DIFFERENTIATION OF KIDNEY MESENCHYME
269
the LDH isozyme pattern was observed after 48 hours contact with the
inducing spinal cord, and, thereafter, the enzyme pattern changed rapidly
toward the more anodal isozyme bands up to the tenth day of culture, by
which time an isozyme spectrum resembling that of the adult mouse
kidney was observed (Fig. 11). In the control cultures, with mesenchyme
explants cultured alone or in contact with noninducing adult mouse liver
tissue, no shift toward the anodal isozyme bands was observed during
cultivation for 5 days. The change in LDH isozyme composition in
differentiating kidney mesenchyme in vitro is markedly similar to that
occurring in vivo, as is shown in Fig. 11. In fact, the shift is even more
rapid in vitro than in vivo.
The shift in the LDH isozyme pattern toward the more anodal bands
in differentiating kidney mesenchyme indicates that the ratio of polypeptides A:B changes in favor of polypeptide B. Because the total LDH
activity is roughly the same throughout the culture period and there is
no evidence to suggest storage of LDH subunits in the cells, it can be
concluded that the synthesis of LDH subunits changes from synthesis of
A subunits to synthesis of B subunits (Koskimies, 1967a). Whether new
genome activation is needed for the shift was further studied, employing
actinomycin D as a tool.
The morphogenesis of metanephrogenic mesenchyme is actinomycinsensitive during a period of about 24 hours subsequent to the inductormesenchyme contact (Jainchill et al., 1964). The LDH isozyme shift
could be inhibited by similar actinomycin treatment if this was given
before 30 hours of transfilter culture. If treatment was given between
28-30 hours of culture, morphogenesis was no longer inhibited and
tubules were formed, but the normal LDH shift was totally prevented.
If the treatment was given later on, at 32 to 35 hours, the LDH shift was
not inhibited and there was a rapid shift similar to that occurring in
untreated cultures. These results show that some irreversible change in
the polypeptide B-synthesizing machinery occurs at around 30 hours. A
low concentration of actinomycin inhibits rRNA and/or messenger ribonucleic acid (mRNA) synthesis, and, thus, the blockage in the synthesis
of the B subunit of LDH has to be correlated with the impairment of
RNA synthesis.
These results indicate the existence of a critical period in LDH isozyme synthesis at around 30 hours of cultivation, prior to the aggregation period. Aggregating mesenchyme cells are found to contain an
increased concentration of RNA as compared to tubule cells or to cells
not embodied in cellular aggregates (Sundelin et al., unpublished), this
269
the LDH isozyme pattern was observed after 48 hours contact with the
inducing spinal cord, and, thereafter, the enzyme pattern changed rapidly
toward the more anodal isozyme bands up to the tenth day of culture, by
which time an isozyme spectrum resembling that of the adult mouse
kidney was observed (Fig. 11). In the control cultures, with mesenchyme
explants cultured alone or in contact with noninducing adult mouse liver
tissue, no shift toward the anodal isozyme bands was observed during
cultivation for 5 days. The change in LDH isozyme composition in
differentiating kidney mesenchyme in vitro is markedly similar to that
occurring in vivo, as is shown in Fig. 11. In fact, the shift is even more
rapid in vitro than in vivo.
The shift in the LDH isozyme pattern toward the more anodal bands
in differentiating kidney mesenchyme indicates that the ratio of polypeptides A:B changes in favor of polypeptide B. Because the total LDH
activity is roughly the same throughout the culture period and there is
no evidence to suggest storage of LDH subunits in the cells, it can be
concluded that the synthesis of LDH subunits changes from synthesis of
A subunits to synthesis of B subunits (Koskimies, 1967a). Whether new
genome activation is needed for the shift was further studied, employing
actinomycin D as a tool.
The morphogenesis of metanephrogenic mesenchyme is actinomycinsensitive during a period of about 24 hours subsequent to the inductormesenchyme contact (Jainchill et al., 1964). The LDH isozyme shift
could be inhibited by similar actinomycin treatment if this was given
before 30 hours of transfilter culture. If treatment was given between
28-30 hours of culture, morphogenesis was no longer inhibited and
tubules were formed, but the normal LDH shift was totally prevented.
If the treatment was given later on, at 32 to 35 hours, the LDH shift was
not inhibited and there was a rapid shift similar to that occurring in
untreated cultures. These results show that some irreversible change in
the polypeptide B-synthesizing machinery occurs at around 30 hours. A
low concentration of actinomycin inhibits rRNA and/or messenger ribonucleic acid (mRNA) synthesis, and, thus, the blockage in the synthesis
of the B subunit of LDH has to be correlated with the impairment of
RNA synthesis.
These results indicate the existence of a critical period in LDH isozyme synthesis at around 30 hours of cultivation, prior to the aggregation period. Aggregating mesenchyme cells are found to contain an
increased concentration of RNA as compared to tubule cells or to cells
not embodied in cellular aggregates (Sundelin et al., unpublished), this