XASH-3, a novel Xenopus achaete-scute homolog, provides an early marker of planar neural induction and position along the mediolateral axis of the neural plate

We have isolated a novel Xenopus homolog of the Drosophila achaete-scute genes, called XASH-3. XASH-3 expression is neural specific and is detected as early as stage 11 1/2, making it one of the earliest markers of neural induction so far described. Moreover, XASH-3 expression within the neural plate is regionally restricted. Transverse bands of XASH-3 mRNA mark discrete positions along the anteroposterior axis, while longitudinal bands mark a discrete position along the mediolateral axis. This latter site of XASH-3 expression appears to demarcate the prospective sulcus limitans, a boundary zone that later separates the functionally distinct dorsal (alar) and ventral (basal) regions of the spinal cord. In sandwich explants lacking any underlying mesoderm, XASH-3 is expressed in longitudinal stripes located lateral to the midline. This provides the first indication that planar or midline-derived inductive signals are sufficient to establish at least some aspects of positional identity along the mediolateral axis of the neural plate. By contrast, the transverse stripes of XASH-3 expression are not detected, suggesting that this aspect of anteroposterior neural pattern is lost or delayed in the absence of vertically passed signals. The restricted mediolateral expression of XASH-3 suggests that mediolateral patterning of the neural plate is an early event, and that this regionalization can be achieved in the absence of inducing signals derived from underlying mesoderm

Crx, a Novel otx-like Homeobox Gene, Shows Photoreceptor-Specific Expression and Regulates Photoreceptor Differentiation

AbstractWe have isolated a novel otx-like homeobox gene, Crx, from the mouse retina. Crx expression is restricted to developing and mature photoreceptor cells. CRX bound and transactivated the sequence TAATCC/A, which is found upstream of several photoreceptor-specific genes, including the opsin genes from many species. Overexpression of Crx using a retroviral vector increased the frequency of clones containing exclusively rod photoreceptors and reduced the frequency of clones containing amacrine interneurons and Müller glial cells. In addition, presumptive photoreceptor cells expressing a dominant-negative form of CRX failed to form proper photoreceptor outer segments and terminals. Crx is a novel photoreceptor-specific transcription factor and plays a crucial role in the differentiation of photoreceptor cells

Mouse labial-like homeobox-containing genes: structure and expression during embryogenesis

While mouse development has been well described at a morphological level, very little is known about how development is regulated. In contrast, the ease of developmental analysis in Drosophila has led to the identification of a large number of developmentally important genes. Molecular characterisation revealed that many of the genes involved specifically in determining the Drosophila body plan contain a conserved sequence called the homeobox. This sequence is highly conserved through evolution and so it can be used to isolate homologous genes in other species. In this way more than 40 homeobox- containing genes have been identified in the mouse. The high level of sequence conservation and the temporally and spatially restricted expression of the mouse genes during development indicate that they are also developmental regulators involved in conferring spatial information within the embryo. Thus, through knowledge of Drosophila development and the techniques of molecular biology, it is now possible to study mouse developmental genes in detail.In this thesis, the characterisation of two mouse homeobox -containing genes, Hox 2.9 and Hox 1.6, is presented. Sequence analysis revealed that these genes are closely related and that, among Drosophila genes, they are most similar to labial in the Antennapedia complex. They are therefore thought to have arisen by duplication of a single ancestral gene. As well as being structurally similar the genes share many features of their expression patterns. Both genes are expressed early in development (71/2 days) and, unlike other known mouse homeobox -containing genes, they are not expressed after 11 days of development. At 8 days the genes share the same anterior boundary of expression in the hindbrain and in the later embryo, with the exception of persistent Hox 2.9 expression in the hindbrain, they have the same anterioposterior restrictions. This indicates that the genes are functionally similar and also that they respond to at least some of the same signals in the embryo.A striking difference between the expression patterns of mouse labial -like genes is the unique expression of Hox 2.9 in a single segmental unit (rhombomere 4) of the hindbrain (from 81/2 days). This expression coincides perfectly with the morphological extent of rhombomere 4 and persists throughout the period that rhombomeres are visible (up to 11 days). It is therefore suggested that Hox 2.9 participates in conferring segment identity. In addition neural crest cells that arise from rhombomere 4 specifically express Hox 2.9 and this supports the idea of neural crest cells being patterned according to their position of origin in the central nervous system. Detailed analysis of the onset of segmental expression of Hox 2.9 and another segmentally expressed gene in the hindbrain, Krox 20, showed that Hox 2.9 expression becomes localised from a broad domain at 81/2 days of development, up to 6 hours before rhombomeres are clearly visible.Retinoic acid is a strong candidate for a natural morphogen in the vertebrate embryo. The effect of in vitro treatment with retinoic acid on segmentation of the mouse hindbrain and on the expression of Hox 2.9 and Krox 20 was therefore analysed. It was found that segmentation in treated embryos is abnormal and that the clear segmental localisation of expression of the two genes is not found. The hindbrain expression domains are shifted rostrally following treatment and while the expression of the two genes remains mutually exclusive there is no longer a single planar boundary between the domains. Instead there is an irregular alternation of cells expressing the two genes at the boundaryTwo differential splicing products of Hox 1.6 were isolated from the developing embryo. A comparison was made of the distribution of these transcripts, only one of which can code for a homeodomain containing protein. It was found that the relative proportion of homeodomain producing message decreases as development proceeds

A novel spalt gene expressed in branchial arches affects the ability of cranial neural crest cells to populate sensory ganglia

Cranial neural crest cells differentiate into diverse derivatives including neurons and glia of the cranial ganglia, and cartilage and bone of the facial skeleton. Here, we explore the function of a novel transcription factor of the spalt family that might be involved in early cell-lineage decisions of the avian neural crest. The chicken spalt4 gene (csal4) is expressed in the neural tube, migrating neural crest, branchial arches and, transiently, in the cranial ectoderm. Later, it is expressed in the mesectodermal, but not neuronal or glial, derivatives of midbrain and hindbrain neural crest. After over-expression by electroporation into the cranial neural tube and neural crest, we observed a marked redistribution of electroporated neural crest cells in the vicinity of the trigeminal ganglion. In control-electroporated embryos, numerous, labeled neural crest cells ([similar]80% of the population) entered the ganglion, many of which differentiated into neurons. By contrast, few ([similar]30% of the population) spalt-electroporated neural crest cells entered the trigeminal ganglion. Instead, they localized in the mesenchyme around the ganglionic periphery or continued further ventrally to the branchial arches. Interestingly, little or no expression of differentiation markers for neurons or other cell types was observed in spalt-electroporated neural crest cells

A MOLECULAR APPROACH TO CALANUS (COPEPODA:CALANOIDA) DEVELOPMENT AND SYSTEMATICS

Production and recruitment measurements in marine copepods of the genus Calanus have been addressed via the study of genes involved in early embryogenesis. The first sequence from a Calanus helgolandicus (C. helgolandicus) developmental gene (Cal-Antp) has been cloned by screening a C. helgolandicus genomic library with a homologous Calanus homeobox probe. Sequencing of an isolated and sub-cloned fragment of this gene, plus further analysis by Inverse Polymerase Chain Reaction (IVPCR), has shown it to be homologous with other Antennapedia homeobox genes. The temporal expression of Cal-Antp was analysed through its messenger RNA (mRNA) complement by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The gene was expressed in tissue taken from eggs over 18 hours old, and in nauplii and copepodite stages, but no expression was detected in eggs less than 18 hours old or adult tissue. Three further homeobox-containing genes have been identified and analysed through their expression in C. helgolandicus eggs. Two of these are caudal homologues, and the third is homologous to the Antennapedia class of genes. The C. helgolandicus developmental gene sequence data provides a means of developing probes to monitor the temporal expression of such genes and their responses to environmental influence. The applicability of such probes to the investigation of key production and recruitment processes, including egg viability measurement, is discussed. A relatively simple and cost effective method has been developed to identify the four Calanus species common to the North Atlantic. This system involves the PCR amplification of a region of the mitochondrial rRNA gene without prior purification of the DNA, followed by Restriction Fragment Length Polymorphism (RFLP) analysis of the amplified product. The versatility of the method is demonstrated by the unambiguous identification to species of any life stage, from egg to adult, and of any individual body parts. The molecular identification technique has for the first time shown the unexpected presence of three different Calanus species in Lurefjorden, Norway and has proved to be consistently accurate for all individuals tested including geographically distinct conspecific populations.Plymouth Marine Laborator

MASH1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identity

We have investigated the genetic circuitry underlying the determination of neuronal identity, using mammalian peripheral autonomic neurons as a model system. Previously, we showed that treatment of neural crest stem cells (NCSCs) with bone morphogenetic protein-2 (BMP-2) leads to an induction of MASH1 expression and consequent autonomic neuronal differentiation. We now show that BMP2 also induces expression of the paired homeodomain transcription factor Phox2a, and the GDNF/NTN signalling receptor tyrosine kinase c-RET. Constitutive expression of MASH1 in NCSCs from a retroviral vector, in the absence of exogenous BMP2, induces expression of both Phox2a and c-RET in a large fraction of infected colonies, and also promotes morphological neuronal differentiation and expression of pan-neuronal markers. In vivo, expression of Phox2a in autonomic ganglia is strongly reduced in Mash1 -/- embryos. These loss- and gain-of-function data suggest that MASH1 positively regulates expression of Phox2a, either directly or indirectly. Constitutive expression of Phox2a, by contrast to MASH1, fails to induce expression of neuronal markers or a neuronal morphology, but does induce expression of c-RET. These data suggest that MASH1 couples expression of pan-neuronal and subtype-specific components of autonomic neuronal identity, and support the general idea that identity is established by combining subprograms involving cascades of transcription factors, which specify distinct components of neuronal phenotype

Amino acid sequence and distribution of mRNA encoding a major skeletal muscle laminin binding protein: an extracellular matrix-associated protein with an unusual COOH-terminal polyaspartate domain.

Two cDNAs encoding an abundant chicken muscle extracellular matrix (ECM)-associated laminin-binding protein (LBP) have been isolated and sequenced. The predicted primary amino acid sequence includes a probable signal peptide and a site for N-linked glycosylation, but lacks a hydrophobic segment long enough to span the membrane. The COOH terminus consists of an unusual repeat of 33 consecutive aspartate residues. Comparison with other sequences indicates that this protein is different from previously described LBPs and ECM receptors. RNA blot analysis of LBP gene expression showed that LBP mRNA was abundant in skeletal and heart muscle, but barely detectable in other tissues. Blots of chicken genomic DNA suggest that a single gene encodes this LBP. The amino acid sequence and mRNA distribution are consistent with the biochemical characterization described by Hall and co-workers (Hall, D. E., K. A. Frazer, B. C. Hahn, and L. F. Reichardt. 1988. J. Cell Biol. 107:687-697). These analyses indicate that LBP is an abundant ECM-associated muscle protein with an unusually high negative charge that interacts with both membranes and laminin, and has properties of a peripheral, not integral membrane protein. Taken together, our studies show that muscle LBP is a secreted, peripheral membrane protein with an unusual polyaspartate domain. Its laminin and membrane binding properties suggest that it may help mediate muscle cell interactions with the extracellular matrix. We propose the name "aspartactin" for this LBP