Takahiro Kunisada

In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: Two distinct waves of c-kit-dependency during melanocyte development

Previous studies on mice bearing various mutations within the c‐kit gene, dominant white spotting (W), indicate the functional role of this tyrosine kinase receptor in the development of melanocytes, germ cells and hematopoietic cells. Despite the availability of mice defective in the c‐kit gene and a respectable understanding of the molecular nature of c‐kit, however, it is not clear at what stage of gestation c‐kit is functionally required for the development of each of these cell lineages. To address this question, we have used a monoclonal anti‐c‐kit antibody, ACK2, as an antagonistic blocker of c‐kit function to interfere with the development of melanocytes during embryonic and postnatal life. ACK2 injected intradermally into pregnant mice entered the embryos where it blocked the proper development of melanocytes. This inhibitory effect was manifested as coat color alteration in the offspring. Furthermore, ACK2 injection also altered the coat color of neonatal and adult mice. Based on the coat color patterns produced by ACK2 administration at various stages before or after birth, the following conclusions are drawn: (i) during mid‐gestation, c‐kit is functionally required during a restricted period around day 14.5 post‐coitum when a sequence of events leading to melanocyte entry into the epidermal layer occurs; (ii) during postnatal life, c‐kit is required for melanocyte activation which occurs concomitantly with the hair cycle which continues throughout life after neonatal development of the first hair.

Cell-cycle-dependent expression of the STK-1 gene encoding a novel murine putative protein kinase

We have cloned a novel putative serine/threonine kinase-encoding gene, designed STK-1, from murine embryonic stem (ES) cell and testis cDNA libraries. The kinase most closely related to STK-1 is Xenopus laevis XLP46 protein kinase which shows 71% amino-acid identity to STK-1 between their kinase domains. Nevertheless, STK-1 is conserved throughout phylogeny with hybridizing sequences being detected in DNA from mammals, amphibians, insects and yeast. STK-1 mRNA is detected in testis, intestine and spleen, tissues that contain a large number of proliferating cells, but not in other tissues. All cell lines tested expressed STK-1 mRNA with levels being dependent upon proliferation rates. In NIH 3T3 cells, STK-1 is expressed in a cell-cycle-dependent fashion. These findings suggest a role for STK-1 in cell growth.

Conditions required for myelopoiesis in murine spleen

While the spleen is an active site for myelopoiesis during the late embryonal and perinatal stages, the activity is gradually lost later. However, myelopoiesis in the adult spleen can be reactivated by irradiation or various stimulants. In this study we investigated factors which determine the myelopoiesis-supporting activity in the adult spleen. To address this question, we used scid mouse because virtually no lymphocytes, which might compete in the splenic microenvironment with hematopoietic progenitors, are present there. The results demonstrated: 1. Even in scid mouse, the myelopoiesis-supporting activity in the spleen is lost within a week after birth as in normal mice. 2. While myelopoiesis does not occur in the spleen of unstimulated scid mouse by bone marrow transfer alone, myelopoiesis in the spleen is reactivated by irradiation or lipopolysaccharide (LPS) application. 3. Myelopoiesis in the spleen induced by irradiation is dependent on c-kit and its ligand steel factor (SLF), because it was suppressed completely by the monoclonal antibody (mAb) against c-kit. 4. The expression of SLF transcripts in the spleen was enhanced after irradiation. These results suggest that the factor which determines myelopoietic activity in the spleen resides primarily in the status of the splenic microenvironment.

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.

Control of Intramarrow B-Cell Genesis by Stromal Cell-Derived Molecules

A remarkable progress has been made in the investigation of B cell-genesis since Whitlock and Witte have reported a long-term culture system of bone marrow B cells (Whitlock and Witte 1982). Although some more stromal cell molecules are yet to be cloned before concluding that we know all molecules involved in the intramarrow B cellgenesis, the basic framework of the molecular requirements for B cell-genesis has been understood to a considerable extent. As a result of this progress, a numbers of methods which use stromal cell lines and recombinant cytokines to control B cell-genesis in vitro are presently available (for review see Kincade et al. 1989; Rolink and Melchers 1991, Kunisada et al. 1992). It is also true, however, that some inconsistency is still present among the previous results on the growth signal requirement of B precursors. Given that control of cell proliferation in higher organisms might be achieved in a redundant and failsafe manner, this redundancy could be a source of the inconsistency among the previous results. Thus, in this article, we would like to re-examine three key propositions which we have been stated in the previous studies and discuss them in detail in light of the previous results of ours and other groups.

Defect of Scid Mouse Revealed in In Vitro Culture Systems

Since a series of analyses by Bosma’s group on the scid mouse demonstrated that there are virtually no lymphocytes in this mutant strain (Bosma et al 1983), it has been attracting those investigating the development of lymphocytes from hemopoietic stem cells. A pioneering study by Whitlock et al. enabled us to follow a considerable part, if not all, of the process during intramarrow B cell development reproducibly in an in vitro culture system (Whitlock and Witte 1982; Whitlock et al. 1984). In this situation, expecting that scid mouse might contribute to the understanding of B cell development, we started to analyze the defect of scid mouse in long-term bone marrow culture. The question which was addressed at the beginning by using the Whitlock-Witte type long-term bone marrow culture (W-LTBC) was whether or not B lineage cells were generated in the scid mouse culture. If the defect of scid mouse affects the commitment process of pluripotent stem cells into lymphoid cells, B lineage cells would not appear in the W-LTBC of scid bone marrow. On the other hand, if the defect affects the later differentiation stage, B lineage cells whose differentiation is arrested would be generated in this culture. In this article, we first briefly describe our previous results on scid mouse which utilized classical long-term bone marrow culture methods, then describe the system which we are currently using for the analysis of B cell differentiation, and finally our analysis on the defect of scid mouse in this culture system.