Ettensohn CA et al. (2003), Alx1, a member of the Cart1/Alx3/Alx4 subfamily...

ECB-ART-38077

Development 2003 Jul 01;13013:2917-28. doi: 10.1242/dev.00511.

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Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins, is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo.

Ettensohn CA , Illies MR , Oliveri P , De Jong DL .

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In the sea urchin embryo, the large micromeres and their progeny function as a critical signaling center and execute a complex morphogenetic program. We have identified a new and essential component of the gene network that controls large micromere specification, the homeodomain protein Alx1. Alx1 is expressed exclusively by cells of the large micromere lineage beginning in the first interphase after the large micromeres are born. Morpholino studies demonstrate that Alx1 is essential at an early stage of specification and controls downstream genes required for epithelial-mesenchymal transition and biomineralization. Expression of Alx1 is cell autonomous and regulated maternally through beta-catenin and its downstream effector, Pmar1. Alx1 expression can be activated in other cell lineages at much later stages of development, however, through a regulative pathway of skeletogenesis that is responsive to cell signaling. The Alx1 protein is highly conserved among euechinoid sea urchins and is closely related to the Cart1/Alx3/Alx4 family of vertebrate homeodomain proteins. In vertebrates, these proteins regulate the formation of skeletal elements of the limbs, face and neck. Our findings suggest that the ancestral deuterostome had a population of biomineral-forming mesenchyme cells that expressed an Alx1-like protein.

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Species referenced: Echinodermata
Genes referenced: alx1 alx1l LOC100887844 LOC115919910 LOC115925415 LOC594353 pmar1
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	Fig. 1. (A) ClustalX alignment of the predicted amino acid sequences of SpAlx1 and LvAlx1. Homeodomains, OAR domains, and charged domains are boxed. Note that in each case the assignment of the start methionine is provisional, as there are three in-frame AUG codons near the 5′ end of the open reading frame (ORF) of each mRNA. There are in-frame stop codons upstream of each ORF. (B) Unrooted neighbor-joining tree showing the relationship of Lv/SpAlx1 to other Paired-class proteins (homeodomains only). The other proteins shown are those with Paired-class homeodomains that are most closely related to those of the Cart1/Alx3/Alx4 subfamily (Galliot et al., 1999). A multiple alignment was generated using ClustalX and tree construction was carried out with PAUP*4.0 (Sinauer Associates) using the Neighbor Joining Method. Numbers indicate the fraction of amino acid substitutions between nodes.
	Fig. 2. Whole-mount in situ hybridizations showing alx1 mRNA expression in S. purpuratus (A-H) and L. variegatus (I-L) embryos. (A) 56-cell stage embryo, the earliest stage at which alx1 mRNA is detectable in the four large micromeres (arrow). Initially, staining is limited to one to two small, intracellular spots in each cell. (B,C) Two focal planes of a 56-cell stage embryo, viewed along the animal-vegetal axis. Alx1 mRNA is present in the large micromeres (B, arrow) but not the small micromeres (C, arrow). (D,E) Lateral and vegetal views, respectively, of∼ 128-cell stage embryos, showing alx1 mRNA in the eight progeny of the large micromeres (arrows). (F) Mid-blastula stage. (G) Mesenchyme blastula. (H) Late gastrula. Alx1 transcript continues to be restricted to large micromere progeny throughout blastula and gastrula stages (arrows). In L. variegatus, alx1 is expressed in a similar pattern, although expression is first detectable, by in situ hybridization, one cell cycle later than in S. purpuratus, after the large micromeres have divided once. (I) Blastula, showing expression in ∼16 large micromere progeny (arrow). (J) Early gastrula. (K) Mid-gastrula. (L) Late gastrula. Alx1-expressing PMCs are indicated by arrows. By the end of gastrulation, levels of Lvalx1 expression have declined in most embryos.
	Fig. 3. Alx1 protein expression in S. purpuratus (A-D) and L. variegatus (E-H) embryos. In both species, Alx1 protein is restricted to the nuclei of large micromere progeny (arrows). Expression is first detectable prior to PMC ingression and is evident throughout gastrulation. (A) 128-cell stage. (B) Mid-blastula stage; vegetal view. (C) Mesenchyme blastula. (D) An Alx1 MO-injected embryo that was allowed to develop until controls had reached the late gastrula stage. No nuclear staining is evident in cells of the vegetal plate (arrow). (E) Blastula. (F) Late gastrula. (G) Late gastrula stained with αAlx1 and mAb 6a9, which recognizes a family of PMC-specific cell surface proteins. There is a one-to-one correspondence between 6a9- and Alx1-positive cells (arrow). (H) Overexpression of Xenopus C-cadherin. This embryo was fixed when sibling controls were late gastrulae. It has an animalized phenotype (Wikramanayake et al., 1998; Logan et al., 1999) and no Alx1 staining is detectable.
	Fig. 6. Activation of Alx1 expression in transfating cells. L. variegatus embryos were fixed 10 hours after PMC removal and immunostained withα Alx1 and mAb 6a9. (A) αAlx1 staining. (B) MAb 6a9 staining. (C) Overlay of A and B. 6a9-positive cells also have nuclear Alx1 (arrows). Some Alx1-positive cells are not stained with 6a9, perhaps because they are at an early stage in the transfating process.
	Fig. 4. Effects of Alx1 MOs in S. purpuratus (A-H) and L. variegatus (I-P). MOs were complementary to non-overlapping regions of the Spalx1 and Lvalx1 mRNAs and produced essentially identical phenotypes in the two species. Control embryos show normal PMC ingression (A,I, arrows), PMC migration (B,J, arrows), and skeletogenesis (C,D,K,L, arrows). Alx1 MO-injected embryos lacked PMCs (E,M) and invaginated in a delayed fashion (F,N, arrows). They failed to form skeletal elements even after prolonged culture (H,P). Eventually they developed a tripartite gut (H,P, arrows), pigment cells (see Fig. 5), blastocoelar cells, and coelomic pouches (P, arrowhead). Ectodermal territories appear to differentiate normally (thickened oral ectoderm is indicated by the arrow in G and the double arrow in P). Arrow in O indicates expanded archenteron tip in L. variegatus embryos injected with Alx1 MO.
	Fig. 5. Effects of Alx1 MOs in S. purpuratus (A-H) and L. variegatus (I-N). MO-injected embryos were examined 36 hours (B,D,F,H) or 54 hours (J,L,N) postfertilization. (A-F) In situ hybridizations with SpMSP130-related 2, SpP19 and SpFRP probes. Control embryos show strong signal in large micromere progeny in the vegetal plate and in PMCs following ingression (A,C,E, arrows) (see also Illies et al., 2002). MO-injected embryos show few or no positive cells. (G,H) Immunostaining with mAb 6a9, showing stained cells (PMCs) in control (G, arrow) but not MO-injected (H) embryos. (I,J) Endo1 expression in the midgut of a control pluteus at 24 hours (note that the hindgut is out of focus) and in the midgut/hindgut of a MO-injected embryo. (K,L) Myosin heavy chain expression in circumesophageal muscle cells (arrows) of a control pluteus (24 hours) and in a MO-injected embryo. (M,N) Pigment cells in the aboral ectoderm of a control (arrow; 48 hours) and a MO-injected embryo.
	Fig. 6. The micromere-PMC gene regulatory network. The total developmental time represented in the diagram is from fertilization (top) to the blastula stage (bottom). Arrows and bars indicate positive and negative interactions, respectively. All genes shown encode transcription factors with the exception of Delta, which encodes a transmembrane protein. There is evidence for a direct interaction between Ets1 and sm50 (Kurokawa et al., 1999) but all other interactions may be indirect. β-catenin and Otx are maternal proteins that become differentially enriched in micromere nuclei at the 16-cell stage (Chuang et al., 1996; Logan et al., 1999). These two proteins are required for the activation of pmar1, which is expressed only by the micromeres and their progeny (Oliveri et al., 2002). Pmar1 may block the expression of a putative repressor (Repressor X) specifically in the micromeres. This repressor (which may be several proteins) blocks PMC fate specification in all non-micromere lineages (Oliveri et al., 2002). Ets1, alx1 and delta are all regulated independently by pmar1 and the repressor. Ets1 regulates the tbr gene (Fuchikami et al., 2002) and Alx1 regulates dri (this study). Alx1, Ets1 and Tbr are all expressed only by the large micromeres and their progeny. Alx1 and Ets1 both regulate genes involved in ingression and skeletogenesis (Kurokawa et al., 1999; this paper). Delta signaling activates genes involved in SMC specification, including gcm (Ransick et al., 2002; Sweet et al., 2002). PMC signals feed into the network upstream of alx1 (this study); dashed bars and dashed arrow show possible inputs.