Isolation and identification of homeobox genes from the human placenta including a novel member of the Distal-less family,DLX4

We have carried out a DNA binding site screen of a 32-week human placental cDNA library using a consensus homeodomain binding site as a probe. This study represents the first library screen carried out to isolate homeobox genes from the human placenta. We have shown that three homeobox genes known to be expressed in the embryo, HB24, GAX and MSX2 are also expressed in the placenta. We have also identified a novel homeobox gene, DLX4, that shows 85% sequence identity with the homeodomain encoded by the Drosophila Distal-less (Dll) gene. DLX4 therefore represents a new member of the Distal-less family of homeobox genes. This is the first evidence that members of the Distal-less family of homeobox genes are expressed in the placenta. Using fluorescence in situ hybridisation (FISH), DLX4 has been assigned to human chromosome 17q21–q22. This places DLX4 in the same region of chromosome 17 as another member of the Distal-less family, DLX3 (Scherer et al., 1995), and the HOX-B homeobox gene cluster (Acampora et al., 1989; Boncinelli et al., 1991). Members of the Distal-less family (DLX1 and DLX2; DLX5 and DLX6) are found as closely linked pairs on human chromosomes (Simeone et al., 1994). We predict that DLX3 and DLX4 are closely linked and have arisen through gene duplication and divergence from a common ancestral precursor.     

optix functions as a link between the retinal determination network and the dpp pathway to control morphogenetic furrow progression in Drosophila

optix, the Drosophila ortholog of the SIX3/6 gene family in vertebrate, encodes a homeodomain protein with a SIX protein–protein interaction domain. In vertebrates, Six3/6 genes are required for normal eye as well as brain development. However, the normal function of optix in Drosophila remains unknown due to lack of loss-of-function mutation. Previous studies suggest that optix is likely to play an important role as part of the retinal determination (RD) network. To elucidate normal optix function during retinal development, multiple null alleles for optix have been generated. Loss-of-function mutations in optix result in lethality at the pupae stage. Surprisingly, close examination of its function during eye development reveals that, unlike other members of the RD network, optix is required only for morphogenetic furrow (MF) progression, but not initiation. The mechanisms by which optix regulates MF progression is likely through regulation of signaling molecules in the furrow. Specifically, although unaffected during MF initiation, expression of dpp in the MF is dramatically reduced in optix mutant clones. In parallel, we find that optix is regulated by sine oculis and eyes absent, key members of the RD network. Furthermore, positive feedback between optix and sine oculis and eyes absent is observed, which is likely mediated through dpp signaling pathway. Together with the observation that optix expression does not depend on hh or dpp, we propose that optix functions together with hh to regulate dpp in the MF, serving as a link between the RD network and the patterning pathways controlling normal retinal development.     

A novel role of BELL1-like homeobox genes, PENNYWISE and POUND-FOOLISH, in floral patterning

Flowers are determinate shoots comprised of perianth and reproductive organs displayed in a whorled phyllotactic pattern. Floral organ identity genes display region-specific expression patterns in the developing flower. In Arabidopsis, floral organ identity genes are activated by LEAFY (LFY), which functions with region-specific co-regulators, UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS), to up-regulate homeotic genes in specific whorls of the flower. PENNYWISE (PNY) and POUND-FOOLISH (PNF) are redundant functioning BELL1-like homeodomain proteins that are expressed in shoot and floral meristems. During flower development, PNY functions with a co-repressor complex to down-regulate the homeotic gene, AGAMOUS (AG), in the outer whorls of the flower. However, the function of PNY as well as PNF in regulating floral organ identity in the central whorls of the flower is not known. In this report, we show that combining mutations in PNY and PNF enhance the floral patterning phenotypes of weak and strong alleles of lfy, indicating that these BELL1-like homeodomain proteins play a role in the specification of petals, stamens and carpels during flower development. Expression studies show that PNY and PNF positively regulate the homeotic genes, APETALA3 and AG, in the inner whorls of the flower. Moreover, PNY and PNF function in parallel with LFY, UFO and WUS to regulate homeotic gene expression. Since PNY and PNF interact with the KNOTTED1-like homeodomain proteins, SHOOTMERISTEMLESS (STM) and KNOTTED-LIKE from ARABIDOPSIS THALIANA2 (KNAT2) that regulate floral development, we propose that PNY/PNF-STM and PNY/PNF-KNAT2 complexes function in the inner whorls to regulate flower patterning events.     

Comprehensive expression analysis of all Wnt genes and their major secreted antagonists during mouse limb development and cartilage differentiation

Wnt signalling plays important roles in patterning and outgrowth of the vertebrate limb. Different mutations in Wnt genes, their antagonists or (co-)receptors result in patterning and outgrowth defects as well as chondrocyte and bone phenotypes in mouse and human. Understanding Wnt activity during mouse limb development and chondrogenesis requires a temporal and spatial overview of Wnt signalling key factor expression. Here we present a comparative expression analysis of all 19 Wnt genes and their major secreted antagonists of the Dickkopf (Dkk), Wisp and the secreted frizzled related protein (Sfrp) families during mouse limb development. Our study reveals new domains of expression for Wnt2, Wnt2b, Wnt5b, Wnt6, Wnt7b, Wnt9a, Wnt10a, Wnt10b, Wnt11 and Wnt16, in the limb. We also identified novel expression domains for the Wnt antagonists Sfrp1, Sfrp3, Sfrp5, Wisp1 as well as Dkk2 and Dkk3. We provide a full expression pattern for Wif1 in limb development, for which no limb expression had been documented so far.     

The zebrafish amyloid precursor protein-b is required for motor neuron guidance and synapse formation

The amyloid precursor protein (APP) is a transmembrane protein mostly recognized for its association with Alzheimer's disease. The physiological function of APP is still not completely understood much because of the redundancy between genes in the APP family. In this study we have used zebrafish to study the physiological function of the zebrafish APP homologue, appb, during development. We show that appb is expressed in post-mitotic neurons in the spinal cord. Knockdown of appb by 50–60% results in a behavioral phenotype with increased spontaneous coiling and prolonged touch-induced activity. The spinal cord motor neurons in these embryos show defective formation and axonal outgrowth patterning. Reduction in Appb also results in patterning defects and changed density of pre- and post-synapses in the neuromuscular junctions. Together, our data show that development of functional locomotion in zebrafish depends on a critical role of Appb in the patterning of motor neurons and neuromuscular junctions.     

Tri-phasic expression of posterior Hox genes during development of pectoral fins in zebrafish: implications for the evolution of vertebrate paired appendages

During development of the limbs, Hox genes belonging to the paralogous groups 9-13 are expressed in three distinct phases, which play key roles in the segmental patterning of limb skeletons. In teleost fishes, which have a very different organization in their fin skeletons, it is not clear whether a similar patterning mechanism is at work. To determine whether Hox genes are also expressed in several distinct phases during teleost paired fin development, we re-analyzed the expression patterns of hox9-13 genes during development of pectoral fins in zebrafish. We found that, similar to tetrapod Hox genes, expression of hoxa/d genes in zebrafish pectoral fins occurs in three distinct phases, in which the most distal/third phase is correlated with the development of the most distal structure of the fin, the fin blade. Like in tetrapods, hox gene expression in zebrafish pectoral fins during the distal/third phase is dependent upon sonic hedgehog signaling (hoxa and hoxd genes) and the presence of a long-range enhancer (hoxa genes), which indicates that the regulatory mechanisms underlying tri-phasic expression of Hox genes have remained relatively unchanged during evolution. Our results suggest that, although simpler in organization, teleost fins do have a distal structure that might be considered comparable to the autopod region of limbs.     

Axial and appendicular skeletal transformations, ligament alterations, and motor neuron loss in Hoxc10 mutants

Vertebrate Hox genes regulate many aspects of embryonic body plan development and patterning. In particular, Hox genes have been shown to regulate regional patterning of the axial and appendicular skeleton and of the central nervous system. We have identified patterning defects resulting from the targeted mutation of Hoxc10, a member of the Hox10 paralogous family. Hoxc10 mutant mice have skeletal transformations in thoracic, lumbar, and sacral vertebrae and in the pelvis, along with alterations in the bones and ligaments of the hindlimbs. These results suggest that Hoxc10, along with other members of the Hox10 paralogous gene family, regulates vertebral identity at the transition from thoracic to lumbar and lumbar to sacral regions. Our results also suggest a general role for Hoxc10 in regulating chondrogenesis and osteogenesis in the hindlimb, along with a specific role in shaping femoral architecture. In addition, mutant mice have a reduction in lumbar motor neurons and a change in locomotor behavior. These results suggest a role for Hoxc10 in generating or maintaining the normal complement of lumbar motor neurons.     

Changes in the Inflammatory Response to Injury and Its Resolution during the Loss of Regenerative Capacity in Developing Xenopus Limbs

Tissue and organ regeneration, unlike development, involves an injury that in postembryonic animals triggers inflammation followed by resolution. How inflammation affects epimorphic regeneration is largely uninvestigated. Here we examine inflammation and its resolution in Xenopus laevis hindlimb regeneration, which declines during larval development. During the first 5 days postamputation, both regeneration-competent stage 53 and regeneration-deficient stage 57 hindlimbs showed very rapid accumulation of leukocytes and cells expressing interleukin-1β and matrix metalloproteinase 9. Expression of genes for factors mediating inflammatory resolution appeared more persistent at stages 55 and 57 than at stage 53, suggesting changes in this process during development. FoxP3, a marker for regulatory T cells, was upregulated by amputation in limbs at all three stages but only persisted at stage 57, when it was also detected before amputation. Expression of genes for cellular reprogramming, such as SALL4, was upregulated in limbs at all 3 stages, but markers of limb patterning, such as Shh, were expressed later and less actively after amputation in regeneration-deficient limbs. Topical application of specific proinflammatory agents to freshly amputated limbs increased interleukin-1β expression locally. With aqueous solutions of the proinflammatory metal beryllium sulfate, this effect persisted through 7 days postamputation and was accompanied by inhibition of regeneration. In BeSO₄-treated limbs expression of markers for both inflammation and resolution, including FoxP3, was prolonged, while genes for cellular reprogramming were relatively unaffected and those for limb patterning failed to be expressed normally. These data imply that in Xenopus hindlimbs postamputation inflammation and its resolution change during development, with little effect on cellular dedifferentiation or reprogramming, but potentially interfering with the expression of genes required for blastema patterning. The results suggest that developmental changes in the larval anuran immune system may be involved in the ontogenetic loss of epimorphic regeneration in this system.     

Involvement of Hox genes in shell morphogenesis in the encapsulated development of a top shell gastropod (Gibbula varia L.)

Regulatory gene expression during the patterning of molluscan shells has only recently drawn the attention of scientists. We show that several Hox genes are expressed in association with the shell gland and the mantle in the marine vetigastropod Gibbula varia (L.). The expression of Gva-Hox1, Gva-Post2, and Gva-Post1 is initially detected in the trochophore larval stage in the area of the shell field during formation of embryonic shell. Later, during development, these genes are expressed in the mantle demonstrating their continuous role in larval shell formation and differentiation of mantle edge that secretes the adult shell. Gva-Hox4 is expressed only late during the development of the veliger-like larva and may also be involved in the adult shell morphogenesis. Additionally, this gene also seems to be associated with secretion of another extracellular structure, the operculum. Our data provide further support for association of Hox genes with shell formation which suggest that the molecular mechanisms underlying shell synthesis may consist of numerous conserved pattern-formation genes. In cephalopods, the only other molluscan class in which Hox gene expression has been studied, no involvement of Hox genes in shell formation has been reported. Thus, our results suggest that Hox genes are coopted to various functions in molluscs.