slouch

EVOLUTIONARY HOMOLOGS

The amino acid sequence of the msh homeobox domain is highly homologous to the homeodomains of the Drosophila NK1/S59 and empty spiracles genes (Lord, 1995).

Sax1 (previously CHox3) is a chicken homeobox gene belonging to the same homeobox gene family as the Drosophila Slouch (NK1) and the honeybee HHO genes. Sax1 transcripts are present from stage 2 H&H until at least 5 days of embryonic development. However, specific localization of Sax1 transcripts could not be detected by in situ hybridization prior to stage 8-, when Sax1 transcripts are specifically localized in the neural plate, posterior to the hindbrain. From stages 8- to 15 H&H, Sax1 continues to be expressed only in the spinal part of the neural plate. The anterior border of Sax1 expression is always in the transverse plane separating the youngest somite from the yet unsegmented mesodermal plate and to regresses with similar dynamics to that of the segregation of the somites from the mesodermal plate. The posterior border of Sax1 expression coincides with the posterior end of the neural plate. In order to study a possible regulation of Sax1 expression by its neighboring tissues, several embryonic manipulation experiments were performed. These manipulations included: removal of somites, mesodermal plate or notochord; transplantation of a young ectopic notochord in the vicinity of the neural plate, or transplantation of neural plate sections into the extraembryonic area. The results of these experiments reveal that the induction of the neural plate by the mesoderm has already occurred in full primitive streak embryos, after which Sax1 is autonomously regulated within the spinal part of the neural plate (Spann, 1994).

The novel murine Sax-1 gene, a member of the NK-1 class of homeobox genes, has been isolated. Reported here is its expression pattern in the developing central nervous system (CNS) in comparison to two other homeobox genes (Evx-1 and Pax-6). Sax-1 is transiently expressed in the developing posterior CNS. First seen in the ectoderm lateral to the primitive streak, the signal later encompasses the neural plate. Posteriorly, the expression domain overlaps with the Evx-1 expression in the streak, while anteriorly it is delimited by the Pax-6 signal in the neural tube. In this early phase, starting at day 9.5 pc, Sax-1 is expressed in distinct areas of spinal cord, hindbrain and forebrain. Particularly strong signals are detected in rhombomere 1 and in the pretectum. In these areas, subsets of neurons may be marked and specified. In addition to the normal pattern of Sax-1 during development, the expression in different mouse mutants was analysed. In Brachyury curtailed homozygotes, the expression of Sax-1 is reduced during neurulation and even lost at day 9.0 pc. Ventral shift and finally loss of the signal in the ventral spinal cord is observed in Danforth's short tail homozygotes (Schubert, 1995).

A novel single-sided specific polymerase chain reaction (PCR) strategy inspired by ligation-mediated PCR has been used to clone fragments of divergent homeobox genes from a flatworm, the planarian Polycelis nigra. Eight homeobox-containing fragments were amplified, belonging to the Hox, msh, NK-1 and NK-2 classes. Together with the results obtained from several genomes of platyhelminths, this screening shows the presence of the same array of homeodomain developmental regulators in planarians, traditionally regarded as primitive metazoans in terms of body plan, as in coelomate organisms. However, the presence of a Ubx/abd-A homolog may indicate that platyhelminths are more closely related to protostomes than to deuterostomes and supports the idea that flatworms have inherited an elaborate HOX cluster (seven or eight genes) from their ancestor. Likely homologs of the fly genes tinman, bagpipe and S59 suggest that the mesoderm might be patterned by the same genes in all bilaterally symmetrical animals. Finally, a msh-like gene, a family known to be involved in inductive mechanisms in vertebrates, has been found. These results support the hypothesis that the tremendous diversity of metazoan body plans is specified by a largely conserved array of homeobox-containing developmental genes (Balavoine, 1996).

A mouse homeobox gene, Nkx-1.2, (previously termed Sax-1) that is closely related to the Drosophila NK-1/S59 gene was cloned, and genomic DNA and cDNA were sequenced. Nine Nkx-1.2 cDNA clones were found that correspond to three species of Nkx-1.2 mRNA that are formed by alternative splicing at conventional 5' donor and 3' acceptor splice sites; however, seven cDNA clones were found that correspond to three species of Nkx-1.2 mRNA from testes that have novel TG/AC 5' and 3' splice sites. The consensus splice sequences are: 5' donor, CC|TGGAAG; 3' acceptor, ACTTAC|. Predicted amino acid sequences suggest that some transcripts may be translated into proteins that lack part or all of the homeodomain. At least three bands of Nkx-1.2 mRNA were found in RNA from the testes. Nkx-1.2 mRNA is present in postmeiotic germ cells of the testis and in mature spermatozoa. Nkx-1.2 mRNA is also found in regions of the adult cerebral cortex, hippocampus, diencephalon, pons/medulla, and cerebellum. Nkx-1.2 mRNA is found in embryos in highest abundance in 10-day embryos; the mRNA levels decrease during further development. Nkx-1.2 mRNA is also found in discrete zones of the embryonic mesencephalon and myelencephalon (Rovescalli, 2000).

A homeobox gene, pnx, a homolog of Drosophila Slouch/S59/NK-1, is expressed in prospective posterior neurogenic regions and later in primary neurons. pnx expression is regulated by a signal from the non-axial mesendoderm and by Notch signaling. Pnx contains an Eh1 repressor domain, which interacts with Groucho and acts as a transcriptional repressor. Misexpression of pnx increases neural precursor cells and postmitotic neurons, which express neurogenin1 and elavl3/HuC, respectively. Expression of an antimorphic Pnx (VP16Pnx) or inhibition of Pnx by antisense morpholino oligonucleotide lead to the reduction in the number of a subset of primary neurons. Misexpression of pnx promotes neurogenesis independent of Notch signaling. Epistatic analyses shows that Pnx also functions downstream of the Notch signal. These data indicate that pnx is a novel repressor-type homeobox gene that regulates posterior neurogenesis (Bae, 2003).

pnx transcripts were detected in two separate regions (medial and lateral domains) that later gave rise to primary motor, inter and Rohon-Beard neurons, indicating that pnx does not simply mark the posterior neuroectoderm but rather defines the posterior neurogenic regions. pnx expression is regulated by a signal from the non-axial mesendoderm and can be upregulated by putative posteriorizing signals, indicating that pnx functions downstream of posteriorizing signals derived from non-axial mesendoderm. It remains to be elucidated how pnx expression is restricted to the neurogenic regions and does not occur between them. swirl mutant embryos display a circular expression of pnx and do not have intermediate regions that lack pnx expression in the posterior neuroectoderm, suggesting that the neurogenic region-specific expression of pnx is regulated by the balance between BMPs and the organizer-derived BMP antagonists (neural inducers). Expression of pnx in medial neurogenic regions and in primary motoneurons was diminished or absent in antivin (lefty1) RNA-injected embryos and squint mutant embryos, which lacked (a part of) axial mesendoderm. This is consistent with the observation that signals from axial mesendoderm, such as notochord and floor plate, are required for development of motoneurons (Bae, 2003 and references therein).

pnx functions in the lateral inhibition mechanism. Gain and loss of Pnx function shows that pnx regulates the expression of the proneural gene ngn1. Misexpression of ngn1, which elicits an increased or ectopic expression of deltaA, deltaD and her4, leads to a reduction in pnx-expressing cells. pnx-expressing cells increase in number within the neurogenic regions in the mib mutant embryos and DN-Delta-expressing embryos at the segmentation (neurula) stages, indicating that pnx expression is negatively regulated by the Notch signal. These data support the idea that Pnx activates proneural gene (such as ngn1)-dependent lateral inhibition machinery that suppresses the expression of pnx in non-neuronal cells and restricts the numbers of neurons (Bae, 2003).

Pnx contains an Eh1 repressor domain and interacts with the transcriptional co-repressor Groucho2, at least in 293T cells. Reporter analysis reveals that Pnx acts as a transcriptional repressor and that the Eh1-mediated interaction with Groucho(s) is involved in this repressor activity. Furthermore, VP16-Pnx functions as an antimorphic molecule in the formation of primary neurons. These data indicate that Pnx functions as a transcriptional repressor and should repress genes that have the ability to repress the proneural genes. Candidates that are repressed by Pnx could include downstream components of the Notch signal, such as the hes/her-family genes. However, this is not the case. Misexpression of Pnx still increases the ngn1- and elavl3-expressing cells in embryos in which Notch signaling is suppressed. Furthermore, Pnx does not inhibit the expression of either her4 or her9, which are the only Hes/Her-family members reported to be expressed in the neural plate. Inhibition of the Notch signal leads to an increase in the density of neuronal cells 'within the neurogenic region', but does not lead to the expansion of neurogenic regions. By contrast, the misexpression of Pnx in either wild-type or mib mutant embryos elicits an 'expansion' of the ngn1-expressing neurogenic regions. pnx is epistatic to the Notch signaling in the formation of primary neurons, providing genetic evidence that the pnx-mediated neurogenesis does not require the Notch signal. These data indicate that Pnx can promote neurogenesis not by inhibiting the Notch signal (lateral inhibition mechanisms), but rather by expanding neurogenic regions within the neuroectoderm. To promote neurogenesis, Pnx represses the expression of certain transcriptional repressor(s), other than those downstream of the Notch signal, which inhibit the proneural gene expression and neurogenesis. The identification of targets for Pnx will shed light on the mechanisms by which the neurogenic regions are established and proneural genes are regulated (Bae, 2003).

The vertebrate neural crest is formed at the border between the neural plate and nonneural ectoderm during neurulation and eventually gives rise to a variety of cell types, including neurons, glia, facial chondrocytes and osteoblasts, and melanocytes. Although several secreted molecules, such as BMP, Wnts, FGF, and Noelin, have been implicated in neural crest formation, little is known about the precise intracellular mechanism underlying neural crest induction and differentiation. A novel NK-1 class homeobox gene Nbx in Xenopus has been defined whose expression is correlated with neural crest formation. Nbx harbors an Eh1 domain and is a transcriptional repressor. Overexpression of Nbx suppresses neural plate makers and causes enhanced expression of the neural crest maker Slug. In contrast, the overexpression of a dominant negative form of Nbx during neurula stages suppresses the expression of the neural crest marker Slug and expands neural markers such as Otx2 and Sox2. Taken together, it is proposed that Nbx is an essential transcriptional repressor required to permit neural crest induction by inhibiting the neural fate (Kurata, 2003).

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