The genome-wide evaluation of the multi-potent progenitor cell mesenchyme and early stage nephron transcriptome after birth showed sequential activation or inactivation of many genes. The use of tissue after birth to obtain expression data eliminates potential artifacts introduced during in vitro
studies of sequential activation after induction. The period after birth allows serial measurements of transcripts early after induction because there is such an abrupt change in the behavior of the progenitors. By P3 all remaining capping mesenchyme has begun conversion into renal vesicles. Because the inductive period and the time to show signs of differentiation after induction each take 12-24 hours (reviewed in Saxen [12
]), the final wave of induction to form nephrons had begun by P2. This strongly suggests, therefore, that the Cited1(+) cells at P2, which still constitute most of the GFP(+) capping mesenchyme (See additional file 1
), were likely to have been induced and to be different from the capping mesenchyme on P0 and P1 when the bulk of the high GFP expressing cells was un-induced. There is clearly a progression of molecular events post-induction, with the down-regulation of Cited1 corresponding to a more advanced state of induction or possibly a state of commitment.
Our results confirmed differences in expression level between capping mesenchyme and renal vesicle found among a series of markers previously reported by Mugford et.al
]. Seven of the nine markers, which distinguished between progenitor cell mesenchyme and vesicles by in situ
(Cited1, Bbx, Eya1, Osr1, Six2, Dpf3
, and Meox1
), showed greater than a 2-fold change in level of expression between P0 and P4. The trend was correct for the remaining two markers (Hoxc5
), but did not reach a 2-fold change in level. Our data extend this list significantly by adding more than 400 genes that change in level between P0, when mesenchyme is abundant, and P4, when GFP primarily marks renal vesicles. This provides a significant resource that can be used when monitoring induction of the mesenchyme.
Discovery of genes that have a spatially restricted pattern of expression during the process of differentiation helps to identify pathways that may be needed for nephron formation. We found that Fat3
was expressed in the cap at P0 and was down regulated by P2. The Fat genes encode cadherin-type proteins with cell-cell adhesion properties. With mutation of Fat genes in Drosophila
, there is overgrowth of tissues [13
]. It is interesting in Drosophila
that Fat is part of a pathway involved in the suppression of wingless, a homolog of a murine gene (Wnt4
) that is expressed after mesenchymal induction and is required for nephron development [14
]. This suggests that the down-regulation of Fat3
may be needed before activation of Wnt4
in the induced mesenchyme.
We also identified early up-regulated expression of genes in several different pathways and examined them by in situ
hybridization. One gene, Bmp2
, was activated early in the induced capping mesenchyme next to one side of the ureteric bud branch tips. The secreted protein encoded by Bmp2
is known to regulate both the branching of the ureteric bud tips and the proliferation of their cells [15
]. With the localized expression, Bmp2
may regulate the regional growth of cells within the tips. Its expression is also of interest because along with Clu
it is activated in the induced cap before morphologic changes are evident. It seems reasonable that capping mesenchymal cells initiating renal vesicle formation would begin expressing genes associated with renal vesicles, although to our knowledge this has not been previously demonstrated.
It is interesting that these induced genes, which are associated with differentiation, are co-expressed with Six2. Six2 is necessary to maintain the population of multi-potential progenitors; however, it has not been shown to be sufficient. The co-expression of genes involved in differentiation in a subset of Six2 (+) cells suggests that Six2 is not sufficient to block transcription of some genes associated with differentiation in the presence of an inductive signal.
We also observed an interesting surge in cell proliferation that preceded the expression of markers of differentiation. The sequence of events suggests the possibility that proliferation promotes the reprogramming of renal progenitors. This type of mechanism has been described during reprogramming of somatic cells into pluri-potent stem cells [16
]. An increase by P2 in proliferation, evidence of a change in the behavior of progenitors, is also consistent with induction of the cells by P2.
Also of note, the gene expression profiles of early forming nephrons differed before and after birth. The genes included those encoding enzymes of the glycolytic pathway. The shift in transcription is compatible with a response by the population of progenitors to a change in the microenvironment, such as a post-natal increase in oxygen levels. Prior to birth the kidney is fed by deoxygenated blood, blood with the same oxygen content as that returning to the placenta. In addition, oxygen delivery to the nephrogenic region is further limited because the tissue is relatively avascular. Relative physiologic hypoxia, such as this, is known to cause an increase in transcription of genes encoding enzymes of the glycolytic pathway [17
]. Changes in levels of expression of other genes that are regulated in an oxygen-dependent manner, such as P4ha1
, and Txnip
, provide further supportive evidence of an increase in oxygenation in the progenitors after birth.
Cellular fates of placental cytotrophoblasts [18
], hematopoietic progenitors [19
], human neural stem cells [20
], bone marrow stromal cells [21
], and human embryonic stem cells [22
] have been shown to be altered by oxygen. The fate of murine embryonic stem cells also appears to be coupled to metabolism [23
]. It seems reasonable, therefore, to speculate that a change in the microenvironment, such as a change in oxygenation within the physiologic range, might also lead to a change in behavior of the multi-potential progenitors in the kidney in vivo
Lastly, the final nephron endowment is clearly a result of regulation of the balance between the rate of progenitor renewal and the rate of differentiation. Simple geometry might play an important role in the balance in both mice and humans. In the early kidney, the capping mesenchyme layer is relatively thick. As branching morphogenesis proceeds, the number of branch tips will expand geometrically, subdividing the capping mesenchyme while also inducing it. Unless renewal of the progenitors similarly expands, the cap around each tip will thin. And, at some point it will no longer be able to promote further branching. After branching ends, if usage of cells to make nephrons exceeds the renewal rate, nephron production will consume the remaining cap mesenchyme. In humans this simple model fits nicely with the completion of nephron production because there is a prolonged period of nephron production without branching. In the mouse, however, there is an abrupt end to both nephron production and branching morphogenesis. The end coincides with birth and with a change in metabolism that is compatible with an increase in oxygenation of the progenitors. Coupled with the known effects of oxygen on cellular fate, the events suggest a possible trigger in mice at birth that shifts the balance between renewal and differentiation of progenitors favoring differentiation. We speculate that the trigger then limits the lifespan of the population of progenitors and causes the production of nephrons in mice to end abruptly.