These data demonstrate that the GGAs are essential proteins in mammalian cells. They also provide two key findings concerning the issue of redundancy. First, while the individual loss of either GGA1 or GGA3 in mice is well-tolerated, the absence of GGA2 causes either embryonic or neonatal lethality, depending on the genetic background of the strain. And second, the combined loss of GGA1 and GGA3 results in a high incidence of mortality during the first day of life. The requirement for GGA2 indicates that this adaptor protein carries out a vital function that cannot be compensated for by GGA1 and GGA3. However, the nature of this critical function(s) is not clear at this time.
It is of interest that the expression level of GGA2 in the rodent brain is highest during embryonic development and early life but decreases substantially two weeks after birth. This is consistent with GGA2 serving a very important function during the embryonic and neonatal stage. This finding, however, does not obviate a role for GGA2 in adult animals since GGA2 is highly expressed in adipose tissue and fibroblasts (; Figure S3A
). In contrast, brain expression of GGA1 and GGA3 remains consistently high from late embryogenesis through adulthood. The fact that the levels of GGA1 and GGA3 are somewhat greater in the brain than the level of GGA2 during development excludes the possibility that GGA2 is the sole GGA present in this organ. However, the GGA levels in the other tissues of the embryo could not be determined. In the C57BL/6NJ background, the Gga2
null newborn mice are small, but histological examination of their organs on day 1 did not reveal any obvious abnormalities. These mice did have quite low blood glucose levels even though they had detectable milk in their stomachs indicating that they are able to suckle and take in milk. At this point the etiology of the low blood glucose is unknown and its possible contribution to the early demise of these mice is not clear.
Interestingly, the one female Gga2
null mouse that survived is now an adult and has no obvious abnormalities to date other than being 25% smaller than its littermates. This indicates that GGA2 may be dispensable after the neonatal period and is consistent with the low expression level seen in rodent and human adult brains. This female mouse has been crossed with a Gga2
het male and produced two small litters (5 and 3 pups, all of which died). The finding that the Gga2
null mice in the mixed C57BL/6J and 129/Ola background (SYA176 strain) die before embryonic day 9 suggested that genetic background may influence the tolerance to the loss of GGA2. However, when the SYA176 mice were backcrossed into the C57BL/6J background, the embryonic lethal phenotype remained, as opposed to the neonatal lethality of the TIGM Gga2
null mice. A closer look at the genetics of the two strains revealed that they are not identical as the SYA176 strain is in the C57BL/6J background while the TIGM strain is in the C57BL/6NJ background. It is thought that the many phenotypic differences among such closely related substrains could be due to as yet undiscovered genetic modifiers 
. Furthermore, changes in gene expression clustered up to 28 MB of several knockout loci have been reported despite eleven generations of backcrosses into the C57BL/6 background, likely due to polymorphisms originating from the parental ES cells that alter gene expression 
. Another remote possibility is that some important nucleotide element, such as a micro RNA, is situated in the 2 kb segment of intronic sequence between the upstream gene-trap insertion site in the SYA176 strain and the downstream insertion site in the TIGM strain, such that this critical element is only lacking in the chimeric GGA2 pre-mRNA in the SYA176 strain, and therefore contributes to its embryonic lethality 
The finding that loss of either GGA1 or GGA3 is well tolerated whereas the combined loss of both these GGAs results in a high incidence of neonatal lethality raises the possibility that these two GGAs share at least one vital function that cannot be carried out by GGA2. In this regard, both GGA1 and GGA3 are more similar to one another than to GGA2 in that both are highly phosphorylated, subject to autoinhibition and bind ubiquitin whereas GGA2 exhibits none of these properties 
. Moreover, crystallographic studies suggest that GGA1 and GGA3 VHS domains are more similar to each other than to the GGA2 VHS domain 
. An alternative possibility for the partially lethal phenotype associated with the combined loss of GGA1 and GGA3 is that the expression level of GGA2 in some critical organ/tissue is not sufficient to overcome the loss of the other GGAs. It is of note that the Gga1/3
null mice that lived beyond one month had a normal lifespan although their weight remained significantly lower than that of wt
mice. Thus, as in the case of GGA2, GGA1 and GGA3 may not be required for survival after the neonatal-period.
While this manuscript was in preparation, two independent studies were published in which the single GGA
gene in Drosophila melanogaster
was knocked-down using GGA transgenic RNAi fly lines 
. Although the study by Hirst and Carmichael did not show any obvious phenotype when greater than 95% of the GGA is depleted in flies, the authors did note a gender specific higher expression of GGA in male flies 
. In contrast, Eissenberg et al
reported that GGA depletion resulted in lethality during early pupation under certain circumstances 
. The basis for this discrepancy is not clear at this point.
The mammalian GGAs have been implicated in the trafficking of a number of transmembrane proteins including the mannose 6-phosphate receptors, sortilin, SorLA and BACE1 in in vitro studies. The knock-out mice described herein should be useful in determining the physiological roles of GGA1 and GGA3 in the intracellular pathways mediated by these receptors.