Tissue-specific regulation and function of Grb10 during growth and neuronal commitment

Abstract

Growth-factor receptor bound protein 10 (Grb10) is a signal adapter protein encoded by an imprinted gene that has roles in growth control, cellular proliferation, and insulin signaling. Additionally, Grb10 is critical for the normal behavior of the adult mouse. These functions are paralleled by Grb10's unique tissue-specific imprinted expression; the paternal copy of Grb10 is expressed in a subset of neurons whereas the maternal copy is expressed in most other adult tissues in the mouse. The mechanism that underlies this switch between maternal and paternal expression is still unclear, as is the role for paternally expressed Grb10 in neurons. Here, we review recent work and present complementary data that contribute to the understanding of Grb10 gene regulation and function, with specific emphasis on growth and neuronal development. Additionally, we show that in vitro differentiation of mouse embryonic stem cells into alpha motor neurons recapitulates the switch from maternal to paternal expression observed during neuronal development in vivo. We postulate that this switch in allele-specific expression is related to the functional role of Grb10 in motor neurons and other neuronal tissues.

Keywords: Grb10; epigenetics; genomic imprinting; motor neurons; neurodevelopment.

Fig. 1.

Grb10 is expressed maternally from most adult tissues and paternally in neuronal tissues. (A) A schematic of the Grb10 locus is shown, with the allelic-specific expression of alternative promoters indicated (maternal and paternal alleles, arrow above the line and below the line, respectively). The imprinting control region (ICR) is designated by a yellow box. Maternal allele-specific DNA methylation, evident in all characterized tissues, is indicated by black lollipops, above the line. The reverse primer location for expression analysis is shown by its name, 4R. The location of the amplicon used for allele-specific (A.S.) analysis is similarly indicated. (B) Expression of transcripts from alternative promoters of Grb10 is shown in adult brain, adult liver, mESCs, and adult spinal cord. (C) Allele-specific expression as measured via SNP-dependent Cac8I restriction enzyme digests is shown in brain and liver from Mus Castaneus/EiJ (Cast) × C57BL/6J (B6) hybrid adult mice. Expression of the maternal allele is indicated by the 199-bp and 112-bp bands after restriction digest whereas the 311-bp band indicates paternal expression.

Fig. 2.

Grb10 protein partners. Shown is a schematic of the five major protein domains of Grb10: the proline-rich (PR) region, the ras-associating domain (RA), the pleckstrin homology domain (PH), the Src homology 2 domain (SH2), and the intervening domain (BPS). The known protein partners defined by in vivo interactions and immunoprecipitation are shown. Confirmed interactions with a specific Grb10 domain are indicated with a solid line whereas inferred interactions are indicated with a dotted line. The amino acids that are phosphorylated by Grb10 kinases are indicated above each protein.

Fig. 3.

Grb10 expression in mouse embryonic stem cells after CTCF knockdown results in the up-regulation of the major isoform. (A) Representative Western blot for CTCF and tubulin comparing cells infected with CTCF shRNA (KD) and scramble control (Scr). Average densitometry of the CTCF bands across three replicates is shown in the bar graph. (B) CTCF knockdown results in loss of CTCF enrichment over background at the Grb10 ICR compared with IgG. (C) CTCF knockdown causes a small but significant increase in the expression of the major Grb10 isoform (1A).

Fig. 4.

Promoter use switches from the maternal to paternal isoform of Grb10 during motor neuron differentiation in vitro. (A) Schematic of motor neuron differentiation. Mouse ESCs that express eGFP under the control of the promoter of an early motor neuron marker, Hb9 (d0), are used to form embryoid bodies (EBs). To induce neuralization, EBs are treated with retinoic acid (RA) on day 3 of differentiation. To induce ventralization of neural tissue and motor neurons, Smoothened agonist (SAG) is added to the EB culture on differentiation day 4. Glial cell-derived neurotrophic factor (GDNF) is added to EB culture post day 5 to promote neuronal survival. On day 7 of differentiation, EBs are dissociated and FACs-sorted based on eGFP expression, with an approximate 20% yield of motor neurons. (B–D) The maternally expressed major (1A) isoform of Grb10 is repressed during the differentiation process whereas the paternally expressed neuronal-specific (1B) isoform is up-regulated. These changes in expression correlate with the maturation of motor neurons as quantified by Hb9 expression, a marker for motor-neuron fate. For comparison, levels of expression of each of these genes in the adult spinal cord are shown.