Figure 1.
The development of a novel Tle4 null mouse model.
(a) Targeting schema for generation of Tle4 null animals. Conditional Tle4 null mice were created by homologous recombination with an original construct containing a pgk-neo positive selection cassette and a Diptheria toxin (Dt) negative selection sequence, The pgk-neo selection cassette was excised between flanking Frt sites (white arrow head) by breeding mice to beta actin-Flp mice, leaving loxp sites flanking exon 2. These conditionally Tle4 null mice were bred to beta-actin cre mice to generate Tle4 null mice with a deleted exon 2 between flanking Loxp sites (black arrowheads). (b) RT-PCR showed exon 2 was cleanly excised. Loss of exon 2 creates a frameshift in the cDNA and a truncated non-functional Tle4 peptide. (c) Loss of Tle4 does not significantly affect the expression of other Tle family members as shown by RT-PCR of cDNA from the bone marrow of 2 week old mice. (d) Loss of Tle4 expression in Tle4 null mice was demonstrated by Western blot with protein from brain and lung.
Figure 2.
Tle4 null mice exhibit growth retardation and under-mineralization of bone.
(a) Tle4 null mice are born of similar size to wild-type littermates, but exhibit severe growth retardation after birth. (b) One day old Tle4 null mice (KO) have decreased mineralization of the trabeculae and cortical bone in the tibiae compared to wild-type littermates (WT) as shown by Von Kossa staining. (c) Alizarin Red/Alcian Blue staining for ossified bone (red) and cartilage (blue) of 1 day old wild-type (WT) and Tle4 null (KO) skeletons shows decreased mineralization in Tle4 null mice of both membranous bone (skull) and endochondral bone (vertebrae and long bones).
Figure 3.
Progressive bone marrow hypoplasia with defective ossification, loss of trabecular bone and thinning of cortical bone is seen by 3 and 4 weeks in Tle4 null mice.
A–D. Hematoxylin and Eosin (H&E) staining of tibiae of 3 and 4 week old WT and Tle4 null (KO) mice demonstrate multiple abnormalities in Tle4 null mice including progressive pancytopenia of the bone marrow (BM), loss of trabecular bone (T), and thinning of the cortical bone layer (C). A higher power view shows a thinner proximal tibial growth plate in Tle4 null mice with a decrease in thickness of the resting (R), proliferative (P), and hypertrophic (H) zones and near complete loss of the trabeculae. E–H. Tartrate-resistant acid phosphatase (TRAP) staining (pink) demonstrates osteoclasts clustering under the hypertrophic zone at the boundary of the bone marrow cavity in Tle4 null mice at 3 and 4 weeks of age.
Figure 4.
Peripheral blood counts are normal in Tle4 null mice at 2 weeks of age, but significant abnormalities are seen in peripheral blood and bone marrow at 4 weeks of age.
(a) At 2 weeks of age there was no significant difference in any of the cell compartments in the peripheral blood of Tle4 null mice (KO) as compared with normal control littermates (WT). However, by 4 weeks of age Tle4 null mice exhibit a marked leukopenia (decreased WBC) and lymphopenia in the peripheral blood, that primarily affects the B-cells (B220+ cells), and not T-cell (CD3+) or myeloid cells (CD11b+). (b) Tle4 null mice exhibit severe bone marrow aplasia with a 2-fold decrease in bone marrow cellularity. Within the remaining population of lymphoid cells, B-cell development appears particularly affected with a significant decrease in the percentage of B-cells (B220+) and relative increase in the percentage of T-cells (CD3+).
Figure 5.
Tle4 null mice develop splenic atrophy with abnormal splenic architecture.
(a) At three weeks of age there is marked splenic atrophy and decreased cellularity especially of the major B-cell (B220+) compartment. (b) H&E staining of the spleen reveals an absence of splenic follicles (dashed oval) in two week old Tle4 null mice (KO). (n = 3–4 per genotype; mean +/−SEM; *: P<.05).
Figure 6.
Tle4 null mice develop thymic atrophy with a block in T-cell differentiation.
(a) There is a dramatic decrease in total thymocytes in 3 week old Tle4 null mice as compared to wild-type littermates. The majority of this decrease is due to loss of double positive CD4+CD8+ cells. Within the double negative (DN) T progenitor populations there appears to be a block between DN1 (CD44+CD25−) and DN2 (CD44+CD25+) with a significant decrement in DN2 cells and an insignificant decrease in DN3 (CD44−, CD25+) cells. (n = 3–4 per genotype; mean +/−SEM; *: P<.05). (b) The thymus of 3 week old Tle4 null mice is atrophied with a loss in the demarcation between cortex and medulla as seen by H&E. (c) TUNEL staining demonstrates thymic apoptosis in Tle4 mice.
Figure 7.
Tle4 null mice have significant aberrations in hematopoietic stem and progenitor cells (HSPC).
Bar graphs with representative flow cytometry plots in two week old littermates (a) show significant loss of LSK and LKS CD34+ cells, though the most immature long-term HSC population (LKS CD34+CD48–CD150+ HSC) is relatively preserved. (b) Examination of CMP, GMP, MEP, and CLP progenitor fractions demonstrated significant decreases in CMP and CLP populations. (c) Amongst the Pre/Pro B cell progenitors the decrement is most prominent in the early Fractions A through C. (n = 3–8 per genotype; mean +/− SEM; *: P<.05, **: P<.001, ***: P<.0001). See methods for gating strategy of stem and progenitor cell compartments.
Figure 8.
The decrease in LKS cells in Tle4 null mice is due to an increase in apoptosis and cell death rather than a decrease in proliferation and is accompanied by abnormalities of the bone marrow stroma.
(a) LKS cells isolated from the bone marrow of two week old mice show no difference in cell cycle distribution. (b) There is however an increase in apoptotic and dead LKS cells in the Tle4 null mice. (c) TUNEL staining of the growth plate of the femur in two week old mice marks the normal zone of cell death between the hypertrophic (H) layer and forming trabecular (T) bone (A, B) with an increase in staining in Tle4 null mice (F, G). Lacunae in the epiphysis are lined with periosteal cells undergoing apoptosis in Tle4 null mice (H), but was not seen in wild type (WT) littermates. Similar periosteal cells undergoing apoptosis and stained by TUNEL are seen under the cortex of diaphyseal bone in Tle4 null mice (I) but absent in wild-type mice (D). An increase in TUNEL staining is also observed in cells of the bone marrow in Tle4 null mice (J) as compared to wild-type bone marrow (E).
Figure 9.
Co-culture of wild-type HSC on Tle4 null stromal cells impairs long term colony forming ability.
(a) Schematic of co-culture assay. Briefly, stromal cells were isolated from bones of 2 week old WT and KO animals and plated in aMEM with dexamethasone, ascorbic acid, and vitamin D3 as described in Methods. WT LKS were plated and used in LTC-IC experiments (b) Bar graph showing a dramatic decreased frequency of Sca-1+c-Kit+ cells after two weeks of co-culture on the stroma from Tle4 null mice with representative flow plots (n = 2 biological replicates per genotype, in triplicate *: P<.01). (c) Long term culture-initiating cell assay (LTC-IC) after four weeks of co-culture with WT or KO stroma showed relatively few colonies obtained after culturing on Tle4 null bone marrow stroma (n = 2 biological replicates, each in 9-plicate, seeded in duplicates for CFU; *: P<.0001).
Figure 10.
LKS cells from TLE4 null mice are impaired in colony forming unit ability.
(a) The number of colonies in methyl cellulose originating from 1000 LKS cells from TLE4 null mice were fewer compared to wild-type LKS cells on Day 7 and again after replating on Day 14. (n = 4 biological replicates per genotype, in triplicate) (b) LKS cells from Tle4 null mice form fewer CFU-GM and CFU-GEMM colonies but similar BFU-E colonies compared to wild-type. (n = 5 biological replicates per genotype, in triplicate) (**: P<.01, ***: P<.0001).
Figure 11.
Tle4 null mice have cell-intrinsic defects in HSPC self-renewal and B-cell development.
Bone marrow transplants of wild-type (WT) or Tle4 null (KO) bone marrow from 2 week old mice into normal recipients were performed to evaluate cell intrinsic defects. (a) Schematic of BMT experimental design. (b) Analysis of peripheral blood at 16 after transplant by flow cytometry and CBC demonstrated insignificant differences in engraftment as measured by the percent CD45.2+ cells, but impaired B-cell numbers (B220+) with relatively increased T-cells (CD3+) and myeloid cells (CD11b+). (c) 32 weeks after transplant mice receiving Tle4 null bone marrow developed leukopenia (decreased WBC) primarily accounted for by a decrease in lymphocytes (c, left panel), which by immunophenotyping represented a decrease in B-cells (B220+). (n = 9–10 per transplant group, *: P<.005 **: P<.0001). (d) Analysis of B-cell differentiation in the BM of recipient mice 32 weeks after transplant by quantitation of ProB Fractions shows an increase in Fraction C, but decreased Fractions D and E indicating a relative block in differentiation between Fractions C and D (n = 5 per genotype, *: P<.05). (e) Analysis of bone marrow 32 weeks after transplant showed no significant differences between KO and WT recipients in the absolute number of LKS, CLP, CMP, or GMP compartments, but did show a decrease in MEPs in KO recipient mice. (f) Competitive homing 18 hours after transplantation using whole BM from two week old WT and KO littermates showed no defect in homing ability (n = 3). The homing index is represented as: Input/Output or .
Figure 12.
Tle4 null fetal liver HSCs have impaired B cell development and exhaust with serial transplantation.
(a) Schematic of serial FL transplantation experimental design. (b) Peripheral blood and LKS analysis of FL transplant recipients 16 weeks after transplant revealed peripheral leukopenia (WBC) and specifically B cell (B220+) lymphopenia in the absence of Tle4 with no difference in LKS, LKS, CD34+, LKS CD34− populations (n = 10 per genotype for blood analysis, n = 6–7 per genotype for LKS analysis, ***: P<.0001). (c) Peripheral blood and LKS analysis 16 weeks after secondary transplantation also indicates leukopenia and lymphopenia in Tle4 null cells, but at this time also a significant decrease in LKS and LKS, CD34− populations (n = 5 KO recipients, n = 10 WT recipients for blood analysis, n = 5 mice per group for LKS analysis, *: P<.05 **: P<.001). (d) Peripheral blood analysis and LKS analysis 16 weeks after tertiary transplantation again showed leukopenia, B-cell lymphopenia, and a profound decrease in all HSC containing populations (n = 4 per genotype for blood and LKS analysis, *: P<.05, **: P<.01, ***: P<.001). (e) Representative flow cytometry plots showing progressive loss of Tle4 null HSCs over successive transplantation.