGTCTCCGTTTG-3 and 5 - in the Engrailed pattern in the posterior compartment, whereas Ј CGCGGATCCACCTTTAAGATGCGAATGTACTG-3 ; Vnd͞NK-2 protein in NBs, located mostly in nuclei, is shown in Ϫ Ϫ Ј 4.7 to 2.9-kb construct, 5 -CGGAATTCACTAAACTCA- red. NBs that express both Vnd͞NK-2 and ␤-Gal proteins were GTTGACCAACTGACC-3Ј and 5Ј-GAGTCCGCATTAGGA- identified by their position and by comparison with previous TCCTTCCTACACAGTTAG-3Ј; Ϫ4.0 to Ϫ2.9-kb construct, studies (15, 16) as 1-2, 6-1, 7-1, and 7-2. As shown in Fig. 1B, 5Ј-CGGAATTCCCTTTTTGACTTTTATACAGCACGG-3Ј incubation of wg-lacZ embryos with anti-Vnd͞NK-2 and anti and 5Ј-GAGTCCGCATTAGGATCCTTCCTACACAGT- ␤-Gal antibodies revealed two NBs per hemisegment that con- TAG-3Ј. All constructs were purified by equilibrium centrifu- tained both Vnd͞NK-2 and ␤-Gal proteins, a small cell that will gation in CsCl-ethidium bromide gradients. become NB 5-1, and NB 5-2. Hkb-lacZ has been shown to be expressed in NBs 2-1, 2-2, 2-4, 4-2, and 5-4 during stage 11 (32). Preparation of Transgenic Fly Lines. P-element constructs contain- As shown in Fig. 1C, only one NB, 2-1, contained both ing vnd͞NK-2 5Ј-flanking region DNA fragments of Ϫ5.3 to Vnd͞NK-2 and ␤-Gal proteins per hemisegment in an hkb5953 ͞ Ϫ2.8 kb, Ϫ5.3 to Ϫ3.5 kb, Ϫ5.3 to Ϫ4.0 kb, Ϫ4.7 to Ϫ2.8 kb, and embryo. Neuroblasts that express vnd NK-2 mRNA were iden- Ϫ4.0 to Ϫ2.8 kb were injected (1 ␮g͞␮l) into ⌬2–3 Sb͞Ser tified previously by in situ hybridization (15, 16). Mellerick and Drosophila Nirenberg (16) reported that most medial NB contained rela- embryos to generate transgenic lines. Transgenic ͞ lines are named Ϫ5.3͞Ϫ2.8, Ϫ5.3͞Ϫ3.5, Ϫ5.3͞Ϫ4.0, tively high amounts of vnd NK-2 mRNA, whereas some inter- Ϫ ͞Ϫ Ϫ ͞Ϫ ͞ Ј mediate or dorsal NBs contained much lower amounts of 4.7 2.8, and 4.0 2.8, according to the vnd NK-2 5 - ͞ flanking DNA fragment carried by the P-element in the Dro- vnd NK-2 mRNA. In this report, an antibody directed against Vnd͞NK-2 protein was used rather than RNA hybridization, sophila genome. Each P-element insertion was mapped to a and fewer intermediate and no dorsal NBs that contain chromosome and made homozygous. Vnd͞NK-2 were found. For example, NBs 2-2, 3-2, 4-2, 6-2, 7-3, and 7-4 previously were found to contain Vnd͞NK-2 mRNA ͞ ¶Shao, X., Koizumi, K., Tan, D.-P., Odenwald, W. & Nirenberg, M. (1999) Soc. Neurosci. (16), but we did not detect Vnd NK-2 protein in these NBs with Abstr. 25, 526. an antibody to Vnd͞NK-2. Chu et al. (15), using a vnd͞NK-2 ʈShao, X., Koizumi, K. Tan, D.-P., Odenwald, W. & Nirenberg, M. (2001) Soc. Neurosci. Abstr. mRNA hybridization assay, also reported that NBs 2-2 and 6-2 27, 1227. contain vnd͞NK-2 mRNA.

114 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.012584599 Shao et al. Fig. 2. Endogenous Vnd͞NK-2 expression. ␤-Gal expression regulated by the Ϫ5.3͞ϩ0.35-kb DNA fragment from the 5Ј-upstream region of the vnd͞NK-2 gene in neuroectoderm compared with NBs. Red, Vnd͞NK-2 protein; green, ␤-Gal protein. (A) Neuroectoderm layer of a double-labeled embryo at early stage 11. A column of neuroectodermal cells, one cell wide, that contain NK-2 protein is seen on each side of the mesectoderm. The arrowhead points to an NB that is close to the neuroectoderm layer (possibly 6-1) that has a higher level of ␤-Gal expression than that of neuroectodermal cells. (B) NB layer of double-labeled embryo; ␤-Gal expression pattern (green), directed by vnd͞NK-2 (Ϫ5.3͞ϩ0.35)-␤-gal construct almost completely overlaps the endogenous Vnd͞NK-2 pattern; however, Vnd͞NK-2 but not ␤-Gal was expressed in NB 7-2 (arrow). The bar corresponds to 5 ␮m.

found in adjacent neuroectodermal cells (not shown in Fig. 2A). Fig. 1. Identification of Vnd͞NK-2 expressing NBs in the ventral nerve cord. Relatively low expression of ␤-Gal in neuroectodermal cells also ͞ To identify the NBs expressing Vnd NK-2, the enhancer trap NB marker was found at earlier stages of development (not shown). The 5953 ␤ Drosophila lines en-lacZ, wg-lacZ, and hkb were used. In each line, -Gal is pattern of ␤-Gal expression directed by the Ϫ5.3͞ϩ0.35-kb expressed in identified NBs. Antibodies to Vnd͞NK-2 protein (red) and ␤-Gal DNA fragment from the 5Ј-upstream region of the vnd͞NK-2 protein (green) were used for double staining. (A) Double-stained en-lacZ ͞ embryo at stage 11; ␤-Gal is expressed in the engrailed pattern in posterior gene was similar to the pattern of Vnd NK-2 protein directed by compartment cells. NBs 1-2, 6-1, 7-1, and 7-2 were shown to express both the endogenous vnd͞NK-2 gene; however, ␤-Gal expression was Vnd͞NK-2 and ␤-Gal proteins. The bar corresponds to 5 ␮m. (B) Double- not detected in NB 7-2 (Fig. 2B) and in some NBs in the cervical stained wg-lacZ embryo at stage 11; NBs 5-1 and 5-2 are Vnd͞NK-2 and ␤-Gal region (not shown). These results show that the Ϫ5.3͞ϩ0.35-kb positive. (C) Double-stained hkb5953 embryo at stage 11; only NB 2-1 contains DNA fragment from the 5Ј-upstream region of the vnd͞NK-2 both Vnd͞NK-2 and ␤-Gal proteins. (D) Schematic summary of the identity of gene is sufficient to regulate Vnd͞NK-2 expression in most, but Vnd͞NK-2 positive NBs. At stage 11, the following nine NBs (orange centers) ͞ ͞ not all, NBs that express Vnd NK-2 and suggests that additional were identified that are Vnd NK-2 positive per hemisegment: 1-2, 2-1, 3-1, regulatory regions in DNA may be required for expression of the 4-1, 5-1, 5-2, 6-1, 7-1, and 7-2. MP-2 and 1-1 also express Vnd͞NK-2 earlier in vnd͞NK-2 gene in the neuroectoderm, NB 7–2, and in some

development and are not shown here. Light blue, en-lacZ positive NBs; green, BIOCHEMISTRY wg-lacZ positive NBs; purple, hkb5953 positive NBs (29). cervical NBs.

Regulatory DNA Required for Vnd͞NK-2 Pattern Formation. The Ϫ5.3 A summary of the nine NBs per hemisegment that express to ϩ0.35-kb DNA fragment from the 5Ј-upstream region of the Vnd͞NK-2 protein at stage 11, 1-2, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, vnd͞NK-2 gene was shown to contain most of the nucleotide and 7-2 is shown in Fig. 1D. In addition, NB MP-2 expresses sequences that are required to generate the normal pattern of Vnd͞NK-2 earlier in development, and NB 1-1 also transiently Vnd͞NK-2 expression in NBs in the embryonic ventral nerve expresses Vnd͞NK-2. Nine of the 11 NBs that express cord, whereas transgenic embryos containing a Ϫ2.8 to ϩ0.35-kb Vnd͞NK-2 homeodomain protein are medial NBs; two, 1-2 and DNA fragment did not express ␤-Gal (17). This suggested that 7-2, are intermediate NBs. major enhancers for Vnd͞NK-2 expression in NBs of the ventral nerve cord are located in a 2.5-kb region of DNA, between Ϫ5.3 Expression of ␤-Gal in Neuroectoderm Cells and NBs Directed by the and Ϫ2.8 kb. Smaller fragments of DNA within this 2.5-kb ؊5.3͞؉0.35-kb DNA Fragment from the 5؅-Upstream Region of the segment of DNA were prepared and subcloned in the P-element vnd͞NK-2 Gene. Transgenic lines of flies were generated that vector, pCaspeRhs43lacZ in the 5Ј-flanking region of the en- contain the Ϫ5.3͞ϩ0.35-kb DNA fragment from the vnd͞NK-2 hancerless ␤-gal gene. Twenty-four transgenic lines were gener- gene ligated to an enhancerless ␤-gal gene. The patterns of ated: five for the Ϫ5.3 to Ϫ2.8-kb DNA fragment (named expression of Vnd͞NK-2 protein resulting from endogenous Ϫ5.3͞Ϫ2.8), two for the Ϫ4.7͞Ϫ2.8 fragment, five containing vnd͞NK-2 gene expression and ␤-Gal in the neuroectoderm cell the Ϫ4.0͞Ϫ2.8 fragment, seven carrying the Ϫ5.3͞Ϫ3.5 frag- layer of a stage 11 embryo (Fig. 2A) are compared with the ment, and five lines carrying the Ϫ5.3͞Ϫ4.0 fragment. patterns of expression in the adjacent neuroblast layer (Fig. 2B). Antibodies directed against Vnd͞NK-2 or ␤-Gal proteins At stage 11, Vnd͞NK-2 and ␤-Gal proteins are expressed in a were used for double labeling of transgenic embryos. As shown medial column of neuroectodermal cells, 1 cell wide on each side in Fig. 3A, a stage 11 transgenic embryo with a wild-type of the ventral mesectoderm (Fig. 2A). The level of expression of vnd͞NK-2 gene and the Ϫ5.3͞Ϫ2.8 kb of DNA from the ␤-Gal in neuroectodermal cells (Fig. 2A) was much lower than 5Ј-flanking region of the vnd͞NK-2 gene ligated in the 5Ј- that of NBs (Fig. 2B), and some ectopic expression of ␤-Gal was flanking region of the ␤-gal gene expressed Vnd͞NK-2 and

Shao et al. PNAS ͉ January 8, 2002 ͉ vol. 99 ͉ no. 1 ͉ 115 Fig. 4. Summary of the analysis of vnd͞NK-2 regulatory DNA. Five DNA fragments from the 5Ј-upstream region of the vnd͞NK-2 gene were subcloned in the 5Ј-flanking region of an enhancerless ␤-gal gene in a P-element. The constructs were used to generate transgenic lines of Drosophila. The number of transgenic lines of flies generated for each construct is shown (enclosed by parentheses). DNA region A, Ϫ4.0 to Ϫ2.8 kb, is sufficient to direct Vnd͞NK-2 expression in the following ten NBs: 1-1 (transient expression, not shown), 1-2, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, 7-1, and MP-2. In addition, region A activates ectopic Vnd͞NK-2 expression in two cells, E1 and E3. DNA region B (B1 and B2, Ϫ5.3 to Ϫ4.0 kb) contains nucleotide sequences that activate the vnd͞NK-2 gene in four NBs as follows: 1-2, 2-1, 3-1, and 4-1. DNA region B2 (Ϫ4.7 to Ϫ4.0 kb) activates the vnd͞NK-2 gene ectopically in cell E2 and represses the vnd͞NK-2 gene in E3. DNA region B1 (Ϫ5.3 to Ϫ4.7 kb) contains nucleotide sequences that repress the ectopic expression of the vnd͞NK-2 gene in E1 and E2. Hypothesized DNA region C, whose location has not been identified, activates the vnd͞NK-2 gene in NB 7-2. ML rep (Ϫ3.6 to Ϫ3.1 kb) corresponds to the region of DNA in the 5Ј-flanking region of the vnd͞NK-2 gene that Estes et al. (18) have shown is required for midline repression of the vnd͞NK-2 gene mediated indirectly by the Sim protein in mesoectodermal cells.

␤-Gal proteins in NBs 1-2, 2-1, 3-1, 4-1, 5-1, 5-2, 6-1, and 7-1. Vnd͞NK-2 protein, but not ␤-Gal, was expressed in NB 7-2. The Ϫ5.3͞Ϫ2.8-kb fragment of DNA also contains sequences required for the expression of ␤-Gal earlier in development in NB MP-2 and for transient expression in NB 1-1. Additional small cells that express both Vnd͞NK-2 and ␤-Gal proteins in Fig. 3A are ganglion mother cells and͞or neural progeny of NBs. Similar results were obtained with transgenic Drosophila lines Ϫ4.7͞Ϫ2.8 (Fig. 3B) and Ϫ4.0͞Ϫ2.8 (Fig. 3C); however, ectopic Fig. 3. Regulatory DNA required for Vnd͞NK-2 pattern formation in NBs. expression of ␤-Gal was observed in two cells (E1 and E2) in Fragments of DNA from the 5Ј-upstream region of the vnd͞NK-2 gene, subcloned in the 5Ј-ﬂanking region of an enhancerless ␤-gal gene in a P- element, were used to generate transgenic lines of Drosophila. Antibodies directed against Vnd͞NK-2 protein (red) or ␤-Gal (green) were used to expression regulated by the Ϫ4.0͞Ϫ2.8-kb DNA fragment from the 5Ј- double-label NBs in the ventral nerve cord of stage 11 transgenic embryos. The upstream region of the vnd͞NK-2 gene. Arrowheads indicate the ectopic neuroblasts are identiﬁed in the panels on the right. An NB with an orange expression of ␤-Gal in E1 and E3 cells. (D) ␤-Gal expression regulated by the center surrounded by a green ring expressed both Vnd͞NK-2 and ␤-Gal. An NB Ϫ5.3͞Ϫ3.5-kb DNA fragment from the 5Ј-upstream region of the vnd͞NK-2 that expressed Vnd͞NK-2, but not ␤-Gal, is shown in red. Cells that ectopically gene. ␤-Gal is expressed strongly in two NB, 1-2 and 3-1, and weakly in NB 2-1 express ␤-Gal are shown with white centers surrounded by green. (A) ␤-Gal and NB 4-1. (E) ␤-Gal expression directed by the Ϫ5.3͞Ϫ4.0-kb DNA fragment expression regulated by the Ϫ5.3͞Ϫ2.8-kb DNA fragment from the 5Ј- from the 5Ј-upstream region of the vnd͞NK-2 gene in a transgenic embryo. upstream region of the vnd͞NK-2 gene in a transgenic embryo. The bar The four NBs that express both ␤-Gal and Vnd͞NK-2 shown in D also express corresponds to 5 ␮m. (B) ␤-Gal expression regulated by the Ϫ4.7͞Ϫ2.8-kb DNA both proteins in E; however, ␤-Gal expression in the embryo shown in E is fragment from the 5Ј-upstream region of the vnd͞NK-2 gene. The arrow and weaker than the expression shown in D. The levels of expression of ␤-Gal in arrowheads indicate cells (E1 and E2) that ectopically express ␤-Gal. (C) ␤-Gal NBs 1-2 and 3-1 are greater than the expression found in NBs 2-1 and 4-1.

116 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.012584599 Shao et al. more lateral positions than medial NBs, aligned with transverse of the ␤-gal gene in E1 and E3, whereas DNA region B2 NB row 2 or 3 (Fig. 3B). In a transgenic embryo with a (Ϫ4.7͞Ϫ4.0 kb) contains nucleotide sequences involved in ec- Ϫ4.0͞Ϫ2.8-kb DNA fragment inserted in the chromosome (Fig. topic activation of the ␤-gal gene in E2 and repression of the gene 3C), ectopic expression of ␤-Gal also was observed in two cells in E3. DNA region B1 (Ϫ5.3͞Ϫ4.7 kb) contains nucleotide per hemisegment, E1 and E3; E3 is aligned with transverse NB sequences that repress ectopic expression of the ␤-gal gene in E1 row 3. In contrast, as shown in Fig. 3D, ␤-Gal was expressed and E2. strongly in only two NBs per hemisegment (NB 1-2 and NB 3-1) Estes et al. (18) have shown that the nucleotide sequence(s) and weakly in NBs 2-1 and 4-1 in a transgenic embryo with a required for repression of the vnd͞NK-2 gene mediated indi- Ϫ5.3͞Ϫ3.5-kb DNA insert. Weaker ␤-Gal expression was ob- rectly by the Sim protein in mesectodermal cells is located served in NBs 1-2, 3-1, 2-1, and 4-1 in a transgenic embryo with between Ϫ3.6 and Ϫ3.1 kb in the 5Ј-flanking region of the a Ϫ5.3͞Ϫ4.0-kb DNA insert (Fig. 3E) compared with ␤-Gal vnd͞NK-2 gene. In addition, they showed that three regions in expression shown in Fig. 3D. However, expression of ␤-Gal in the 5Ј-upstream region of the vnd͞NK-2 gene, Ϫ5.3 to Ϫ4.2 kb, NBs 1-2 and 3-1 in the Ϫ5.3͞Ϫ4.0 embryo (Fig. 3E) was higher Ϫ4.2 to Ϫ3.1 kb, and Ϫ3.1 to Ϫ2.8 kb, are required for expression than expression in NB 2-1 and NB 4-1. of the vnd͞NK-2 gene in the ventral neuroectoderm. Our results A summary of results is shown in Fig. 4. The fragments of agree with and extend their findings with regard to regulation of DNA from the 5Ј-flanking region of the vnd͞NK-2 gene are the vnd͞NK-2 gene. The results also suggest that other regions shown that were inserted upstream of an enhancerless ␤-gal gene of DNA that have not been identified may be required for in the P-element used to generate transgenic fly lines. The boxes expression of the vnd͞NK-2 gene in some cephalic cells and in represent regions of DNA required for the expression of ␤-Gal the neuroectodermal precursors of the 11 neuroblasts per he- and Vnd͞NK-2 proteins in NBs that normally express misegment that express the vnd͞NK-2 gene. The regulatory Vnd͞NK-2 from the endogenous vnd͞NK-2 gene as well as mechanism(s) involved in activating the vnd͞NK-2 gene in DNA regions involved in activation and repression of ectopic region B DNA was found only in medial or near medial NBs of expression of the vnd͞NK-2 gene. DNA region A, which corre- transverse rows 1 through 4, but not in NBs of transverse rows sponds to the Ϫ4.0 to Ϫ2.8-kb fragment of DNA from the 6 and 7, which raises the possibility that the expression of the 5Ј-flanking region of the vnd͞NK-2 gene, contains one or more corresponding enhancer(s) or repressor(s) proteins may be strong enhancers that are required for expression of the restricted to the anterior or the posterior part of each segment, vnd͞NK-2 gene in nine medial and one intermediate (1-2) NBs respectively. as follows: 1-1 (transient expression), 1-2, 2-1, 3-1, MP-2, 4-1, 5-1, We conclude that nucleotide sequences in at least three 5-2, 6-1, and 7-1. DNA region B, which extends from Ϫ5.3 to regions of DNA regulate the expression of the vnd͞NK-2 gene, Ϫ4.0 kb of DNA from the 5Ј-flanking region of the vnd͞NK-2 that the pattern of vnd͞NK-2 gene expression in NBs of the gene, contains one or more enhancers of moderate strength for ventral nerve cord is formed from partial patterns, and that three vnd͞NK-2 gene expression in NBs 1-2 and 3-1 and weaker subsets of NBs were identified that differ in the mechanism of expression in NBs 2-1 and 4-1. One or more enhancers that activation of the vnd͞NK-2 gene. DNA region A is involved in activate vnd͞NK-2 gene expression in NB 7-2 resides elsewhere activation of the vnd͞NK-2 gene in NBs 1-1, 1-2, 2-1, 3-1, MP-2, in DNA (hypothesized DNA region C) whose location has not 4-1, 5-1, 5-2, 6-1, and 7-1; DNA region B is involved in activation been identified. Ectopic expression of the ␤-gal gene was de- of this gene in NBs 1-2, 2-1, 3-1, and 4-1; and hypothesized DNA tected in three large cells per hemisegment, possibly NBs, region C is involved in activation the vnd͞NK-2 gene in NB 7-2. termed E1, E2, and E3. DNA region A contains one or more nucleotide sequences involved in activation of ectopic expression We thank Tom Brody for helpful suggestions concerning the manuscript.

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