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Human Molecular Genetics Pages 85-93
Developmental expression of the Fac gene correlates with congenital defects in Fanconi anemia patients
Introduction
Results
   Fac expression in mesenchymal tissues
   Expression of Fac in osteogenic tissues
   Fac expression in non-skeletal tissues
Discussion
Materials And Methods
   Postimplantation embryos
   RNA probes
   In situ hybridization
Acknowledgements
References

Developmental expression of the Fac gene correlates with congenital defects in Fanconi anemia patients

Developmental expression of the Fac gene correlates with congenital defects in Fanconi anemia patients Flora Krasnoshtein and Manuel Buchwald*

Research Institute, Hospital for Sick Children and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1X8, Canada

Received August 14, 1995; Revised and Accepted October 17, 1995

Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder characterized by a variety of congenital and skeletal malformations, progressive pancytopaenia and predisposition to malignancies. While the basic defect in this disease is not known, the cloning of the gene defective in FA group C patients (FAC) allows analysis of its expression pattern, which may provide clues about the functional properties of the protein. This paper describes the distribution of Fac transcripts during murine development (8-19.5 days p.c.), using RNA in situ hybridization. Fac is initially expressed (8-10 days p.c.) in the mesenchyme and its derivatives with osteogenic potential. The transcript is also apparent at later stages of bone development (13-19.5 days p.c.), localized to cells of the inner perichondrium, periosteum and zone of endochondral ossification. In the latter, Fac transcripts are seen in cells from both osteogenic and hematopoietic lineages. Fac mRNA is also seen in intramembranous cranial and facial bones. In addition, Fac signal is detected in non-skeletal tissues: brain, whisker follicles, lung, kidney, gut and stomach. Fac expression is high in progenitor cell populations but is downregulated in differentiating cells that give rise to connective tissue. The pattern of Fac expression is consistent with the skeletal and non-skeletal congenital abnormalities in FA patients. As well, expression in rapidly dividing progenitors is consistent with hypotheses regarding the nature of the basic defect in FA: a role of the protein in DNA repair or protection from oxygen toxicity.

INTRODUCTION

Fanconi anemia (FA) is a rare, phenotypically and genetically heterogeneous, autosomal recessive disorder characterized by progressive pancytopenia and an increased susceptibility to the development of malignancy (1 -8 ). Approximately 50% of FA patients also have one or more of a varied set of congenital abnormalities (9 ,10 ). Skeletal anomalies of the hand and the forearm, such as radial ray deformities, are the most commonly encountered, but affected regions can include the head and face, spine and lower limbs. Although the abnormalities encountered in FA patients are usually listed separately, multiple anomalies are commonly seen in many affected individuals and some, such as growth retardation, microcephaly, microphthalmia and skeletal abnormalities, tend to occur together. Other congenital anomalies seen in FA patients include skin hyperpigmentation, hypogenitalia, ear malformations and hearing loss, kidney anomalies, cardiac/cardiopulmonary involvement and learning disabilities (9 -11 ).

The congenital defects seen in FA patients imply a role for the defective gene(s) in normal development; it has been suggested that structures undergoing high rates of cell replication may be most likely to be affected (12 ,13 ). Defects in at least five genes can cause Fanconi anemia; five complementation groups (FA-A-E) have been defined by somatic cell hybridization studies (7 ,8 ). The cloning of FAC (14 ) provided the opportunity to define its normal function and, subsequently, an understanding of the basic defect in the disease. However, the predicted protein does not show homology to known entities nor does it have clearly recognizable functional motifs, precluding immediate identification of its function. As one of the steps towards defining the role of FAC, the pattern of Fac expression in murine development can be analyzed. For this purpose, we first cloned the murine homologue of human FAC and found that the predicted murine protein shares 79% amino acid sequence similarity and 67% amino acid identity with its human counterpart. Notwithstanding this degree of sequence diversity, function is conserved, since expression of the mouse cDNA in FA(C) cells corrects their sensitivity to mitomycin C (15 ).

Adult murine (and human) tissues express Fac (and FAC) ubiquitously as shown by RT-PCR and Northern analysis (14 ,15 ). Preliminary studies of the expression pattern of Fac during mouse embryogenesis indicate that the gene is present in head mesenchyme (8-9 days p.c.), perichondrium of developing bones (14-16 days p.c.) and the outer root sheath of hair follicles (16 days p.c.). However, these studies did not define the precise developmental stages of Fac expression and, because the hybridization signal was weak, only tissues with the highest levels of expression were identified (15 ).


Figure 1. In situ hybridization analysis of embryonic Fac expression in mesenchymal tissue in frontal section of 9 days p.c. (A-C) and in parasaggital sections of 10 days p.c. hind limb bud (D-F), 11 days p.c. tail (G-I), 13 days p.c. gut (J-L) and 15 days p.c. lung (M-O), stained with hematoxylin and eosin. A, D, G, J, M bright field, antisense strand hybridization; B, E, H, K, N dark field, antisense strand hybridization; C, F, I, L, O dark field, sense strand. M, mesenchyme; Lu, lung; Li, liver; H, heart. The scale bar represents 133 [mu]m.Here we describe the temporal and spatial pattern of Fac expression during murine development through the use of RNA in situ hybridization. Fac expression is localized to areas undergoing osteogenesis by both endochondral and intramembranous ossification, as well as in non-skeletal regions containing actively dividing and/or differentiating cells. The relationship of these results to the phenotypes of FA patients and to the hypotheses regarding the basic defect are discussed.

RESULTS

Fac expression in mesenchymal tissues

Mesenchyme is the basic cellular organization exclusive to the embryo that gives rise to skeletal and connective tissues (16 ). The expression of Fac in mesenchyme can be detected at 8 days p.c. and persists until 17.5 days p.c. (Fig. 1 A-C). Fac mRNA in the limb bud at 10 days p.c. (Fig. 1 D-F) and tail at 11 (Fig. 1 G-I) and 12 (data not shown) days p.c., appears to be limited to undifferentiated mesenchyme, presumably in those cells that give rise to skeletal tissues. However, Fac mRNA is not restricted to mesenchymal osteoprogenitors, since non-skeletal mesenchyme also expresses the gene. These regions include mesenchyme of the gut at 13 days p.c. (Fig. 1 J-L), lung at 13-17.5 days p.c. (Fig. 1 M-O), kidney at 13-19.5 days p.c. (data not shown) and mesenchyme of the craniofacial region at the site of future whisker follicles at 10-14 days p.c. (see below, Fig. 6 A-C). Although Fac expression in the mesenchyme is seen throughout the course of embryonic development, the mRNA becomes less abundant as embryogenesis progresses. Fac transcripts were not examined in mesenchymal condensations during early stages of bone development.

Expression of Fac in osteogenic tissues


Figure 2. In situ hybridization analysis of Fac expression in the perichondrial/periosteal (P) regions and sites of osteogenesis (zone of endochondral ossification, EO), containing osteoprogenitors and actively forming bone, respectively. Bones formed by endochondral ossification express Fac. Fac mRNA is expressed in the occipital bone of 14 (P) and 16 (EO) days p.c.(A-F); vertebrae of 13 (P) days p.c. (G-I), in embryonic parasaggital sections, stained with hematoxylin and eosin. A, D, G bright field, antisense strand hybridization; B, E, H dark field, antisense strand hybridization; C, F, I dark field, sense strand. -> :Inner perichondrium/periosteum; C, cartilage; EO, zone of endochondral ossification; site of osteogenesis; HC, hypertrophic chondrocytes; PC, proliferative chondrocytes. The scale bar represents 133 [mu]m. As described in more detail below, a characteristic pattern of Fac expression is observed during bone development, particularly in those formed by endochondral ossification (see Figs 2 , 3 and 4 ). The transcript is first detected in the actively dividing and differentiating chondroprogenitor cells of the inner perichondrium and, later, in the osteoprogenitors of the inner periosteal layer as well as at the site of osteogenesis, the zone of endochondral bone formation. In contrast, no Fac expression is seen in the outer, or fibrous layer, chief constituents of which are collagen and fibroblasts. Fac mRNA is also absent from relatively mature and differentiated cells of the chondro- and osteo-genic lineages, as exemplified by the hypertrophic and proliferative chondrocytes (Fig. 3 A-I), as well as by osteocytes in the zone of endochondral ossification.


Figure 3. In situ hybridization analysis of Fac expression in the perichondrial/periosteal (P) regions and sites of osteogenesis (zone of endochondral ossification, EO), containing osteoprogenitors and actively forming bone respectively. Bones formed by both the endochondral (EO) and intramembranous (IO) ossification express Fac. Fac mRNA is expressed in the femur (EO) of 16 days p.c. (A-C); tibia and fibula (EO) of 16 days p.c. (D-F); and the alveolar bone (IO) of the maxilla of 19.5 days p.c. (G-I) embryonic parasaggittal sections, stained with hematoxylin and eosin. A, D, G bright field, antisense strand hybridization; B, E, H dark field, antisense strand hybridization; C, F, I dark field, sense strand. Small straight arrow: inner perichondrium/periosteum; large curved arrow: bone; AB: alveolar bone; C: cartilage; EO: zone of endochondral ossification; HC: hypertrophic chondrocytes; PC: proliferative chondrocytes; TB: tooth bud. The scale bar represents 133 [mu]m.


Figure 4. In situ hybridization analysis of Fac expression in the zone of calcification of the developing bone of 19.5 days p.c. embryo. A, C bright field, antisense strand hybridization; B, D bright field, sense strand hybridization. A and B represent Fac signal in osteogenic cells. C and D represent Fac signal in hematopoietic cells. Solid arrows, osteogenic cells; open arrows, hematopoietic cells; B, bone. The scale bar represents 14.3 [mu]m.


Bones formed by endochondral ossification. In the axial skeleton, Fac is expressed in the occipital bone, which forms the base of the cranium (13-19.5 days p.c.) (Fig. 2 A-F). Fac mRNA is initially distributed in the inner layer of the perichondrium at 14 days p.c. (Fig. 2 A-C) and later, in the periosteum and at the site of osteogenesis in the zone of endochondral ossification at 16 days p.c. (Fig. 2 D-F). The same pattern of Fac expression is observed during development of both the vertebrae (14-19.5 days p.c.) (Fig. 2 G-I) and the ribs (13-19.5 days p.c.) (data not shown).

A similar pattern of Fac expression is seen in the appendicular skeleton. For example, in the scapula and the ilium, Fac is expressed in cells of the inner perichondrium (14-15 days p.c.) (data not shown), periosteum and the zone of endochondral ossification (16-19.5 days p.c.) (data not shown). In the extremities, Fac mRNA is expressed in the developing long bones of both the forelimbs and the hindlimbs. Beginning at 13 days p.c. through birth, Fac mRNA is present in moderate amounts, first in the inner perichondrium and, later, in the inner periosteum and the osteogenic diaphysis (the zone of endochondral ossification) of bones of the forelimb (data not shown) and hindlimb (Fig. 3 A-F). In the scapula, ilium and bones of the extremities, as in the other endochondral bones, Fac is expressed by neither the proliferating nor the hypertrophic chondrocytes, both relatively differentiated and mature cell types (Fig. 3 ).

Although the in situ hybridization analysis performed in this study defines regions where Fac is expressed, it does not precisely identify the specific cell types expressing the gene. This is particularly evident in the zone of endochondral ossification of long bones, where cells derived from both osteogenic (mesenchymal, chondroblasts and osteoblasts) and hematopoietic (osteoclasts and blood cells occupying the marrow cavity) lineages are present. Our initial results suggest that Fac is expressed in cells of both lineages that is osteogenic (Fig. 4 A,B) and hematopoietic (Fig. 4 C,D).Bones formed by intramembranous ossification. The pattern of Fac expression in intramembranous bones is limited, first, to the undifferentiated mesenchyme and, later, to the actively differentiating osteogenic cells involved in deposition of new bone. Intramembranous cranial bones of the axial skeleton express Fac. Fac mRNA is present in moderate amounts in the temporal bone region at 14 and 15 days p.c. (data not shown). The osteogenic cells of the sphenoid bone, an irregular bone making up the base of the skull and parts of the orbit and nose, express Fac at 15-19.5 days p.c. (data not shown). Fac is also expressed in the intramembranous facial bones. A moderate hybridization signal is detected in both the alveolar bone of the maxillary region (Fig. 3 G-I) as well as the mandible from 10 days p.c. until birth. In the pectoral girdle of the appendicular skeleton, Fac is expressed in the clavicle at 13-19.5 days p.c. (data not shown), which undergoes a combination of intramembranous followed by endochondral bone formation.

Fac expression in non-skeletal tissues

In addition to being detected in the mesenchyme and osteogenic regions of developing skeletal structures, Fac mRNA is also seen in a number of non-skeletal tissues, particularly in those containing undifferentiated progenitor cells (e.g. non-skeletal mesenchyme, ependymal cell layer of the brain). In the central nervous system, Fac transcripts are detected in the mesenchyme of the spinal cord at 11-13 and 16 days p.c. (data not shown) and in the ependymal cell layer of the brain. This layer consists of simple cuboidal epithelial cells surrounding the central canal that give rise to precursors with the potential to differentiate into neuronal and glial cells (9-16 days p.c.) (Fig. 5 A-F) (17 ). As development proceeds and ependymal cells differentiate, the ependymal layer diminishes in size, which probably accounts for a signal of lower intensity. A consistent pattern of Fac expression is seen in the ependymal cell layer of all brain sections examined (fore-, mid- and hind-brain regions). Representative sections are shown in Figure 5 .


Figure 5. In situ hybridization analysis of Fac expression in the developing brain of nine (A-C) embryonic transverse sections and 11 (D-F) days p.c. parasaggital sections, stained with hematoxylin and eosin. A, D bright field illumination, antisense strand hybridization; B, E dark field, antisense strand hybridization; C, F dark field, sense strand. -> , actively dividing ependymal layer; M, mesenchyme. The scale bar represents 133 [mu]m.


Figure 6. In situ hybridization analysis of Fac expression initially seen in the frontonasal mesenchyme at 11 (A-C) days p.c. and later in the whisker follicles at 16 (D-F) and 19.5 (G-I) days p.c. of embryonic parasaggital sections, stained with hematoxylin and eosin. Expression is seen in the outer root sheath of the whisker follicles only. The signal within the follicles is non-specific. A, D, G bright field, antisense strand hybridization; B, E, H dark field, antisense strand hybridization; C, F, I dark field, sense strand. M, mesenchyme; WF, whisker follicles; -> , outer root sheath. The scale bar represents 133 [mu]m.

Table 1 . Summary of results and their relationship to defects in FA patients
Tissue expressing Fac

 Relevance to Fanconi anemia phenotypes
Undifferentiated mesenchyme

 Mesenchyme gives rise to tissues affected in FA patients (e.g. bone)
Lung mesenchyme

 Lung lobe absence and abnormal pulmonary drainage
Kidney mesenchyme

 Numerous kidney abnormalities (hypoplastic or dysplastic)
Gut mesenchyme

 Gastrointestinal problems (atresia - esophagus, duodenum, jejunum, anus)
Developing bones

 Skeletal abnormalities (e.g. deformities of radial ray, lower limbs, ribs, vertebrae, head and face)

In the craniofacial region, Fac is expressed initially in differentiating cells of the frontonasal mesenchyme at 10-14 days p.c. (Fig. 6 A-C) and, later, as the embryo develops, in the outer root sheath of the whisker follicles at 15-19.5 days p.c. (Fig. 6 D-I). The observed expression of Fac follows a specific pattern through different developmental stages, beginning with a high intensity, uniform expression over the entire frontonasal mesenchyme (10 and 11 days p.c.) and a diminishing intensity as the embryo develops and mesenchyme differentiates into other cell types (13 and 14 days p.c.), until only one region of the hair follicles (outer root sheath) express Fac (15-19.5 days p.c.).

DISCUSSION

The pattern of Fac expression during murine development is consistent with the skeletal abnormalities observed in FA patients, which include radial ray deformities, metacarpal hypoplasia, abnormalities of the lower limbs, ribs, vertebrae, head and face (11 ). For example, the signal detected in the developing cranial bones of the mouse embryo echoes the sloped forehead and microcephaly in FA patients while the facial anomalies in patients may correspond to Fac expression in the bones of the murine face. The presence of Fac mRNA in lung and kidney mesenchyme is consistent with the lung lobe absence and abnormal pulmonary drainage and numerous kidney abnormalities respectively, encountered in FA patients. The correspondence of Fac expression to the FA phenotype is summarized in Table 1 .

As defined by in situ hybridization, the overall pattern of Fac expression in skeletal as well as non-skeletal tissues appears to be tightly coupled with cellular proliferation and/or differentiation. During bone formation, Fac expression follows a characteristic pattern. Transcripts are first abundant in mesenchyme, the early connective tissue of the embryo. Subsequently, undifferentiated chondro- and osteo-progenitor cells express Fac, while relatively mature and differentiated cells of these lineages do not, as exemplified by the presence of Fac mRNA in perichondrium and periosteum and its absence in chondrocytes and osteocytes. Although proliferative chondrocytes are in active mitosis, they are terminally differentiated cells of the chondrogenic lineage (16 ). Fac expression in bones formed by intramembranous ossification is also most abundant in undifferentiated progenitors. The presence of Fac mRNA is limited, first, to the regions of undifferentiated mesenchyme and, later, to the actively differentiating osteogenic cells involved in deposition of new bone.

In non-skeletal tissues, Fac mRNA is also localized to actively dividing and differentiating tissues. For example, in the central nervous system Fac expression is detected in the mesenchyme of the spinal cord and in the actively dividing ependymal cell layer of the developing brain. Fac is also expressed in mesenchyme of the lung, kidney and gut. Fac transcripts are also seen in tissues undergoing inductive interactions between the mesenchyme and epithelium, such as the actively dividing inner epithelial layer of the stomach and the developing whisker follicles (18 ).

Thus, in general, high levels of transcripts are first localized to relatively undifferentiated cells (e.g. mesenchymal, ependymal) and these diminish as cells mature and differentiate. If Fac expression is related to proliferation of undifferentiated cells, this hypothesis can be studied in vitro by correlating cell differentiation and Fac expression in multipotential mesenchymal cells (19 -21 ), osteoblast-like cells (22 ) and peripheral blood monocytes. Based on the results of the in situ hybridization analysis, we can predict that Fac expression will decline as cells mature and differentiate.

The pattern of Fac expression can also be considered in light of hypotheses about the basic defect: DNA repair and oxygen toxicity, proposed to explain the complex and variable phenotype seen in FA (reviewed in refs 23 ,24 ). With regard to the first hypothesis, expression of Fac in actively dividing and differentiating cells, a stage when replicating DNA is most prone to damage, is consistent with a role for Fac in processes essential for faithful replication, such as DNA repair. With regard to the second hypothesis, it is known that bone is continually resorbed by osteoclasts and remodelled by osteoblasts, a process that generates oxygen-derived free radicals (25 ). Since Fac is expressed by cells in the zone of endochondral ossification, if defective Fac is unable to remove oxygen radicals from the resorbing site, this could ultimately lead to defects in bone formation. These speculations suggest areas of research that may help elucidate the role(s) of the Fac gene product.

In the zone of endochondral bone formation, Fac transcripts are seen in cells of both osteogenic and hematopoietic lineages. Our analysis does not define which specific hematopoietic cells express the gene or whether Fac is expressed in osteoclasts, also present in the zone of endochondral ossification. This question can be answered by analyzing Fac expression in conjunction with markers specific for cells of these lineages, either at the RNA or protein level.

The role of Fac cannot be inferred based solely on the in situ hybridization analysis of the expression pattern of the gene. However, comparison of the pattern of Fac expression with published data shows striking similarities to some genes known to be important for the development and differentiation of mesenchymal tissues. These include genes coding for transcription factors [e.g. hXBP-1 (26 ) and Gli (27 )], growth factors (e.g. TGF-[beta] and BMPs) (28 ), collagenous and non-collagenous bone matrix proteins (e.g. TIMP) (29 ) and cytokine receptors (e.g. Etl-2) (30 ). The coincident expression patterns could reflect either a functional or regulatory interaction of Fac with genes or proteins implicated in bone formation and remodelling.

MATERIALS AND METHODS

Postimplantation embryos

Using the copulation plug timing method (31 ), embryos of gestation stages ranging from 8.5-19.5 days p.c. (newborn pups) were collected from matings of inbred C57BL/6J black mice of at least 6-8 weeks of age. Embryos were fixed overnight in 4% (w/v) paraformaldehyde (Fisher Scientific) in 1 * PBS, embedded in paraffin and sectioned (5-7 [mu]m). In addition to the C57BL/6J specimens, the hybrid-ready tissue of Mus musculus NIH Swiss (outbred strain) were obtained from Novagen. The Novagen preparations were fixed, paraffin embedded 7 [mu]m parasaggital embryonic sections with limbs ranging from 8 to 16 days p.c. No significant differences were observed between the results obtained with the two strains. For each gestation stage and each region, 4-20 embryonic sections were analyzed.

RNA probes

Two separate mouse probes generated from a full-length Fac cDNA were used for in situ RNA hybridization studies. Though the larger one gave a somewhat better signal, no significant differences were observed between the results obtained with the two probes. The probe 6EH, cloned into EcoRI and HindIII sites in the multiple cloning site of the pBluescript vector (Stratagene), is a 376 bp subclone from position 720 (exon 4) to position 1096 (exon 7) of the full-length murine Fac cDNA (15 ). The probe 2BX, cloned into BamHI and XbaI sites in pBluescript, is a 1288 bp subclone from position 647 (exon 4) to position 1940 (end of the coding region). The probes were labelled with [[alpha]-35S]UTP (Amersham Canada Ltd) by in vitro transcription from the T7 (6EH anti-sense; 2BX sense strand) and T3 (6EH sense; 2BX anti-sense strand) promoters of pBluescript using the Stratagene in vitro transcription kit.

In situ hybridization

The paraffin-embedded embryonic sections were dewaxed in three 5 min changes of xylene and rehydrated through decreasing concentrations of ethanol (100, 100, 95, 80, 50 and 30%) in diethylpyrocarbonate (DEPC) (Sigma) -treated water. This was followed by in situ hybridization as previously described (32 ). Briefly, the sections were treated with 0.001% Proteinase K (Boehringer-Mannheim) for 8 min, hybridized for 16-20 h at 56-60oC and a final 0.1 * SSC wash was performed at 60-65oC. To obtain high-resolution images, the slides were coated with a Kodak Nuclear Tracking (NTB-2) liquid emulsion and exposed in a light- and radiation-tight container for 2-3 weeks at 4oC. After exposure, the slides were developed, rinsed in water and fixed. The tissues were counter stained with hematoxylin and eosin and photographed using both bright-field and dark-field illumination.

ACKNOWLEDGEMENTS

We thank R. R. McInnes, S. J. Tang and C. C. Hui for helpful comments about the manuscript, M. Chen for providing a Fac cDNA subclone, C. Duff for help with microscopy and J. Aubin and U. Bhargava for identification of cell types. This research was supported by the Medical Research Council of Canada and the National Cancer Institute of Canada, supported by the Canadian Cancer Society. F.K. was partially supported by the Fanconi Anemia Research Fund, Eugene, OR. Support from Childcan is gratefully acknowledged.

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31 Cockroft,D.L. (1990) Dissection and culture of postimplantation embryos. In Copp,A.J. and Cockroft,D.L. (eds), Postimplantation Mammalian Embryos: A Practical Approach. IRL Press, Oxford, p. 20.

32 Trezise,A.E.O. and Buchwald,M. (1991) In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator. Nature, 353, 434-437. MEDLINE Abstract

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