Disruption of the cyclin-dependent kinase-inhibitory domain of p27 enhances growth of mice. Growth is attributed to an increase in cell number, due to increased cell proliferation, most obviously in tissues that ordinarily express p27 at the highest levels. Disruption of p27 function leads to nodular hyperplasia in the intermediate lobe of the pituitary. However, increased growth occurs without an increase in the amounts of either growth hormone or IGF-I. In addition, female mice were infertile. Luteal cell differentiation is impaired, and a disordered estrus cycle is detected. These results reflect a disturbance of the hypothalamic-pituitary-ovarian axis. The phenotypes of these mice suggest that loss of p27 causes an alteration in cell proliferation that can lead to specific endocrine dysfunction.

We generated mice with a null mutation of the forebrain-restricted transcription factor BF-1 to examine its function in brain development. Heterozygous animals have an apparently normal phenotype. Homozygous null BF-1 mutants die at birth and have a dramatic reduction in the size of the cerebral hemispheres. The development of the ventral telencephalon is more severely affected than that of the dorsal telencephalon. Telencephalic neuroepithelial cells are specified in the BF-1 mutant, but their proliferation is reduced. Dorsal telencephalic neuroepithelial cells also differentiate prematurely, leading to early depletion of the progenitor population. These results suggest that BF-1 controls the morphogenesis of the telencephalon by regulating the rate of neuroepithelial cell proliferation and the timing of neuronal differentiation.

Metanephric mesenchyme gives rise to both the epithelial cells of the nephron and the stromal cells of the mature kidney. The function of the stroma. in kidney morphogenesis is poorly understood. We have generated mice with a null mutation in the Winged Helix (WH) transcription factor BF-2 to examine its function during development. BF-2 expression within the developing kidney is restricted to the stromal cell lineage. Homozygotes die within the first 24 hr after birth with abnormal kidneys. Mutant kidneys are small, fused longitudinally, and rotated 90 degrees ventrally. Histological examination reveals a smaller collecting system, numerous large condensations of mesenchyme, and a decrease in the number of nephrons. Using molecular markers we show that induction and condensation of the nephrogenic mesenchyme occurs normally in mutant. The disruption of BF-2 reduces the rate of differentiation of the condensed mesenchyme into tubular epithelium, as well as the rate of growth and branching of the ureter and collecting system. Our findings demonstrate that BF-2 and stromal cells have essential functions during kidney morphogenesis. Furthermore, they suggest that BF-2 controls the production, by the stroma, of signals or factors that are required for the normal transition of induced mesenchyme into tubular epithelium and full growth and branching of the collecting system.

Acute promyelocytic leukemia (APL) is associated with reciprocal chromosomal translocations involving the retinoic acid receptor ␣ (RAR␣) locus on chromosome 17. In the majority of cases, RAR␣ translocates and fuses with the promyelocytic leukemia (PML) gene located on chromosome 15. The resulting fusion genes encode the two structurally unique PML͞RAR␣ and RAR␣͞PML fusion proteins as well as aberrant PML gene products, the respective pathogenetic roles of which have not been elucidated. We have generated transgenic mice in which the PML͞RAR␣ fusion protein is specifically expressed in the myeloid-promyelocytic lineage. During their first year of life, all the PML͞RAR␣ transgenic mice have an abnormal hematopoiesis that can best be described as a myeloproliferative disorder. Between 12 and 14 months of age, 10% of them develop a form of acute leukemia with a differentiation block at the promyelocytic stage that closely mimics human APL even in its response to retinoic acid. Our results are conclusive in vivo evidence that PML͞RAR␣ plays a crucial role in the pathogenesis of APL.

The basic-helix-loop-helix (bHLH) proteins encoded by the E2A gene are broadly expressed transcription regulators which function through binding to the E-box enhancer sequences. The DNA binding activities of E2A proteins are directly inhibited upon dimerization with the Id1 gene product. It has been shown that disruption of the E2A gene leads to a complete block in B-lymphocyte development and a high frequency of neonatal death. We report here that nearly half of the surviving E2A-null mice develop acute T-cell lymphoma between 3 to 10 months of age. We further show that disruption of the Id1 gene improves the chance of postnatal survival of E2A-null mice, indicating that Id1 is a canonical negative regulator of E2A and that the unbalanced ratio of E2A to Id1 may contribute to the postnatal death of the E2A-null mice. However, the E2A/Id1 double-knockout mice still develop T-cell tumors once they reach the age of 3 months. This result suggests that E2A may be essential for maintaining the homeostasis of T lymphocytes during their constant renewal in adult life.Helix-loop-helix (HLH) proteins belong to a family of transcription factors which play important roles in cell fate determination and cellular differentiation (for reviews, see references 29 and 42). They contain a highly conserved sequence motif known as the basic helix-loop-helix (bHLH) domain. The bHLH motif can be further divided into two subdomains, the HLH domain and an adjacent basic region. The HLH domain, consisting of two amphipathic ␣-helices separated by a loop of amino acid sequence of variable length, is responsible for the dimerization between HLH proteins. The basic region,

Paroxysmal nocturnal hemoglobinuria (PNH) develops in patients who have had a somatic mutation in the X-linked PIG-A gene in a hematopoietic stem cell; as a result, a proportion of blood cells are deficient in all glycosyl phosphatidylinositol (GPI)-anchored proteins. Although the PIG-A mutation explains the phenotype of PNH cells, the mechanism enabling the PNH stem cell to expand is not clear. To examine this growth behavior, and to investigate the role of GPI-linked proteins in hematopoietic differentiation, we have inactivated the pig-a gene by homologous recombination in mouse embryonic stem (ES) cells. In mouse chimeras, pig-a Ϫ ES cells were able to contribute to hematopoiesis and to differentiate into mature red cells, granulocytes, and lymphocytes with the PNH phenotype. The proportion of PNH red cells was substantial in the fetus, but decreased rapidly after birth. Likewise, PNH granulocytes could only be demonstrated in the young mouse. In contrast, the percentage of lymphocytes deficient in GPI-linked proteins was more stable. In vitro, pig-a Ϫ ES cells were able to form pig-a Ϫ embryoid bodies and to undergo hematopoietic (erythroid and myeloid) differentiation. The number and the percentage of pig-a Ϫ embryoid bodies with hematopoietic differentiation, however, were significantly lower when compared with wild-type embryoid bodies. Our findings demonstrate that murine ES cells with a nonfunctional pig-a gene are competent for hematopoiesis, and give rise to blood cells with the PNH phenotype. pig-a inactivation on its own, however, does not confer a proliferative advantage to the hematopoietic stem cell. This provides direct evidence for the notion that some additional factor(s) are needed for the expansion of the mutant clone in patients with PNH. ( J.

Membrane growth factors that are processed to produce soluble ligands may function both as soluble factors and as membrane factors. The membrane growth factor Kitligand (KL), the ligand of the Kit receptor tyrosine kinase, is encoded at the Sl locus, and mice carrying Sl mutations have defects in hematopoiesis, gametogenesis, and melanogenesis. Two alternatively spliced KL transcripts encode two cellassociated KL protein products, KL-1 and KL-2. The KL-2 protein lacks the major proteolytic cleavage site for the generation of soluble KL, thus representing a more stable cellassociated form of KL. We investigated the consequences of exclusive expression of KL-2 in vivo. The KL gene in embryonic stem cells was modified and KL exon 6 was replaced with a PGKneoNTRtkpA cassette by homologous recombination, and mice carrying the Sl KL2 allele were obtained. Sl KL2 ͞Sl KL2 mice had only slightly reduced levels of soluble KL in their serum, suggesting that in vivo KL-2 may be processed to produce soluble KL-2S. The steady-state characteristics of the hematopoietic system and progenitor numbers were normal, and the mutant animals were not anemic. However, mast cell numbers in the skin and peritoneum were reduced and the mutant animals displayed increased sensitivity to sublethal doses of ␥-irradiation. Therefore, KL-2 may substitute for KL-1 in most situations with the exception of the production of mast cells, and induced proteolytic cleavage of KL-1 to produce soluble KL may have a role in the regeneration of hematopoietic tissue after radiation injury.

The primitive streak is the defining feature of the gastrulating mouse embryo. Currently, little is known in the mouse about the mechanisms that mediate the assembly of the primitive streak or about the signaling pathways that specify the different types of mesoderm and endoderm generated from the streak. To gain insight into primitive streak assembly and function, we have conducted a detailed phenotypic characterization of amnionless, a transgene-induced insertional mouse mutation that arrests embryonic development during gastrulation. Our histological and molecular analyses, when examined in the context of the mouse gastrula fate map, lead to the model that middle streak formation is specifically impaired in the amnionless mutant. Significantly, these observations argue that the formation of the middle streak is mediated by a pathway that is genetically separable from those that direct the specification of the distal and proximal streak regions. Intriguingly, our findings from wt ES cell left and right arrow amnionless-/- blastocyst chimeras indicate that this proposed separate pathway for middle streak formation is dependent on amnionless gene functions in the visceral endoderm.