The leech hunchback protein is expressed in the epithelium and CNS but not in the segmental precursor lineages

We are interested in identifying the regulatory genes involved in segmental pattern formation in annelids. The Drosophila segmentation gene hunchback (hb) is critical for the proper anteroposterior development of the fly embryo, but its function outside the diptera is currently unknown. Here, the protein expression pattern of Leech Zinc Finger II (LZF2), a leech orthologue of hb is characterized. In early embryogenesis, LZF2 protein is expressed in a subset of micromeres and is later expressed in the micromere-derived epithelium of the provisional epithelium and prostomium. LZF2 protein is detected in the ventral nerve cord during organogenesis, first in interganglionic muscle cells and later in subsets of neurons in each neuromere of the CNS. The location of immunoreactive cells during development and the similarity of the expression pattern of LZF2 to the expression of the Caenhorhabditis elegans hb homologue hbl-1 suggests that LZF2 plays a role in the morphogenetic movements of leech gastrulation and later in CNS specification but not in anteroposterior pattern formation. Received: 28 May 1999 / Accepted: 21 December 1999     

Lox6, a leech Dfd ortholog, is expressed in the central nervous system and in peripheral sensory structures

 Sequence analysis of a newly isolated Hirudo medicinalis cDNA containing an Antennapedia (Antp)-class homeobox suggests that the corresponding gene, Lox6, is an ortholog of the Drosophila Deformed (Dfd) gene. In situ hybridization of whole-mounted preparations shows that the major sites of Lox6 expression during embryogenesis are the central nervous system (CNS) and the peripheral sensory system. Lox6 mRNA can be detected in a subset of neurons in each ganglion from the subesophageal ganglion (RG2) to the most posterior ganglion, with the highest level of expression seen in RG3. Peripherally, Lox6 is expressed principally in the primordia of the sensillae and in the eyes. This pattern of expression of Lox6 suggests that one of its functions may be to contribute to the diversification of neuronal phenotypes. Received: 16 August 1997/Accepted: 20 December 1997     

Segmental diversification of an identified leech neuron correlates with the segmental domain in which it expresses lox2, a member of the hox gene family

The cellular colocalization of LOX2 protein and small cardioactive peptide (SCP)-like immunoreactivity was studied in the nerve cord of the glossiphoniid leech Helobdella triserialis. Of the six neurons that express SCP in the midbody segments 7 to 17, only one, the MPS neuron, expresses LOX2 protein. The medial paired SCP (MPS) neurons are segmentally repeated and can be divided into three contiguous segmental domains according to cell body size and the timing and level of SCP expression. MPS neurons located in the anterior and middle segmental domains express LOX2 protein. In the middle domain, large MPS neurons begin to accumulate SCP shortly after the end of embryonic development, whereas in the anterior domain the MPS neurons are smaller and begin to express SCP at a later stage. In the posterior domain the MPS neurons exhibit a third phenotype—they have large cell bodies, express low levels of SCP starting from the midjuvenile stage, and do not show detectable LOX2 expression. Lineage tracer injections showed that the MPS neurons arise from a stereotyped cell lineage and are descended from the O teloblast stem cell. In midbody ganglia 2 to 6 and 18 to 21, there are lineally homologous neurons that do not express either LOX2 protein or SCP. Thus, the boundaries of LOX2 expression coincide precisely with two of the segmental boundaries of MPS differentiation, suggesting that expression of Lox2 at the level of this single identified neuron governs some, but not all, aspects of the neuron's segmental diversification. © 1996 John Wiley & Sons, Inc.     

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease,is expressed by specific cell lineages during mouse embryonic development and in adult tissues

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.     

Developmental origin of segmental differences in the leech ectoderm: survival and differentiation of the distal tubule cell is determined by the host segment

The body plan of the adult leech is metameric, with each hemisegmental complement of ectodermal and mesodermal tissues being produced from a set of seven serially repeated embryonic blast cells. Previous studies have shown that homologous o blast cells give rise to an almost identical complement of descendant cells in each of the 21 abdominal segments, but that one o blast cell derivative--the distalmost cell of the nephridial tubule--is only present in 15 abdominal segments in the mature leech. Here we show that all o blast cells generate a presumptive distal tubule cell and that this cell migrates to its normal position in all abdominal segments. However, in segments which normally do not contain the mesodermal portion of the nephridium, the distal tubule cell dies before undergoing its terminal morphological differentiation. To ascertain whether the fate of the distal tubule cell is determined by its lineage history or by the segmental environment into which it is born, we utilized a previously described procedure for altering the segmental register between different embryonic cell lines. This procedure allowed us to effectively transplant o blast cells into more posterior segments prior to the cell divisions which generate their descendant clones. The results indicate that the survival or death of the distal tubule cell is determined by the identity of the host segment and that a given distal tubule cell could be effectively murdered or rescued by slipping its blast cell precursor into an appropriate segment. These findings suggest that the segment-specific pattern of distal tubule cell survival is not inherent to the O cell line, but arises from interactions with surrounding tissues.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

The putative peroxisomal gene Pxt1 is exclusively expressed in the testis

Genes reported to be crucial for spermatogenesis are often exclusively expressed in the testis. We have identified a novel male germ cell-specific expressed gene named peroxisomal testis specific 1 (Pxt1) with expression starting at the spermatocyte stage during mouse spermatogenesis. The putative amino acid sequence encoded by the cDNA of the Pxt1 gene contains a conserved Asn-His-Leu (NHL)-motif at its C-terminal end, which is characteristic for peroxisomal proteins. Pxt1-EGFP fusion protein is co-localized with known peroxisomal marker proteins in transfected NIH3T3 cells. In addition, we could demonstrate that the peroxisomal targeting signal NHL is functional and responsible for the correct subcellular localization of the Pxt1-EGFP fusion protein. In male germ cells peroxisomes were reported only in spermatogonia. The Pxt1 gene is so far the first gene coding for a putative peroxisomal protein which is expressed in later steps of spermatogenesis, namely in pachytene spermatocytes.     

A conserved nucleotide sequence, coding for a segment of the C-propeptide, is found at the same location in different collagen genes.

The nucleotide sequence of a segment of the chick alpha 1 type III collagen gene which codes for the C-propeptide was determined and compared with the corresponding sequence in the alpha 1 type I and alpha 2 type I collagen genes. As in the alpha 2 type I gene the coding information for the C-propeptide of the type III collagen gene is subdivided in four exons. Similarly, the amino proximal exon contains sequences for both the carboxy terminal end of the alpha-helical segment of collagen and for the beginning of the C-propeptide in both genes. Therefore, this organization of exons must have been established before these two collagen genes arose by duplication of a common ancestor. In several subsegments the deduced amino acid sequence for the C-propeptide of type III collagen shows a strong homology with the corresponding amino acid sequence in alpha 1 and alpha 2 type I. For one of these homologous amino acid sequences, however, the nucleotide sequence is much better conserved than for the others. It is possible that a mechanism of gene conversion has maintained the homogeneity of this nucleotide sequence among the interstitial collagen genes. Alternatively, the conserved nucleotide sequence may represent a regulatory signal which could function either in the DNA or in the RNA.     

Bmp4 gene is expressed at the putative site of fusion in the midfacial region

The molecular mechanisms by which the primordia of the midface grow and fuse to form the primary palate portion of the craniofacial region are not well characterized. This is in spite of the fact that failure of growth and/or fusion of these primordia leads to the most common craniofacial birth defect in humans (i.e. clefts of the lip and/or palate). Bmp4 plays a critical role during early embryonic development and has previously been shown to play a role in epithelial-mesenchymal interactions in the craniofacial region of chicks. We analyze the expression of bmp4 in mouse as the midfacial processes undergo fusion to form the primary palate. We show that bmp4 is expressed in a very distinct manner in the three midfacial processes (lateral nasal, LNP, medial nasal, MNP, and maxillary processes, MxP) that ultimately fuse to form the midface. Prior to fusion of the midfacial processes, bmp4 is expressed in the ectoderm of the LNP, MNP, and MxP in a distinct spatial and temporal manner near and at the site of fusion of the midface. Bmp4 appears to demarcate the cells in the LNP and MNP that will eventually contact and fuse with each other. As fusion of the three prominences proceeds, some bmp4 expressing cells are trapped in the fusion line. Later, the expression of bmp4 switches to the mesenchyme of the midface underlying its initial expression in the ectoderm. The switch occurs soon after fusion of the three processes. The pattern of expression in the midfacial region implicates the important role of bmp4 in mediating the fusion process, possibly through apoptosis of cells in the putative site of fusion, during midfacial morphogenesis.     

nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain.

The nitrogen regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. The positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. The complete nucleotide sequence of the nit-2 gene was determined. The nit-2 mRNA is 4.3 kilobases long and has a long nontranslated sequence at both its 5' and 3' ends. The nit-2 gene nucleotide sequence can be translated to yield a protein containing 1,036 amino acid residues with a molecular weight of approximately 110,000. Deletion analyses demonstrated that approximately 21% of the NIT2 protein at its carboxy terminus can be removed without loss of function. The nit-2 protein contains a single putative Cys2/Cys2 zinc finger domain which appears to function in DNA binding and which has striking homology to a mammalian trans-acting factor, GF-1.     

Integrin-alphavbeta6, a putative receptor for foot-and-mouth disease virus, is constitutively expressed in ruminant airways.

Evolved functions of integrin-alpha(v)beta(6) include roles in epithelial cell-extracellular matrix protein interactions and in the binding and activation of latent TGF-beta(1). Integrin-alpha(v)beta(6) is also exploited as a receptor by foot-and-mouth disease virus (FMDV) and may play a significant role in its transmission and pathogenesis. The ovine beta(6) integrin subunit was cloned and sequenced (EMBL accession no. AJ439062). Screening of normal ovine tissues by RT-PCR and immunocytochemistry confirmed that integrin-alphavbeta6 is restricted to sheep epithelial cells. Integrin-alphavbeta6 expression was detected in epithelia of the airways, oral cavity, gastrointestinal tract, kidney, sweat glands, hair follicle sheaths, and the epidermis of pedal coronary band (PB) but not of normal skin. Consistent with FMDV tropism, integrin-alphavbeta6 was detected within the basal layers of the stratified squamous epithelium of the oral mucosa and PB. In addition, integrin-alphavbeta6 appears to be constitutively expressed in the normal airways of both cattle and sheep. The latter finding suggests that ruminant airway epithelium presents a highly accessible target for initiation of infection with FMDV by inhalation.