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First published online 11 June 2008
doi: 10.1242/dev.018101

Development 135, 2445-2454 (2008)
Published by The Company of Biologists 2008
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LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo

Ryan C. Range*, Thomas D. Glenn, Esther Miranda and David R. McClay{dagger}

Department of Biology, Duke University, Durham, NC 27708, USA


Figure 1
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  		Fig. 1. Alignment of the predicted amino acid sequence of LvNumb and Human Numb1 proteins. (A) Identical residues are denoted by an asterisk and conserved by `.' or `:'. The four conserved domains are labeled. (B) A molecular phylogeny of the Numb and Numb-like family members. Abbreviations: Ce, C. elegans; Mm, mouse; Rn, rat; Hs, human; Tn, Tetraodon (puffer fish); Dr, zebrafish; Xt, Xenopus tropocalis; Gg, chicken; Ci, Ciona; Lv, Lytechinus; Dm, Drosophila.

 

Figure 2
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  		Fig. 2. lvnumb is transcribed beginning at hatched blastula and remains expressed until the end of embryogenesis in the endomesoderm. The protein is localized to the presumptive NSM. (A) Northern blot of LvNumb expression during early development (5 µg/lane). Ethidium bromide staining of the 18s rRNA bands used as a loading control. A single transcript accumulates beginning at hatched blastula stage and is expressed throughout the rest of development. 16, 16-cell stage; HB, hatched blastula; MB, mesenchyme blastula; EG, early gastrula; LG, late gastrula; PL, pluteus larvae. (B) lvnumb expression is upregulated at the vegetal plate beginning at hatched blastula stage. (a,b) lvnumb transcripts are not localized in the egg or 60-cell stage embryos. (c) Beginning at hatched blastula, lvnumb is upregulated in the vegetal plate (region of thickened epithelia). (d) lvnumb maintains high expression in the vegetal plate region of mesenchyme blastula embryos. (e) lvnumb is expressed in involuting endoderm, but not delaminating secondary mesenchyme cells in early gastrula embryos (arrowheads indicate SMCs). (f) By late gastrula, lvnumb is expressed primarily in the hindgut and the foregut (arrowheads), but is largely excluded from the midgut. (C) (a-c) In the blastula, LvNumb protein is apically localized to the presumptive NSM that has been cleared of LvNotch. LvNotch (a) and LvNumb (b) staining and merge (c) showing overlap at the edge of the NSM field (arrowhead). (d-f) Apical LvNumb expression expands with the presumptive NSM territory in early gastrula embryos. LvNotch (d) and LvNumb (e) staining and merge (f) showing overlap at the edge of the NSM field (arrowheads). (g-i) In gastrula embryos, the apical expression of LvNumb has expanded into the endoderm, significantly overlapping with dorsal LvNotch expression. (g) LvNotch staining. (h) LvNumb staining. (i) Merged image of LvNotch and LvNumb protein expression. Arrowhead indicates where LvNumb and LvNotch overlap.

 

Figure 3
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  		Fig. 3. Differentiation of primary and non-skeletogenic mesoderm cells after manipulation of the LvNumb levels. (A-C) Pigment cells. (D-F) Embryos stained with SMC2, which stains blastocoelar cells (indicated by arrowheads; there is background staining in the ectoderm and endoderm). (A,D) Control pluteus larvae show normal levels of pigment cells and blastocoelar cells. (B,E) Embryos injected with LvNumb1 morpholino (1.8 mM) have few pigment cells or blastocoelar cells. (C,F) Following injection with the lvnumb mRNA (0.5-1 pg/pl), there are increases in pigment cells and blastocoelar cells. (G) Control embryo stained with {alpha}-myosin heavy chain antibody showing esophageal muscle cell fibers. (J) The number of muscle cell fibers is reduced in embryos injected with LvNumb2 morpholino (0.5 mM). (H,I) Control embryos stained with the PMC marker 1d5 (H), and the general mesenchyme marker Meso1, which stains both NSM and PMCs (I). (K,L) Images of the same embryo injected with lvnumb mRNA showing that the large increase in mesenchyme is seen in L comes largely from an increase in NSM cells, not the PMCs (K).

 

Figure 4
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  		Fig. 4. Numb functions in Notch bearing cells that receive the Delta signal. (A) Control pluteus larva with pigment cells, a necessary consequence of presentation of the Delta ligand on micromeres to Notch on the receiving macromeres. (B) A pluteus larva with injected LvNumb2 morpholino. (C) The experiment in D. Micromeres bearing a LvNumb2 morpholino (red) were transplanted to a host micromereless embryo. (D) Numb morpholino in the micromeres does not affect the transfer of the Delta signal as the embryo has pigment cells. (E) Experiment in F. At the 60-cell stage, two embryos were cut in half from the animal to vegetal pole. A half bearing LvNumb2 morpholino was combined with a control half (green). (F) A fluorescent overlay shows that pigment cells produced by the control half embryo migrate into the LvNumb morpholino half embryo, indicating that if control pigment cells are provided to the Numb half they develop normally and migrate to the correct position in the ectoderm.

 

Figure 5
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  		Fig. 5. Expression of the PTB domain, but not the PRR domain, affects non-skeletogenic mesoderm specification. (A-C) Pigment cells. (D-F) Pluteus larvae stained with SMC2. (A,D) Control embryos. (B,E) Injection of ptb domain mRNA (2 pg/pl) increases the formation of pigment cells and blastocoelar cells (arrowheads indicate large aggregations of blastocoelar cells). (C,F) Pigment cell and blastocoelar cell specification is unaffected by expression of Myc-PRR domain (3-6 pg/pl) (image shown) or untagged LvPRR domain (3-6 pg/pl) (data not shown). (G) Western blot showing expression of Myc-PRR domain protein in early gastrula-stage embryos. The protein is the predicted size of 52 kDa. (H) Schematics showing the LvNumb constructs used for experiments. The PTB domain, full-length LvNumb, the PRR domain and EH domains are indicated.

 

Figure 6
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  		Fig. 6. Expression of the PTB domain rescues the LvNumb morpholino phenotype. (A) Embryos injected with control morpholino have normal levels of pigment cells. (B) Injection ptb domain mRNA (2 pg/pl) increases pigment cell specification. (C) Pluteus larvae injected with LvNumb morpholino (1.7 mM) showed pigment cells specification in less than 9% of the embryos examined. (D) Injection of ptb domain mRNA with LvNumb morpholino showed pigment cells specification in 77% of the embryos examined. Inset, high magnification of embryo in D.

 

Figure 7
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  		Fig. 7. LvNumb requires a functional Notch signal. High levels of Notch signaling override the requirement for LvNumb during NSM specification and LvNumb is necessary for the expression of the earliest known Notch target. (A) (a) Pluteus larvae injected with control morpholino have normal numbers of pigment cells. (b) Injection of either ptb domain mRNA (2 pg/pl) or (f) LvNACT mRNA (2 pg/pl) increases pigment cell specification. (c) Embryos injected with either LvNNEG (3 pg/pl) or (g) LvNumb morpholino display a lack of pigment cells. (d) Injection of ptb domain mRNA in embryos expressing LvNNEG does not rescue pigment cell specification. (h) Expression of LvNACT (2 pg/pl) rescues pigment cell specification in 86% of the embryos injected with the LvNumb morpholino (1.8 mM). (B) (a) A mesenchyme blastula stage embryo injected with control morpholino shows gcm expression in the NSM. (b,c) Embryos injected with LvNumb2 morpholino show reduced (b) or no expression of gcm (c).

 

Figure 8
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  		Fig. 8. Model for NSM specification in the sea urchin embryos. (A) During the first Notch signal, the blastula stage (seventh to ninth cleavage) embryo expresses LvNotch (blue) and Fringe (orange) on the membranes of all cells of the embryo. Micromeres (pink) express the LvDelta protein (green arrows) and signal to adjacent macromeres (light blue), specifying pigment cells and blastocoelar cells. LvFringe is necessary for Delta to activate the LvNotch receptor in the macromere lineage. (B) After LvNotch signals in the macromere lineage, the receptor is cleared from the presumptive NSM territory and presumptive endoderm cells begin to express high levels of apical Notch. The presumptive NSM expresses apical LvNumb (red), partially overlapping with LvNotch expression at the boundary of the territory. LvNumb works with LvNotch, either directly or synergistically, to specify pigment and blastocoelar cells at this time. (C) During the second Notch signal, the micromeres have ingressed as PMCs and no longer express Delta. Delta is expressed in the presumptive NSM where they signal to Notch to specify blastocoelar cells and muscle cells (double headed green arrow). Delta may also activate Notch signaling in neighboring cells (green arrows). Similar to the first signal, Delta requires LvFringe to activate Notch signaling. In addition, LvNumb is necessary for specification of blastocoelar cells and muscle cells in the NSM, and perhaps it is necessary in neighboring cells as well. Notch signaling also activates a secondary signal that is necessary for some aspect of endoderm specification (black arrows). (D) By gastrula stage, LvDelta is no longer expressed. However, LvNotch is apically expressed on the dorsal side of the archenteron and LvNumb is apically expressed in the NSM territory (light blue) and the archenteron. LvFringe is not expressed in the archenteron. We speculate that these expression patterns suggest a role for LvNotch and LvNumb in later aspects of NSM and gut specification. It is possible that an as yet unidentified Notch ligand, Serrate, which cannot activate LvNotch in the presence of LvFringe, activates LvNotch at this time.

 

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© The Company of Biologists Ltd 2008