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The role of TGFß signaling in the formation of the dorsal nervous system is conserved between Drosophila and chordates

Tor Erik Rusten¹, Rafael Cantera², Fotis C. Kafatos¹ and Rosa Barrio^1,*,

¹ European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
² Zoology Department, Stockholm University, 10691 Stockholm, Sweden
^* Present address: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain

View larger version (146K): [in a new window]   Fig. 1. Dorsal sensory neurons develop within the Dpp signaling region in the dorsal ectoderm. (A,B) Merged confocal images of PNCs (cell clusters) and SOPs (single cells) visualized by immunostaining using anti-Ato (green in A) or anti-Ac (green in B) antibodies in two consecutive abdominal segments of staged 11 embryos. Anterior is towards the left and dorsal is upwards. The Dpp signal receiving cells in a dad-lacZ enhancer trap line are visualized by anti-ß-galactosidase antibodies (red). Orthogonal sections are indicated by the blue lines (I and II in A, and III and IV in B) and shown in A', A'' and B', B'', respectively. Merged images are shown in color; black and white images represent the separate red (middle) and green (right) channels. In A, the C1 SOP, which contributes to one of the chordotonal organs, is marked by a stippled line (A') and originates within the dad-lacZ domain (red). The SOP giving rise to the dorso-bipolar neuron (dbp) is in the process of delamination (A'') and also originates within the dad-lacZ domain. The more ventrally located PNC cluster, from which the C2-C5 chordotonal SOPs will be singled out, is only partially within the dad-lacZ domain. In B, the Ac-positive PNCs 1 and 3 originate within and outside the dad-lacZ domain, respectively (B'). The delaminating SOPs 2 and 4 form within the dad-lacZ domain (B',B'').

View larger version (54K): [in a new window]   Fig. 2. The second phase of Dpp signaling is required for normal PNS formation. For clarity, in A-G the dorsal (d), lateral (l) and ventral (v) regions encompassing PNS clusters are colored blue, yellow and pink, respectively. (A-C) Whole-mount stage 16 embryos stained with the 22C10 antibody that reveals PNS neurons. (A) Wild type (WT) embryo. (B,C) Homozyogous shn1 (B) and shnk04412 (C) mutants have fewer neurons and present serious disorganization in the dorsal and lateral PNS clusters. shn1 mutants fail to undergo dorsal closure. As a result of this failure, the gut is extruded through the top, the embryo is twisted and the ventral nerve cord is pulled inwards. Neurons belonging to the other side of the embryo are visible most ventrally. (D-G) Flat preparations showing one thoracic (T3) and four abdominal (A1-A4) segments (upper panels). Cuticle preparations of late stage embryos (lower panels) show no clear expansion of the ventral denticles in any genotype. Fewer neurons can be observed in shnk04412 (E), Kr-Gal4;UAS-brk (F) and HS-ssog (G) embryos, especially in the dorsal and lateral clusters. In alleles of shn and overexpression of brk, the pentascolopodial organ (brackets and yellow in D-G) frequently has fewer neurons and is positioned close to the dorsal cluster. (H) Mean values, standard error (bars) and range (numbers to the right of the genotype) of the number of neurons per dorsal, lateral and ventral clusters in abdominal segments (A1-A7) in different genetic backgrounds. Abdominal segments A1-A7 on both sides of 10 embryos were quantified for each genotype. The inhibition of Dpp signaling results in the reduction in number of neurons, principally in the dorsal and lateral PNS clusters. WT, n=63; shnk04412, n=116; shnk00401, n=90; Kr-Gal4;UAS-brk, n=53; HS-ssog, n=182.

View larger version (88K): [in a new window]   Fig. 3. Proneural gene expression is reduced when Dpp signaling is impeded. Expression of the proneural gene products, Ato (green) and Ac (red), in PNCs and SOPs at stage 11 (A-H). (A,B) The number and position of serially homologous PNCs and SOPs are similar in A1-A7 wild-type abdominal segments. (C-H) Proneural gene expression is lost in some segments in shnk04412 homozygous embryos (C,D), embryos expressing HS-ssog (E,F) and embryos overexpressing brk in segments A1-A3 (G,H). Arrows point to SOPs and PNCs that are not present in certain other segments. (I) Visualization of apoptotic cell death with Acridine Orange reveals no preferential cell death in segments A1-A3 versus other abdominal segments in embryos overexpressing UAS-brk under Kr-Gal4. (J,K) Active TGFß signaling is visualized by anti-p-Mad antibody at stage 9 embryos (Tanimoto et al., 2000). While WT embryos show a broad expression domain of p-Mad (J), embryos where ssog is expressed under heatshock show a severe reduction in p-Mad levels and expression domain (K). The effect varies among embryos: some embryos have no p-Mad after the heat shock treatment.

View larger version (50K): [in a new window]   Fig. 4. Opposing genetic roles of shn and brk in formation of PNS neurons. (A-D) Flat preparations of stage 16 embryos showing one thoracic (T3) and four abdominal (A1-A4) segments with PNS neurons visualized with the 22C10 antibody. Regions that harbor neurons that belong to the dorsal, lateral or ventral clusters are color-coded blue, yellow and red, respectively. (A) brkm68 homozygous embryo with normal numbers of abdominal PNS neurons in dorsal and lateral clusters, but fewer in ventral clusters (compare with Fig. 2D). (B) Homozygous shn1 embryos display a dramatic reduction in neuronal numbers and disorganization in the dorsal and lateral clusters, but less so in ventral clusters (compare with Fig. 2B). The compact appearance of the PNS is due to a lack of dorsal closure (dorsal extension of the epidermis) in these animals. Double homozygous embryos for brkm68;shnk04412 (C) and brkm68;shn1 (D) display a clear rescue in neuronal numbers relative to the single shn mutant (A); this rescue can be observed in dorsal and lateral but not in ventral clusters. The correct positioning of the pentascolopodial organ and dorsal closure are also restored in these double mutants (brackets; compare with Fig. 2E,H). (E) Mean values and standard error of the number of neurons in the dorsal, lateral and ventral clusters in abdominal segments with different genetic backgrounds. shn and brk have opposite roles in the dorsal and lateral clusters as brk;shn double mutants have more neurons than shn mutants alone. In the ventral cluster, brk appears to act independently of shn as brk;shn double mutants have comparable numbers of neurons to brk single mutants. WT, n=63; shnk04412, n=116; brkm68, n=121; brkm68;shnk04412, n=74; shn1, n=37; brkm68;shn1, n=27.

View larger version (24K): [in a new window]   Fig. 5. (A) Summary of genetic data for the roles of TGFß signaling in Drosophila PNS development. Dpp signaling in the dorsal ectoderm promotes the formation of the PNS and requires the co-factor Shn to keep Brk expression low in this domain. Thus, Shn and Brk have opposing effects on the formation of dorsal and lateral PNS clusters. In the ventral cluster, Brk expression is high, while Dpp signaling is low or absent. Brk promotes neuronal formation, probably by a double-negative mechanism that is independent of Shn. (B) Schematic representation of the similarities between the Drosophila nervous system (summarizes stages 8-11 of development) and the developing neural tube in mouse, in terms of the key molecules involved in their patterning (Arendt and Nubler-Jung, 1999; D’Alessio and Frasch, 1996; Gowan et al., 2001; Tanabe and Jessell, 1996). Regions of proneural gene expression are shown on the left half of the schemes while lateral column genes are shown on the right half. The proneural and lateral column genes are largely expressed in the neural progenitors. For simplicity, regions containing differentiating neurons are also colored according to their progenitors, although frequently they have lost the expression of the corresponding genes by this stage. Further references can be found in the main text. Similar proneural genes and lateral column genes specify the dorsoventral pattern of the nervous system. The dorsal nervous system fates are induced by TGFß signaling in both Drosophila and chordates. TGFßs expressed in the roof plate in mouse include BMP4, BMP7 and BMP5, Dorsalin, Activin B and GDF7 (reviewed by Lee and Jessell, 1999; Liem et al., 1997). Neural crest originates from a cell population in the roof plate (Echelard et al., 1994; Lee et al., 2000; Selleck and Bronner-Fraser, 1995). Homologous gene pairs are shown in the same color. Achaete-scute complex (AS-C)/Mash1; Absent solo-MD neurons and olfactory sensilla (Amo)/Math1; Atonal (Ato)/Math1; Ventral nervous system defective (Vnd)/Nkx-2.2; Intermediate neuroblasts defective (Ind)/Gsh-1; and Muscle segment homeobox Msh/Msx1/2/3.