- Email: [email protected]
β1-Integrin-bright stem cells in human skin
MOLECULA. MED,C,NETOOA
Round The
Will the real SMA gene please stand up! Identification and characterization of a spinal muscular atrophy-determining gene Lefebvre, S. et aL (1995) Cell80, 156-165
The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy Roy, N. et aL (1995) Cell80, 167-178
A novel cDNA detects homozygous microdeletions in greater than 50% of type I spinal muscular atrophy patients Thompson, T.G. et aL (1995) Nature Genet. 9, 56-61
SMA genes: deleted and duplicated
proposed to encode for a neuronal apoptosisinhibitory protein, which fits in well with the observed pathophysiology of SMA. Thompson et al. report prematurely on the identification of an incompletely characterized cDNA fragment that maps in the SMA region and may, in fact, be part of NAIP (Mahadevan et aL). Comparing the three maps will be essential to resolve the positions of the genes and the deletion breakpoints. This might be easier if Roy and Thompson had presented clearly the pedigree of their yeast artificial chromosomal (YAC) clones. In a discerning editorial, Lewin discusses that SMN fulfils the criteria for assigning a gene to a disease: it has mutations that appear to be confined to a single gene (itself) that are not present in controls. However, the presence of a second, non-allelic copy of SMN with no apparent inactivating mutations has, at least for now, delayed SMN being crowned champion. While the genetics of this complex disease continue to be resolved, these discoveries should already enable clinical diagnosis of >98% of SMA carriers and patients. For gene sleuths, the articles serve as a textbook of clues on how to identify and navigate the obstacles that nature has planted in the genome.
Mahadevan, M.S. eta/. (1995) Nature Genet. 9, 112-113
Genes for SMA: multum in parvo Lewin, B. (1995)
CD34 gene regulation an entry point to stem cell biology
Cell80, 1-5
With the new year came a trilogy of papers describing the identification of two genes and a cDNA fragment that were found to be deleted in patients with spinal muscular atrophy (SMA). SMA is an autosomal recessive neurodegenerative disease that affects 1 in 6000 individuals, and is characterized by depletion of the motor neurons in the spinal cord, leading to paralysis and death in childhood. The search for elusive genes in the SMA region on chromosome 5q13 has been complicated by the presence of expressed pseudogenes, inverted duplications, and the instability of cloned DNA. Notwithstanding, Lefebvre et al. have isolated a novel gene, the survival motor neuron (SMN) gene, which is deleted in 226 out of 229 patients with SMA examined. The remaining three patients carry subtler mutations that are not observed in unaffected individals. Roy et al. describe a nearby, but different, gene, NAIP, which is absent in 67% of SMA and 2% of non-SMA chromosomes, and is
Targeting gene expression to haematopoietic stem calls: a chromatindependent upstream element mediates call type-specific expression of the stem call antigen CD34 May, G. and Enver, T. (1995)
up
literature
Luciferase reporter plasmids were constructed to test the role of upstream DNase I HSS in haematopoietic-cell-specific expression. In transient transfection of murine 416B cells, inclusion of the CD34 upstream HSS increased transcription by only 1-2.5-fold over the CD34 promoter alone. However, when the same plasmids were used to generate stably transfected 416B cell populations, a much more striking 75-fold enhancement of luciferase activity was observed. This enhancement in activity was specific to 416B cells and was not observed in stably transfected Swiss 3T6 fibroblasts. Have the authors uncovered a CD34 locus control region (LCR)? Probably. The firstdescribed and best-characterized LCR was discovered in the human 13-globin locus, and comprises four DNase I HSS. The ability of this element to direct position-independent expression of co-linked promoters is only observed when stably integrated into chromatin. Much work remains to be done to delineate the exact DNA sequences of the CD34 gene that confer stem-cell-specific gene expression, and to identify the key proteins that interact with the CD34 LCR and promoter. The May and Enver paper is that most unusual of gene-expression papers: one that describes something novel. The CD34 gene harbours a DNA sequence that has important implications for stem-cell biology and gene therapy.
131-1ntegrin-bright stem cells in human skin Stem call patterning and fate in human epidermis Jones, P.H., Harper, S. and Watt, EM. (1995)
EMB01 14, 564-574 Cell 80, 83-93
Antibodies specific for the human stem-cell antigen CD34 are widely used for the identification and purification of haematopoietic stem cells. May and Enver undertook a detailed analysis of the murine CD34 gene in an attempt to define regions of the gene that direct expression of CD34 to haematopoietic stem cells. Regions of chromatin of the CD34 gene were found to be hypersensitive to digestion by the enzyme DNase I in nuclei from a murine haematopoietic cell line. Three DNase I hypersensitive sites (HSS), which were present only in the nuclei of haematopoietic cells and not in the nuclei of fibroblasts expressing CD34, were identified.
Kinetics and regulation of human keratinocyte stem calls in short term primary ex vivo culture Bata-Csorgo, Z., Hammerberg, C., Voorhees, J.J. and Cooper, K.D. (1995) J. Clin. Invest.95, 317-327
The distribution of proliferative and non-proliferative cells in human skin is a familiar textbook image. Close to the collagen IV-containing basement membrane, a layer of relatively undifferentiated basal cells divides to feed columns of increasingly differentiating cells m
© 1995. Elsevier Science Ltd 1357 - 4310/95/$9.50
5
Round The
up
MOLECULAR
literature
upwards to the surface. Studies of the capacity for basal-layer cells to proliferate in response to the elimination of adjacent partners has important implications for the treatment of burns and other wounds, diseases such as psoriasis, and prospects for long-term gene therapy through keratinocyte transfection. Two recent papers increase our knowledge of the properties of the proliferative, or stem, cells in human epidermis. Jones et al. report a continuation of their studies using fluorescently labelled antibodies to 131'-integrins, the cell surface receptors for extracellular matrix components found in the basement membrane. Fresh human epidermal cells with only twofold more ~l-integrin than their morphologically similar partners show rapid adhesion to collagen IV and have a higher
BIOPROBE BV NETHERLANDS HYBRIDOMA-BROKERAGE For Russian, Tsjech, Slovak, Hungarian, German, French, English, Italian, American, Australian and Dutch Institutes,
B I O T E C H FIRMS A N D L A R G E RESEARCH INSTITUTIONS buy and sell hybridomas producing useful diagnostic researchers and their institutesor universities to our clients:
MAJOR AND MINOR BIOTECH FIRMS IN EUROPE, USA, JAP~
SEEKING
TO~J.
THE
GAPSI N THEIR PRODUCT LISTS OF MONOCI~NAL ANTIBODIES AND KITS AND I/X)KING FOR NEW OPPORTUNITIKS
Fax: +31-20-6476794 Bioprobe BV The Netherlands Dr. Schaepmanlaan 1 1182 GM Amstelveen
6
proliferative potential. Interesting patterns of the distribution of these !31-integrin-bright cells are described in the epidermis from different sites. A measure of inherent pattern formation was evident even when fresh epidermal cells were cultured in vitro on plastic. The paper by Bata-Csorgo et aL also reports the correlation of I~-integrin-bright staining with a high proliferative potential. An observation of some significance was then made on epidermal cells from patients with psoriasis. When culture medium was transferred between cultures of T cells isolate(t from active lesions and cultures of epidermal cells obtained from uninvolved areas of skin, it was specifically the ~l-integrin-bright, stem cell population that was stimulated to divide. By contrast, epidermal cells from non-psoriatic individuals showed no such response. This suggests either a genetic sensitivity to T-cell-elaborated cytokines, or some kind of priming of the epidermal stem cells in the psoriatic patients. Now that human epidermal stem cells, or at least their immediate descendants, have been confidently identified, the hunt will be on for additional molecular markers that define this important cell population.
The regenerating
offers over 200
hybridomas for non-exclusive sales worldwide producing antibodies against nuclear, cytoplasmic, membrane and
,m
MEDICINE TODAY
Schwann cell and the axon - who follows who? Schwann cell processes guide regeneration of peripheral axons Son, Y.J. and Thompson, W.J. (1995) Neuron 14, 125-132
Upon denervation, specialized terminal Schwann cells (which are normally confined to a single motor endplate) sprout processes that grow to interconnect adjacent motor endplates. The authors have shown immunohistochemically that axons regenerate down the old Schwann cell tubes that lead to the endplates. However, instead of ending with reinnervation of the endplate, many axons continued to grow along new processes extended by the Schwann cells, forming the so-called 'escape fibres', and were thus guided to adjacent endplates, resulting in polyneuronal innervation. In separate experiments, similar immunohistochemical analysis was used to examine the gila-neurone relationship following axotomy. In this case, Schwann cells and axons extended together across the surface of a muscle at a rate of 0.2 mm day-~. The axons were always accompanied by Schwann cells, but the reverse was not always true. When axonal regeneration was temporarily suspended, Schwann cell migration alone proceeded at the same rate. Furthermore, axons were shown to regenerate over established Schwann cells at a much faster rate (1 mmday-1), implying Schwann cell migration limits the rate of axonal regeneration. This is consistent with the known fact that, when Schwann cell migration is prevented, there is marked impairment of axonal regeneration. If Schwann cells are the rate-limiting leaders in axonal regeneration, a better understanding of their cellular biology will assist manipulation of the microenvironment, facilitating nerve repair.
Contributors David R. Greave= Sir William Dunn Schoolof Pathology, South ParksRoad,Oxford, UK OX1 3RE.
Nerve sprouting in muscle is induced and guided by processes extended by Schwann cells Son, Y.J. and Thompson, W.J. (1995)
P.obin Martin Blonde MclndoeCentre,QueenVictoria Hospital, EastGrinstead, UK RH193DZ,
Neuron 14, 133-141
Steldma IN. kkerer Departmentof Genetics, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
The role of the Schwann cell in peripheral nerve regeneration has important therapeutic implications in the treatment of peripheral nerve injury. One area of interest is the role of the Schwann cell as a follower, or leader, of axonal regeneration. These papers highlight some of the known facts and lend support to the theory that Schwann cells lead and regulate axonal regeneration.
Guy Sterne BlondeMclndoeCentre, Queen Victoria Hospital,EastGrinstead, UK RH193DZ.
© 1995, Elsevier Science Ltd 1357 - 4310/95/$9.50
Round The
Will the real SMA gene please stand up! Identification and characterization of a spinal muscular atrophy-determining gene Lefebvre, S. et aL (1995) Cell80, 156-165
The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy Roy, N. et aL (1995) Cell80, 167-178
A novel cDNA detects homozygous microdeletions in greater than 50% of type I spinal muscular atrophy patients Thompson, T.G. et aL (1995) Nature Genet. 9, 56-61
SMA genes: deleted and duplicated
proposed to encode for a neuronal apoptosisinhibitory protein, which fits in well with the observed pathophysiology of SMA. Thompson et al. report prematurely on the identification of an incompletely characterized cDNA fragment that maps in the SMA region and may, in fact, be part of NAIP (Mahadevan et aL). Comparing the three maps will be essential to resolve the positions of the genes and the deletion breakpoints. This might be easier if Roy and Thompson had presented clearly the pedigree of their yeast artificial chromosomal (YAC) clones. In a discerning editorial, Lewin discusses that SMN fulfils the criteria for assigning a gene to a disease: it has mutations that appear to be confined to a single gene (itself) that are not present in controls. However, the presence of a second, non-allelic copy of SMN with no apparent inactivating mutations has, at least for now, delayed SMN being crowned champion. While the genetics of this complex disease continue to be resolved, these discoveries should already enable clinical diagnosis of >98% of SMA carriers and patients. For gene sleuths, the articles serve as a textbook of clues on how to identify and navigate the obstacles that nature has planted in the genome.
Mahadevan, M.S. eta/. (1995) Nature Genet. 9, 112-113
Genes for SMA: multum in parvo Lewin, B. (1995)
CD34 gene regulation an entry point to stem cell biology
Cell80, 1-5
With the new year came a trilogy of papers describing the identification of two genes and a cDNA fragment that were found to be deleted in patients with spinal muscular atrophy (SMA). SMA is an autosomal recessive neurodegenerative disease that affects 1 in 6000 individuals, and is characterized by depletion of the motor neurons in the spinal cord, leading to paralysis and death in childhood. The search for elusive genes in the SMA region on chromosome 5q13 has been complicated by the presence of expressed pseudogenes, inverted duplications, and the instability of cloned DNA. Notwithstanding, Lefebvre et al. have isolated a novel gene, the survival motor neuron (SMN) gene, which is deleted in 226 out of 229 patients with SMA examined. The remaining three patients carry subtler mutations that are not observed in unaffected individals. Roy et al. describe a nearby, but different, gene, NAIP, which is absent in 67% of SMA and 2% of non-SMA chromosomes, and is
Targeting gene expression to haematopoietic stem calls: a chromatindependent upstream element mediates call type-specific expression of the stem call antigen CD34 May, G. and Enver, T. (1995)
up
literature
Luciferase reporter plasmids were constructed to test the role of upstream DNase I HSS in haematopoietic-cell-specific expression. In transient transfection of murine 416B cells, inclusion of the CD34 upstream HSS increased transcription by only 1-2.5-fold over the CD34 promoter alone. However, when the same plasmids were used to generate stably transfected 416B cell populations, a much more striking 75-fold enhancement of luciferase activity was observed. This enhancement in activity was specific to 416B cells and was not observed in stably transfected Swiss 3T6 fibroblasts. Have the authors uncovered a CD34 locus control region (LCR)? Probably. The firstdescribed and best-characterized LCR was discovered in the human 13-globin locus, and comprises four DNase I HSS. The ability of this element to direct position-independent expression of co-linked promoters is only observed when stably integrated into chromatin. Much work remains to be done to delineate the exact DNA sequences of the CD34 gene that confer stem-cell-specific gene expression, and to identify the key proteins that interact with the CD34 LCR and promoter. The May and Enver paper is that most unusual of gene-expression papers: one that describes something novel. The CD34 gene harbours a DNA sequence that has important implications for stem-cell biology and gene therapy.
131-1ntegrin-bright stem cells in human skin Stem call patterning and fate in human epidermis Jones, P.H., Harper, S. and Watt, EM. (1995)
EMB01 14, 564-574 Cell 80, 83-93
Antibodies specific for the human stem-cell antigen CD34 are widely used for the identification and purification of haematopoietic stem cells. May and Enver undertook a detailed analysis of the murine CD34 gene in an attempt to define regions of the gene that direct expression of CD34 to haematopoietic stem cells. Regions of chromatin of the CD34 gene were found to be hypersensitive to digestion by the enzyme DNase I in nuclei from a murine haematopoietic cell line. Three DNase I hypersensitive sites (HSS), which were present only in the nuclei of haematopoietic cells and not in the nuclei of fibroblasts expressing CD34, were identified.
Kinetics and regulation of human keratinocyte stem calls in short term primary ex vivo culture Bata-Csorgo, Z., Hammerberg, C., Voorhees, J.J. and Cooper, K.D. (1995) J. Clin. Invest.95, 317-327
The distribution of proliferative and non-proliferative cells in human skin is a familiar textbook image. Close to the collagen IV-containing basement membrane, a layer of relatively undifferentiated basal cells divides to feed columns of increasingly differentiating cells m
© 1995. Elsevier Science Ltd 1357 - 4310/95/$9.50
5
Round The
up
MOLECULAR
literature
upwards to the surface. Studies of the capacity for basal-layer cells to proliferate in response to the elimination of adjacent partners has important implications for the treatment of burns and other wounds, diseases such as psoriasis, and prospects for long-term gene therapy through keratinocyte transfection. Two recent papers increase our knowledge of the properties of the proliferative, or stem, cells in human epidermis. Jones et al. report a continuation of their studies using fluorescently labelled antibodies to 131'-integrins, the cell surface receptors for extracellular matrix components found in the basement membrane. Fresh human epidermal cells with only twofold more ~l-integrin than their morphologically similar partners show rapid adhesion to collagen IV and have a higher
BIOPROBE BV NETHERLANDS HYBRIDOMA-BROKERAGE For Russian, Tsjech, Slovak, Hungarian, German, French, English, Italian, American, Australian and Dutch Institutes,
B I O T E C H FIRMS A N D L A R G E RESEARCH INSTITUTIONS buy and sell hybridomas producing useful diagnostic researchers and their institutesor universities to our clients:
MAJOR AND MINOR BIOTECH FIRMS IN EUROPE, USA, JAP~
SEEKING
TO~J.
THE
GAPSI N THEIR PRODUCT LISTS OF MONOCI~NAL ANTIBODIES AND KITS AND I/X)KING FOR NEW OPPORTUNITIKS
Fax: +31-20-6476794 Bioprobe BV The Netherlands Dr. Schaepmanlaan 1 1182 GM Amstelveen
6
proliferative potential. Interesting patterns of the distribution of these !31-integrin-bright cells are described in the epidermis from different sites. A measure of inherent pattern formation was evident even when fresh epidermal cells were cultured in vitro on plastic. The paper by Bata-Csorgo et aL also reports the correlation of I~-integrin-bright staining with a high proliferative potential. An observation of some significance was then made on epidermal cells from patients with psoriasis. When culture medium was transferred between cultures of T cells isolate(t from active lesions and cultures of epidermal cells obtained from uninvolved areas of skin, it was specifically the ~l-integrin-bright, stem cell population that was stimulated to divide. By contrast, epidermal cells from non-psoriatic individuals showed no such response. This suggests either a genetic sensitivity to T-cell-elaborated cytokines, or some kind of priming of the epidermal stem cells in the psoriatic patients. Now that human epidermal stem cells, or at least their immediate descendants, have been confidently identified, the hunt will be on for additional molecular markers that define this important cell population.
The regenerating
offers over 200
hybridomas for non-exclusive sales worldwide producing antibodies against nuclear, cytoplasmic, membrane and
,m
MEDICINE TODAY
Schwann cell and the axon - who follows who? Schwann cell processes guide regeneration of peripheral axons Son, Y.J. and Thompson, W.J. (1995) Neuron 14, 125-132
Upon denervation, specialized terminal Schwann cells (which are normally confined to a single motor endplate) sprout processes that grow to interconnect adjacent motor endplates. The authors have shown immunohistochemically that axons regenerate down the old Schwann cell tubes that lead to the endplates. However, instead of ending with reinnervation of the endplate, many axons continued to grow along new processes extended by the Schwann cells, forming the so-called 'escape fibres', and were thus guided to adjacent endplates, resulting in polyneuronal innervation. In separate experiments, similar immunohistochemical analysis was used to examine the gila-neurone relationship following axotomy. In this case, Schwann cells and axons extended together across the surface of a muscle at a rate of 0.2 mm day-~. The axons were always accompanied by Schwann cells, but the reverse was not always true. When axonal regeneration was temporarily suspended, Schwann cell migration alone proceeded at the same rate. Furthermore, axons were shown to regenerate over established Schwann cells at a much faster rate (1 mmday-1), implying Schwann cell migration limits the rate of axonal regeneration. This is consistent with the known fact that, when Schwann cell migration is prevented, there is marked impairment of axonal regeneration. If Schwann cells are the rate-limiting leaders in axonal regeneration, a better understanding of their cellular biology will assist manipulation of the microenvironment, facilitating nerve repair.
Contributors David R. Greave= Sir William Dunn Schoolof Pathology, South ParksRoad,Oxford, UK OX1 3RE.
Nerve sprouting in muscle is induced and guided by processes extended by Schwann cells Son, Y.J. and Thompson, W.J. (1995)
P.obin Martin Blonde MclndoeCentre,QueenVictoria Hospital, EastGrinstead, UK RH193DZ,
Neuron 14, 133-141
Steldma IN. kkerer Departmentof Genetics, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8.
The role of the Schwann cell in peripheral nerve regeneration has important therapeutic implications in the treatment of peripheral nerve injury. One area of interest is the role of the Schwann cell as a follower, or leader, of axonal regeneration. These papers highlight some of the known facts and lend support to the theory that Schwann cells lead and regulate axonal regeneration.
Guy Sterne BlondeMclndoeCentre, Queen Victoria Hospital,EastGrinstead, UK RH193DZ.
© 1995, Elsevier Science Ltd 1357 - 4310/95/$9.50