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The Mesp bHLH genes play a conserved role during segmental patterning of the mesoderm in the vertebrate embryo by specifying segmental boundaries and anteroposterior (A-P) segmental polarity. Here we use a xenotransgenic approach to compare the transcriptional enhancers that drive expression of the Mesp genes within segments of the presomitic mesoderm (PSM) of different vertebrate species. We find that the genomic sequences upstream of the mespb gene in the pufferfish Takifugu rubripes (Tr-mespb) are able to drive segmental expression in transgenic Xenopus embryos while those from the Xenopus laevis mespb (Xl-mespb) gene drive segmental expression in transgenic zebrafish. In both cases, the anterior segmental boundary of transgene expression closely matches the expression of the endogenous Mesp genes, indicating that many inputs into segmental gene expression are highly conserved. By contrast, we find that direct retinoic acid (RA) regulation of endogenous Mesp gene expression is variable among vertebrate species. Both Tr-mespb and Xl-mespb are directly upregulated by RA, through a complex, distal element. By contrast, RA represses the zebrafish Mesp genes. We show that this repression is mediated, in part, by RA-mediated activation of the Ripply genes, which together with Mesp genes form an RA-responsive negative feedback loop. These observations suggest that variations in a direct response to RA input may allow for changes in A-P patterning of the segments in different vertebrate species.

Noelins comprise a family of extracellular proteins with proposed roles in neural and neural crest development. Here, we show that a previously uncharacterized family member, Noelin-4, functions to maintain neural precursors in an undifferentiated state and biases ectoderm toward a neural fate. We show that Noelin-4 is induced by the neurogenic genes X-ngnr-1 and XNeuroD. Over-expression of Noelin-4 causes expansion of the neural plate at the expense of neural crest and epidermis. Although there is an apparent increase in the neural precursor pool, no increase was noted in differentiated neurons. Later, derivatives such as the neural tube and retina appear enlarged. We show biochemically that Noelin-4 protein is glycosylated and secreted and that it interacts with Noelin-1, an isoform previously found to promote differentiation in neuralized animal caps. Accordingly, the neural precursor expansion activity of Noelin-4 is reversed by co-expression of Noelin-1. Our finding that Noelin isoforms can bind to and antagonize one another suggests that interacting Noelin isoforms may play a role in regulating timing of differentiation.

Neurogenesis in Xenopus neural ectoderm involves multiple gene families, including basic helix-loop-helix transcription factors, which initiate and control primary neurogenesis. Equally important, though less well understood, are the downstream effectors of the activity of these transcription factors. We have investigated the role of a candidate downstream effector, Noelin-1, during Xenopus development. Noelin-1 is a secreted glycoprotein that likely forms large multiunit complexes. In avians, overexpression of Noelin-1 causes prolonged and excessive neural crest migration. Our studies in Xenopus reveal that this gene, while highly conserved in sequence, has a divergent function in primary neurogenesis. Xenopus Noelin-1 is expressed mainly by postmitotic neurogenic tissues in the developing central and peripheral nervous systems, first appearing after neural tube closure. Its expression is upregulated in ectopic locations upon overexpression of the neurogenic genes X-ngnr-1 and XNeuroD. Noelin-1 expression in animal caps induces expression of neural markers XBrn-3d and XNeuroD, and co-expression of secreted Noelin-1 with noggin amplifies noggin-induced expression of XBrn-3d and XNeuroD. Furthermore, in animal caps neuralized by expression of noggin, co-expression of Noelin-1 causes expression of neuronal differentiation markers several stages before neurogenesis normally occurs in this tissue. Finally, only secreted forms of the protein can activate sensory marker expression, while all forms of the protein can induce early neurogenesis. This suggests that the cellular localization of Noelin-1 may be important to its function. Thus, Noelin-1 represents a novel secreted factor involved in neurogenesis.