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First published online 14 January 2009
doi: 10.1242/dev.029959

Development 136, 541-549 (2009)
Published by The Company of Biologists 2009
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Two ParaHox genes, SpLox and SpCdx, interact to partition the posterior endoderm in the formation of a functional gut

Alison G. Cole1,*, Francesca Rizzo1,*, Pedro Martinez2, Montserrat Fernandez-Serra1 and Maria I. Arnone1,{dagger}

1 Stazione Zoologica Anton Dohrn di Napoli, Villa Comunale, 80121 Napoli, Italy.
2 Department de Genètica, Universitat de Barcelona, Avenida Diagonal, 645, 08028 Barcelona, Spain.


Figure 1
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  		Fig. 1. Expression pattern of the SpLox gene from blastula through pluteus larva in Strongylocentrotus purpuratus. (A) Mesenchyme blastula showing no SpLox expression. (B) Midgastrula. First detectable expression appears in cells on one side of the blastopore (*). (C-E) Mid to late gastrula. Expression is detected in all cells surrounding the blastopore and the posterior-most endodermal tube. (F) End of gastrulation: prism stage larva. The entire posterior endodermal tube exhibits SpLox expression, from the posterior midgut through to the anus. (G,H) Early pluteus larva collected 4 hours after that shown in F shows expression restricted to the posterior midgut/hindgut boundary. (G) Oral view; (H) lateral view, oral end upwards. (I) 72-hour pluteus larva, in lateral view, oral end upwards. Expression is retained at the posterior sphincter.

 

Figure 2
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  		Fig. 2. Morphological phenotype resulting from disruption of SpLox in Strongylocentrotus purpuratus. (A,B) Stage I pluteus larvae developed from rhodamine dextran-KCl-injected eggs show a well-defined tripartite gut with anterior (gray arrows) and posterior (white arrows) sphincters separating the midgut (m) from foregut (f) and hindgut (h), respectively. (A) Lateral and (B) oral views. (D,E) SpLox-MASO injected larvae show disorganized structure of the endodermal epithelia and are missing the posterior sphincter between the midgut and hindgut (white arrows), exhibiting only a mild posterior constriction of the gut. (D) Lateral and (E) oral views. Gray arrows indicate anterior sphincter (C,F) Posterior regions of the developing gut from prism stage larvae, wherein the distinct reduction of the posterior constriction (white arrows) is first apparent in SpLox-MASO-injected embryos (F) compared with control injected embryos (C). (G-I,K-M) Three-dimensional maximal projection of confocal image stacks from phalloidin stained (green) control (G-I) and SpLox-MASO (K-M) injected larvae. (G,K) Lateral view of a stage I pluteus larva showing staining of the midgut and hindgut by an Endo1 antibody (red). Note the muscle fibers that define the foregut anteriorly (*) in the control larva (G) and are less conspicuous in the experimental larva (K). (H,L) Oral views, phalloidin staining (green) with nuclear counterstain (purple). (I,M) Reconstructions of phalloidin staining in endoderm from the same larvae imaged in H and L. Phalloidin fibers collect at the level of the midgut-hindgut sphincter (white arrows) and are present in the control larvae G-I, but are absent from SpLox-MASO injected larvae K-M. (J,N) A single optical section from the same larvae shown in H,I and L,M taken at the level of the midgut-hindgut junction; bright-field above, fluorescence below. The smooth surface of stomach lumen in control (J) when compared with the SpLox-MASO larva (N) where many phalloidin-positive projections are evident (white arrowheads).

 

Figure 3
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  		Fig. 3. Silencing of SpLox results in disruption of feeding abilities in Strongylocentrotus purpuratus larvae. (A-C) Control MASO-injected (D-F) and SpLox-MASO-injected larvae. (A,D) Bright-field images with overlaid fluorescence excited through a 488 wavelength filter showing degraded chlorophyll (green) and intact algal cells (red). (B,E) Higher magnification of the gut of the same larvae shown in A,D imaged under UV excitation to illustrate degraded (blue) and intact (red) chlorophyll. Control injected larvae (A,B) show incorporation of chlorophyll within the midgut, whereas SpLox-MASO injected larvae (D,E) show only undigested algal pellets within the digestive tract. (C,F) Alkaline phosphatase staining. The control injected larva imaged in C shows high levels of alkaline phosphatase activity when compared with the SpLox-MASO-injected larva imaged in F.

 

Figure 4
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  		Fig. 4. Changes in gene expression levels assessed by qPCR in SpLox-MASO-injected Strongylocentrotus purpuratus larvae when compared with levels in control injected larvae. Total RNA was collected from control and SpLox-MASO injected larvae at two time points [72 (yellow) and 96 (green) hours post fertilization]. Experiments were repeated at least twice, with replicates from different batches separated by semicolons. Data are expressed as a fold difference from control larvae and are illustrated graphically on the left, with the numerical data presented on the right. We assume an amplification efficiency of 1.9, and all samples varied from one another by no more than 0.3 cycles. For all data, the cycle threshold (CT) was first normalized to ubiquitin expression levels in each sample. Fold difference is calculated as 1.9{Delta}Ct ; thus, fold differences greater than ±1.8 (gray) are considered significantly different and are in bold. Genes of the endomesodermal GRN are presented first, followed by the two ParaHox genes, genes that were chosen because of their role in the pancreatic GRN in vertebrates, downstream structural genes from the echinoderm endomesodermal GRN and, finally, two genes involved in muscle development. We find no differences in expression of endomesodermal GRN genes, assumed to be upstream from SpLox, whereas two transcription factors from the vertebrate pancreas network show altered levels of expression (isl and myt), as do the endodermal structural genes (Endo16 and ap) and those involved in muscle formation (actM and sum1).

 

Figure 5
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  		Fig. 5. SpLox clears expression of the endoderm gene Endo16 from the hindgut region. (A,B) Co-expression of Endo16 (blue) and SpLox (green) in a late gastrula stage embryo showing the overlapping domains of expression (blue-green). The fluorescent channels are shown separately in B. (C,D) Endo16 expression in control pluteus larvae is restricted to the midgut; (C) oral and (D) lateral view. (E,F) Endo16 expression is expanded into the hindgut region in SpLox-MASO injected pluteus larva. (E) Oral and (F) lateral views.

 

Figure 6
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  		Fig. 6. SpLox binds to the regulatory region of Endo16 in vitro. (A) Sequences used for the binding assay shown in B (see Materials and methods for details of origin of these sequences). The core homeobox DNA-binding site is underlined, and nucleotide alignment of the A1 Pdx-binding site of the insulin promoter (InsA1) (Liberzon et al., 2004Go) with two putative Lox-binding sites included in the Endo16 13/44 promoter element (Endo13/44) (Yuh et al., 1994Go) are shown in bold. Altered nucleotides in the mutated oligonucleotide (Endo13/44M) are indicated (*). (B) In vitro binding assay of synthesized SpLox protein shows effective binding to putative Lox-binding site from the Endo16 promoter (lane 1). Binding to labeled oligonucleotides is reduced in the presence of increasing concentration (50 and 100-fold molar excess) of unlabeled competitor DNA (E13/44; lanes 2,3), whereas binding efficiency is unchanged if the competing unlabeled oligonucleotide contains a mutation (E13/44M; lanes 4,5). The SpLox protein binding is also effectively inhibited by competition with unlabeled oligonucleotides containing the A1 Pdx1-binding site from the insulin promoter (InsA1; lanes 6,7).

 

Figure 7
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  		Fig. 7. Expression pattern of the SpCdx gene from blastula through pluteus larvae in Strongylocentrotus purpuratus. Embryos correspond to the same batch as those shown in Fig. 1 for SpLox expression. (A-C) Mesenchyme blastulae through mid-late gastrulae show no SpCdx expression. (D) Late gastrula. First detectable expression appears in cells surrounding the blastopore (*). (E) Late gastrula. Expression is detected in all cells surrounding the blastopore and the posterior-most endodermal tube. (F) End of gastrulation: prism stage larva shown in oral view. The posterior-most endodermal tube exhibits SpCdx expression. (G,H) Early pluteus larvae collected 4 hours after that shown in F show continued high levels of expression throughout the hindgut region. (G) Oral view; (H) lateral view. (I) 72-hour pluteus larva in lateral view showing retention of the high expression levels throughout the hindgut.

 

Figure 8
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  		Fig. 8. ParaHox genes SpLox and SpCdx interact to regulate hindgut formation, as illustrated with double in situ hybridization. (A,D,G,J) Embryos photographed using DIC imaging, with the fluorescent gene expression patterns overlaid, overlapping expression appears white. (B,E,H,K) The green channel only (SpLox expression). (C,F,I,L) The red channel (SpCdx expression). (A-I) Double in situ of expression domains in normal embryos. (J-L) Double in situ illustrating expression domains in SpLox-MASO-injected embryos. (A-C) Late gastrula stage embryo showing largely overlapping expression domains of both genes, although the SpLox domain extends further anteriorly (green), and the SpCdx domain extends further posteriorly (purple). (D-F) Prism stage larva showing a greater region of non-overlapping expression domains. (G-I) Control injected pluteus larva showing mutually exclusive, non-overlapping expression domain for both genes. (J-L) Pluteus larva developed from an egg from the same batch as the larva shown in G-I, injected with the SpLox-MASO. SpLox expression is maintained throughout the hindgut (K) when compared with control (G,H), whereas SpCdx is absent in the posterior hindgut in injected larvae (L) compared with control (I).

 

Figure 9
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  		Fig. 9. Model of hindgut specification involving two ParaHox genes. (A) Summary of expression data for SpLox, SpCdx and the endodermal marker Endo16. Expression is shown schematically on the left, with confocal reconstruction of a triple in situ illustrating the expression of all three genes in a late gastrula stage embryo shown on the right. At the onset of SpLox expression in the late mid-gastrula stage embryo, Endo16 is expressed throughout the presumptive mid and hindgut territories (blue). SpLox (green) represses the expression of Endo16 in the area of overlapping expression, eliminating Endo16 expression from the hindgut region. SpCdx (purple) expression begins in the posterior-most region of the endodermal tube, in the presumptive hindgut. We propose that SpLox is involved in the activation of SpCdx in this region, and that once activated SpCdx inhibits further expression of SpLox in the hindgut. (B) Gene network diagram summarizing these interactions. Repression of Endo16 by SpLox is thought to be a direct interaction based upon the data presented in this paper. Interactions between SpLox and SpCdx, the two ParaHox genes, may be either direct or indirect. (C) Preliminary data showing the expression of SpLox (green) in embryos injected with a MASO targeting the donor splice site between the first and second SpCdx exons (5'-TAGCTTTTGGTTAAATACCTGTTT). SpCdx-MASO injected larvae (right) show expanded expression of SpLox into the hindgut when compared with control (Rho-KCl)-injected larvae (left).

 

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© The Company of Biologists Ltd 2009